U.S. patent application number 17/312247 was filed with the patent office on 2022-02-03 for generating capture probes for spatial analysis.
This patent application is currently assigned to 10x Genomics, Inc.. The applicant listed for this patent is 10x Genomics, Inc.. Invention is credited to Zachary Bent, Rajiv Bharadwaj, Erik Leonard Henrik Borgstrom, Joshua Delaney, Meghan L. F. Frey, Alexander Gagnon, Stefania Giacomello, Adam Lowe, Geoffrey McDermott, Tarjei Sigurd Mikkelsen, Eswar Prasad Ramachandran Iyer, Michael Schnall-Levin, Preyas Shah, Augusto Manuel Tentori, Yifeng Yin.
Application Number | 20220033888 17/312247 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220033888 |
Kind Code |
A1 |
Schnall-Levin; Michael ; et
al. |
February 3, 2022 |
GENERATING CAPTURE PROBES FOR SPATIAL ANALYSIS
Abstract
The present disclosure relates to compositions and methods for
generating capture probes on a substrate for identifying the
location of analytes in a biological sample.
Inventors: |
Schnall-Levin; Michael; (San
Francisco, CA) ; Ramachandran Iyer; Eswar Prasad;
(Sunnyvale, CA) ; Yin; Yifeng; (Elk Grove, CA)
; Mikkelsen; Tarjei Sigurd; (Pleasanton, CA) ;
Shah; Preyas; (Pleasanton, CA) ; McDermott;
Geoffrey; (Pleasanton, CA) ; Lowe; Adam;
(Mountain House, US) ; Delaney; Joshua;
(Pleasanton, CA) ; Tentori; Augusto Manuel;
(Pleasanton, CA) ; Bharadwaj; Rajiv; (Pleasanton,
CA) ; Giacomello; Stefania; (Stockholm, SE) ;
Gagnon; Alexander; (Pleasanton, CA) ; Frey; Meghan L.
F.; (Pleasanton, CA) ; Bent; Zachary;
(Pleasanton, CA) ; Borgstrom; Erik Leonard Henrik;
( rsta, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
10x Genomics, Inc. |
Pleasanton |
CA |
US |
|
|
Assignee: |
10x Genomics, Inc.
Pleasanton
CA
|
Appl. No.: |
17/312247 |
Filed: |
December 6, 2019 |
PCT Filed: |
December 6, 2019 |
PCT NO: |
PCT/US2019/065013 |
371 Date: |
June 9, 2021 |
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International
Class: |
C12Q 1/6837 20060101
C12Q001/6837; C12Q 1/6881 20060101 C12Q001/6881 |
Claims
1-253. (canceled)
254. A method for generating a spatial array comprising: (a)
providing a substrate comprising a plurality of first
oligonucleotides, wherein a first oligonucleotide of the plurality
of first oligonucleotides is attached to a region of the substrate;
and (b) attaching a second oligonucleotide to an unattached end of
the first oligonucleotide, wherein the second oligonucleotide
comprises a spatial barcode.
255. The method of claim 254, wherein the method further comprises
after (b): (c) attaching a third oligonucleotide to an unattached
end of the second oligonucleotide.
256. The method of claim 254, wherein the first oligonucleotide is
attached to the substrate through its 5'-end and the second
oligonucleotide is generated using spatially directed in situ
synthesis on the 3'-end of the first oligonucleotide.
257. The method of claim 254, wherein the second oligonucleotide
has a length selected from 8 nucleotides, 9 nucleotides, 10
nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14
nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18
nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22
nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26
nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30
nucleotides.
258. The method of claim 256, wherein the in situ synthesis is
light-directed synthesis.
259. The method of claim 254, wherein two or more second
oligonucleotides are attached to two or more first
oligonucleotides, wherein a member of the two or more second
oligonucleotides differs from another member of the two or more
second oligonucleotides.
260. The method of claim 254, wherein the first oligonucleotide
and/or the second oligonucleotide comprises a protected
phosphodiester and exocyclic amine moieties.
261. The method of claim 254, wherein the substrate further
comprises a substantially complementary oligonucleotide hybridized
to the first oligonucleotide.
262. The method of claim 254, wherein the first oligonucleotide has
a length of at least about 2 nucleotides, 5 nucleotides, 8
nucleotides, 10 nucleotides, 15 nucleotides, 18 nucleotides, 20
nucleotides, 22 nucleotides, 25 nucleotides, 27 nucleotides, 30
nucleotides, 33 nucleotides, 35 nucleotides, or 40 nucleotides.
263. The method of claim 254, wherein the first oligonucleotide is
synthesized and subsequently attached to the substrate.
264. The method of claim 260, wherein the phosphodiester and
exocyclic amine moieties of the first oligonucleotide are protected
prior to attachment of the first oligonucleotide to the substrate
or the phosphodiester and exocyclic amine moieties of the first
oligonucleotide are protected after attachment of the first
oligonucleotide to the substrate.
265. The method of claim 254, wherein the first oligonucleotide
comprises a cleavage site.
266. The method of claim 254, wherein the first oligonucleotide is
attached to the substrate through a linker and wherein the linker
comprises a chemical group comprising: silane, amine, peptide,
polyethylene glycol, biotin, desthiobiotin, streptavidin, thiol,
acrydite, azide, digoxigenin, hexynyl, or a combination
thereof.
267. The method of claim 266, wherein the linker is a branched
linker or a dendrimeric linker.
268. The method of claim 255, wherein the third oligonucleotide is
attached to the second oligonucleotide by ligation.
269. The method of claim 268, wherein any protected moieties of the
first and second oligonucleotides are deprotected prior to ligating
the third oligonucleotide to the second oligonucleotide.
270. The method of claim 255, wherein the third oligonucleotide
comprises a capture domain of at least 6 nucleotides in length at
the 3'-end, wherein the capture domain comprises a poly-T sequence
or a random sequence of 6 to 20 nucleotides in length.
271. A method of generating a spatial array comprising: (a)
providing an array comprising a plurality of oligonucleotides,
wherein the 3' end of an oligonucleotide of the plurality of
oligonucleotides is attached to a substrate; (b) providing a
plurality of primers, wherein a primer of the plurality of primers
is substantially complementary to a portion of the oligonucleotide;
(c) extending the primer using the oligonucleotide as a template,
thereby generating a first oligonucleotide with a free 3' end (d)
extending the first oligonucleotide to produce a 3' overhang; (e)
providing a splint oligonucleotide that hybridizes to the 3' end of
the first oligonucleotide; and (f) ligating a second
oligonucleotide to the 3' end of the first oligonucleotide, wherein
the second oligonucleotide comprises a sequence that is
substantially complementary to a portion of the splint
oligonucleotide, and wherein the second oligonucleotide comprises a
capture domain, thereby generating a spatial array.
272. The method of claim 271, wherein the oligonucleotide of the
plurality of oligonucleotides comprises a spatial barcode, a first
constant sequence, a second constant sequence, and combinations
thereof.
273. The method of claim 272, wherein the second oligonucleotide
further comprises a unique molecular identifier, a spatial barcode,
a constant sequence, wherein the constant sequence is substantially
complementary to a portion of the splint oligonucleotide, and
combinations thereof.
274. The method of claim 272, wherein the capture domain comprises
a poly(T) sequence or a gene-specific sequence.
275. The method of claim 271, wherein the 3' end overhang of step
(d) is produced by a template-independent polymerase.
276. A method of generating an array comprising: (a) providing a
substrate comprising a plurality of features immobilized on the
substrate, wherein a feature of the plurality of features comprises
a first oligonucleotide, and wherein the first oligonucleotide
comprises a first spatial barcode; (b) delivering a second
oligonucleotide to the first oligonucleotide, wherein the second
oligonucleotide comprises a second spatial barcode and a capture
domain; and (c) attaching the first oligonucleotide and the second
oligonucleotide, thereby generating a capture probe comprising the
first spatial barcode, the second spatial barcode, and the capture
domain.
277. The method of claim 276, wherein the first oligonucleotide
comprises a unique molecular identifier and is associated with one
of a number of different first spatial barcodes and the second
oligonucleotide is associated with one of a number of different
second spatial barcodes.
278. The method of claim 276, wherein the attaching in step (c)
comprises: (i) ligating the first oligonucleotide and the second
oligonucleotide, wherein the ligating comprises a ligase; or (ii)
the first oligonucleotide and the second oligonucleotide are
attached by a chemical coupling reaction.
279. The method of claim 276, wherein the first oligonucleotide
comprises a 3' end that is masked by a protective group, wherein
the protective group is removed prior to delivering the second
oligonucleotide to the first oligonucleotide.
280. The method of claim 279, wherein the protective group is a
photocleavable protective group, wherein the photocleavable
protective group is protected by applying a photomask, and wherein
the photocleavable protective group is removed by exposing the
first oligonucleotide to light.
281. The method of claim 276, wherein a first feature is separated
from other features of the plurality by one or more physical
barriers, wherein the one or more physical barriers comprise: (i) a
hydrophobic surface; (ii) form one or more chambers, wherein the
first oligonucleotide is delivered to a chamber of the one or more
chambers; and (iii) one or more chamber wall comprise a tapered end
in contact with the substrate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/777,521, filed Dec. 10, 2018, U.S. Provisional
Patent Application No. 62/779,342, filed Dec. 13, 2018, U.S.
Provisional Patent Application No. 62/779,348, filed Dec. 13, 2018,
U.S. Provisional Patent Application No. 62/788,867, filed Jan. 6,
2019, U.S. Provisional Patent Application No. 62/788,871, filed
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The contents of each of these applications are incorporated herein
by reference in their entireties.
BACKGROUND
[0002] Cells within a tissue of a subject have differences in cell
morphology and/or function due to varied analyte levels (e.g., gene
and/or protein expression) within the different cells. The specific
position of a cell within a tissue (e.g., the cell's position
relative to neighboring cells or the cell's position relative to
the tissue microenvironment) can affect, e.g., the cell's
morphology, differentiation, fate, viability, proliferation,
behavior, and signaling and cross-talk with other cells in the
tissue.
[0003] Spatial heterogeneity has been previously studied using
techniques that only provide data for a small handful of analytes
in the contact of an intact tissue or a portion of a tissue, or
provide a lot of analyte data for single cells, but fail to provide
information regarding the position of the single cell in a parent
biological sample (e.g., tissue sample).
[0004] Arrays that capture spatial information of analytes within a
biological sample can be prepared in various ways. Arrays can be
designed with pluralities of capture probes. Capture probes can be
designed to capture a substantial population of analytes (e.g.,
mRNA) or to capture specific (e.g., gene specific) analytes. The
methods described herein provide for ways of preparing arrays with
capture probes and capturing analytes.
SUMMARY
[0005] Provided herein are methods of generating a spatial array
including: (a) providing an array including a plurality of
oligonucleotides, where the 3' end of an oligonucleotide of the
plurality of oligonucleotides is attached to a substrate, (b)
providing a plurality of primers, where a primer of the plurality
of primers is substantially complementary to a portion of the
oligonucleotide, (c) extending the primer using the oligonucleotide
as a template, thereby generating a first oligonucleotide with a
free 3' end, (d) extending the first oligonucleotide to produce a
3' overhang, (e) providing a splint oligonucleotide that hybridizes
to the 3' end of the first oligonucleotide, and (f) ligating a
second oligonucleotide to the 3' end of the first oligonucleotide,
where the second oligonucleotide includes a sequence that is
substantially complementary to a portion of the splint
oligonucleotide, and where the second oligonucleotide includes a
capture domain, thereby generating a spatial array.
[0006] In some embodiments of methods of generating a spatial
array, the oligonucleotide of the plurality of oligonucleotides
includes a spatial barcode. In some embodiments, the
oligonucleotide of the plurality of oligonucleotides includes a
first constant sequence. In some embodiments of methods of
generating a spatial array, the oligonucleotide of the plurality of
oligonucleotides further includes a second constant sequence. In
some embodiments of methods of generating a spatial array, the
oligonucleotide of the plurality of oligonucleotides has a length
of between 20 and 60 nucleotides.
[0007] In some embodiments of methods of generating a spatial
array, the second oligonucleotide further includes a constant
sequence, and where the constant sequence is substantially
complementary to a portion of the splint oligonucleotide. In some
embodiments of methods of generating a spatial array, the second
oligonucleotide further includes a unique molecular identifier. In
some embodiments of methods of generating a spatial array, the
second oligonucleotide further includes a spatial barcode.
[0008] In some embodiments of methods of generating a spatial
array, the capture domain includes a poly-T sequence. In some
embodiments of methods of generating a spatial array, the capture
domain includes a gene-specific sequence.
[0009] In some embodiments of methods of generating a spatial
array, the 3' end overhang of step (d) is produced by an enzyme. In
some embodiments of methods of generating a spatial array, the
enzyme is a template-independent polymerase. In some embodiments of
methods of generating a spatial array, the template-independent
polymerase is terminal deoxynucleotidyl transferase (TdT) or
poly(A) polymerase.
[0010] In another aspect, provided herein are methods for
determining the location of an analyte in a biological sample
including (a) contacting the biological sample with a spatial array
produced by any of the variety of methods herein (b) permeabilizing
the biological sample to allow the analyte in the biological sample
to specifically bind with a capture domain on the spatial array,
and (c) identifying the analyte specifically bound to the capture
domain, thereby determining the spatial location of the analyte in
the biological sample.
[0011] In some embodiments of methods for determining the location
of an analyte in a biological sample, a second analyte in the
biological sample interacts with a second capture domain on the
spatial array, and step (c) includes identifying the second analyte
specifically bound to the second capture domain, thereby
determining the spatial location of the analyte in the second
biological sample.
[0012] In another aspect, provided herein are methods for preparing
a spatial array, including (a) contacting an array including one or
more pluralities of first oligonucleotides with a hydrogel
including one or more pluralities of second oligonucleotides, where
a first oligonucleotide of the one or more pluralities of first
oligonucleotides includes a capture domain, a second
oligonucleotide of the one or more pluralities of second
oligonucleotides includes a capture domain, or both, (b) attaching
the first oligonucleotide to the second oligonucleotide to generate
a capture probe including a capture domain, (c) reducing the size
of the hydrogel including the capture probe by removing water to
form a shrunken hydrogel including the capture probe, thereby
generating the spatial array.
[0013] In some embodiments of methods for preparing a spatial
array, the one or more pluralities of second oligonucleotides are
immobilized in the hydrogel. In some embodiments of methods for
preparing a spatial array, the immobilized second oligonucleotides
are partitioned such that a first population of the immobilized
second oligonucleotides is partitioned away from another population
of immobilized second oligonucleotides.
[0014] In some embodiments of methods for preparing a spatial
array, the immobilized second oligonucleotides are partitioned into
a plurality of second oligonucleotide partitions such that
different populations of the immobilized second oligonucleotides
are partitioned away from each other. In some embodiments of
methods for preparing a spatial array, a first immobilized second
oligonucleotide of a partitioned population of immobilized second
oligonucleotides is different from a second immobilized second
oligonucleotide of another partitioned population of immobilized
second oligonucleotides. In some embodiments of methods for
preparing a spatial array, the first immobilized second
oligonucleotide, the second immobilized second oligonucleotide, or
both include a spatial barcode.
[0015] In some embodiments of methods for preparing a spatial
array, the first immobilized second oligonucleotide of a
partitioned population of immobilized second oligonucleotides
includes a spatial barcode that is different from a spatial barcode
of the second immobilized second oligonucleotide of another
partitioned population of immobilized second oligonucleotides.
[0016] In some embodiments of methods for preparing a spatial
array, one or more second oligonucleotide partitions include a
population of immobilized capture probes including a spatial
barcode different from a spatial barcode of the population of
immobilized capture probes of another second oligonucleotide
partition.
[0017] In some embodiments of methods for preparing a spatial
array, the one or more pluralities of first oligonucleotides are
partitioned. In some embodiments of methods for preparing a spatial
array, the one or more pluralities of first oligonucleotides are
partitioned into a first oligonucleotide partitions. In some
embodiments of methods for preparing a spatial array, one or more
first oligonucleotide partitions include a population of first
oligonucleotides that is different from first oligonucleotides in
another first oligonucleotide partition. In some embodiments of
methods for preparing a spatial array, a first oligonucleotide of a
population of first oligonucleotides in one or more first
oligonucleotide partitions includes a spatial barcode. In some
embodiments of methods for preparing a spatial array, the first
oligonucleotide of a population of first oligonucleotides in one or
more first oligonucleotide partitions includes a spatial barcode
that is different from the spatial barcode of a first
oligonucleotide of a population of first oligonucleotides in
another first oligonucleotide partition. In some embodiments of
methods for preparing a spatial array, a spatial barcode of the
first oligonucleotide of a population of first oligonucleotides in
one or more first oligonucleotide partitions is different from a
spatial barcode of a second oligonucleotide of a population of
second oligonucleotides in one or more second oligonucleotide
partitions. In some embodiments of methods for preparing a spatial
array, a first oligonucleotide partition includes a first
population of first oligonucleotides including a spatial barcode
that is different from a spatial barcode of a second population of
first oligonucleotides in another first oligonucleotide
partition.
[0018] In some embodiments of methods for preparing a spatial
array, the reduction in the size of the hydrogel in step (c) is
isometric. In some embodiments of methods for preparing a spatial
array, the removal of water in step (c) includes a dehydrating
solvent. In some embodiments of methods for preparing a spatial
array, the solvent includes at least one of an alcohol, a ketone,
an aldehyde, acetonitrile, or a combination thereof. In some
embodiments of methods for preparing a spatial array, the solvent
includes at least one of glycerol, ethanol, acetonitrile, acetone,
2-propanol, and a combination thereof. In some embodiments, the
removal of water in step (c) includes the use of a salt. In some
embodiments of methods for preparing a spatial array, the salt
includes an inorganic salt. In some embodiments of methods for
preparing a spatial array, the removal of water in step (c)
includes an acid. In some embodiments of methods for preparing a
spatial array, the removal of water in step (c) includes a
dehydrating process.
[0019] In some embodiments of methods for preparing a spatial
array, the shrunken hydrogel is about 2 to about 10 fold smaller in
a linear dimension than the size of the hydrogel prior to step (c)
pre-shrunk hydrogel. In some embodiments of methods for preparing a
spatial array, the shrunken hydrogel is at least 2-fold smaller in
a linear dimension than the pre-shrunk hydrogel. In some
embodiments of methods for preparing a spatial array, the shrunken
hydrogel is at least 4-fold smaller in a linear dimension than the
pre-shrunk hydrogel. In some embodiments of methods for preparing a
spatial array, the shrunken hydrogel is at least 8-fold smaller in
a linear dimension than the pre-shrunk hydrogel.
[0020] In some embodiments of methods for preparing a spatial
array, attaching includes a polymerase chain reaction. In some
embodiments of methods for preparing a spatial array, the hydrogel
includes a DNA polymerase, a plurality of primers, and a plurality
of nucleotides. In some embodiments of methods for preparing a
spatial array, members of a plurality of first oligonucleotides are
cross-linked to the hydrogel. In some embodiments of methods for
preparing a spatial array, the capture probe is amplified. In some
embodiments of methods for preparing a spatial array, the capture
probe is amplified prior to the reduction of the size of the
hydrogel.
[0021] In some embodiments of methods for preparing a spatial
array, the hydrogel is prepared from hydrogel subunits including at
least one of: acrylamide, bis-acrylamide, a poly(ethylene glycol),
a polyaliphatic polyurethane, a polyether polyurethane, a polyester
polyurethane, a polyethylene copolymer, a polyamide, a polyvinyl
alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl
pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate),
poly(hydroxyethyl methacrylate), collagen, hyaluronic acid,
chitosan, dextran, agarose, gelatin, alginate, a protein polymer,
methylcellulose, and a combination thereof.
[0022] In some embodiments of methods for preparing a spatial
array, the first oligonucleotide of the one or more pluralities of
first oligonucleotides includes a spatial barcode. In some
embodiments of methods for preparing a spatial array, the first
oligonucleotide of the one or more pluralities of first
oligonucleotides includes a unique molecular identifier. In some
embodiments of methods for preparing a spatial array, the first
oligonucleotide of the one or more pluralities of first
oligonucleotides includes a spatial barcode and a capture
domain.
[0023] In another aspect, provided herein are methods for
determining the location of an analyte in a biological sample,
including (a) providing a spatial array of any of the variety of
methods described herein, (b) contacting a biological sample with a
spatial array of any one of the spatial arrays described herein
under conditions that allow the analyte to specifically bind to a
capture probe of the spatial array, and (c) determining the
identity of the analyte specifically bound to the capture probe,
thereby determining the location of the analyte in the biological
sample.
[0024] In some embodiments of methods for determining the location
of an analyte in a biological sample, the biological sample is a
tissue sample. In some embodiments of methods for determining the
location of an analyte in a biological sample, the tissue sample is
a fresh-frozen tissue sample. In some embodiments of methods for
determining the location of an analyte in a biological sample, the
tissue sample includes a tumor cell. In some embodiments of methods
for determining the location of an analyte in a biological sample,
the tissue sample includes a tissue section.
[0025] In some embodiments, methods for determining the location of
an analyte in a biological sample further includes migrating the
analyte in the biological sample to the capture probe. In some
embodiments of methods for determining the location of an analyte
in a biological sample, migrating includes passive migration. In
some embodiments of methods for determining the location of an
analyte in a biological sample, migrating includes active
migration.
[0026] In some embodiments of methods for determining the location
of an analyte in a biological sample, the analyte includes at least
one of RNA, DNA, protein, lipid, peptide, metabolite, small
molecule, and a analyte capture agent.
[0027] In another aspect, provided herein are methods of generating
an array including (a) providing a substrate including a plurality
of features immobilized on the substrate, where a feature of the
plurality of features includes a first oligonucleotide, and where
the first oligonucleotide includes a first spatial barcode, (b)
delivering a second oligonucleotide to the first oligonucleotide,
where the second oligonucleotide includes a second spatial barcode
and a capture domain, and (c) attaching the first oligonucleotide
and the second oligonucleotide, thereby generating a capture probe
including the first spatial barcode, the second spatial barcode,
and the capture domain.
[0028] In some embodiments of methods of generating an array, the
first oligonucleotide is associated with one of a number of
different first spatial barcodes. In some embodiments of methods of
generating an array, the second oligonucleotide is associated with
one of a number of different second spatial barcodes.
[0029] In some embodiments of methods of generating an array, step
(c) includes ligating the first oligonucleotide and the second
oligonucleotide. In some embodiments of methods of generating an
array, the ligating includes the use of a ligase. In some
embodiments of methods of generating an array, the first
oligonucleotide and the second oligonucleotide are attached to one
another by a chemical coupling reaction.
[0030] In some embodiments of methods of generating an array, the
first oligonucleotide includes a 3' end that is masked by a
protective group. In some embodiments of methods of generating an
array, the protective group is removed prior to delivering the
second oligonucleotide to the first oligonucleotide. In some
embodiments of methods of generating an array, the protective group
is a photocleavable protective group. In some embodiments of
methods of generating an array, the photocleavable protective group
is removed by exposing the first oligonucleotide to light. In some
embodiments of methods of generating an array, the photocleavable
protective group coupled to the first oligonucleotide is protected
by applying a photomask.
[0031] In some embodiments of methods of generating an array, a
first feature is separated from other features of the plurality by
one or more physical barriers, where the one or more physical
barriers form one or more chambers, and where the first
oligonucleotide is delivered to a chamber of the one or more
chambers.
[0032] In some embodiments of methods of generating an array, the
one or more physical barriers include a hydrophobic surface. In
some embodiments of methods of generating an array, the one or more
physical barriers include one or more chamber walls having a
tapered end in contact with the substrate. In some embodiments of
methods of generating an array, the first oligonucleotide includes
a unique molecular identifier.
[0033] In another aspect, provided herein are methods of generating
an array including (a) providing a substrate including two or more
partitions, where a first partition of the two or more partitions
includes a feature immobilized on the substrate, where the feature
includes a first oligonucleotide, and where the first
oligonucleotide includes a first spatial barcode, (b) delivering a
second oligonucleotide to the first partition, where the second
oligonucleotide includes a second spatial barcode and a capture
domain, and (c) attaching the first oligonucleotide and the second
oligonucleotide in the first partition, thereby generating a
capture probe including the first spatial barcode, the second
spatial barcode, and the capture domain in the first partition.
[0034] In some embodiments, methods of generating an array further
include (d) delivering a third oligonucleotide to a second
partition of the two or more partitions, where the third
oligonucleotide includes a third spatial barcode and a second
capture domain, where the second partition includes a second
feature, where the second feature includes a fourth oligonucleotide
including the first spatial barcode, and (e) attaching the third
oligonucleotide and the fourth oligonucleotide, thereby generating
a second capture probe including the first spatial barcode, the
third spatial barcode, and the second capture domain in the second
partition.
[0035] In some embodiments of methods of generating an array, the
two or more partitions are separated by one or more physical
barriers. In some embodiments of methods of generating an array,
the one or more physical barriers are printed on the substrate. In
some embodiments of methods of generating an array, the one or more
physical barriers form one or more chambers including one or more
chamber walls. In some embodiments of methods of generating an
array, the one or more chamber walls include a hydrophobic surface.
In some embodiments, the one or more chamber walls include a
tapered end in contact with the substrate. In some embodiments of
methods of generating an array, the second oligonucleotide is
delivered using a microfluidic device.
[0036] In another aspect, provided herein are methods for enriching
a population of capture probes including (a) providing a plurality
of capture probes where: (i) members of the plurality of capture
probes include a plurality of different spatial barcodes, and (ii)
a target capture probe of the population of capture probes to be
enriched includes a target spatial barcode, and (b) enriching a
population of target capture probes including the target spatial
barcode from the plurality of capture probes.
[0037] In another aspect, provided herein are methods for
determining the location of an analyte in a biological sample
including (a) providing a substrate including a plurality of
capture probes, where a subset of the plurality of capture probes
is attached at a location on the substrate, and where two or more
capture probes of the subset of capture probes includes a first
common spatial barcode, (b) contacting the biological sample with
the substrate such that a capture probe of the subset of capture
probes specifically binds to the analyte from the biological
sample, where the analyte that is specifically bound to the capture
probe includes the first common spatial barcode or a complement
thereof, (c) identifying a region of interest within the biological
sample, where the region of interest includes the analyte
specifically bound to the capture probe, and (d) enriching the
analyte specifically bound to the capture probe to determine the
location of the analyte in the biological sample.
[0038] In some embodiments of methods for determining the location
of an analyte in a biological sample, the substrate further
includes a second subset of capture probes attached at a second
location on the substrate, where two or more capture probes of the
second subset of capture probes include a second common spatial
barcode that is different from the first common spatial
barcode.
[0039] In some embodiments of methods for determining the location
of an analyte in a biological sample, the substrate further
includes a third subset of capture probes attached at a third
position on the substrate, where two or more capture probes of the
third subset of capture probes includes a third common spatial
barcode that is different from the first common spatial barcode and
the second common spatial barcode. In some embodiments of methods
for determining the location of an analyte in a biological sample,
the capture probe includes a capture domain.
[0040] In some embodiments of methods for determining the location
of an analyte in a biological sample, the analyte includes at least
one of RNA, DNA, and a analyte capture agent. In some embodiments
of methods for determining the location of an analyte in a
biological sample, the analyte capture agent includes an antigen
binding molecule. In some embodiments of methods for determining
the location of an analyte in a biological sample, the analyte
capture agent includes a region configured to couple or
specifically bind to the capture domain of the capture probe.
[0041] In some embodiments of methods for determining the location
of an analyte in a biological sample, the region of interest is
identified by performing shallow sequencing of at least a subset of
a plurality of analytes specifically bound to capture probes, and
identifying the first common spatial barcode associated with the
region of interest. In some embodiments of methods for determining
the location of an analyte in a biological sample, the region of
interest is identified by at least one of immunostaining,
immunohistochemistry, microscopy, visual inspection, and
combinations thereof.
[0042] In some embodiments of methods for determining the location
of an analyte in a biological sample, step (d) includes performing
a nucleic acid extension reaction using (i) the analyte
specifically bound to the capture probe and (ii) a primer including
a sequence specific for the first common spatial barcode or a
complement thereof to generate an enriched plurality of the analyte
specifically bound to the capture probe(s) including the first
common barcode or a complement thereof.
[0043] In some embodiments of methods for determining the location
of an analyte in a biological sample, the analyte is amplified by
performing a polymerase chain reaction (PCR).
[0044] In some embodiments, methods for determining the location of
an analyte in a biological sample further include enriching a
population of capture probes present in a subregion of interest (a)
where the subregion of interest is located within the region of
interest, where the subregion of interest includes a sub-plurality
of capture probes, and where: (i) two or more capture probes of the
sub-plurality of capture probes include a first common sub-spatial
barcode, and (ii) the first common sub-spatial barcode sequence is
associated with the subregion of interest, and (b) enriching a
population of capture probe including the first common sub-spatial
barcode.
[0045] In some embodiments, methods for determining the location of
an analyte in a biological sample further include enriching a
population of capture probes present in a sub-subregion of interest
(a) where the sub-subregion of interest is located within the
subregion of interest, where the sub-subregion of interest includes
a sub-sub-plurality of capture probes, and where: (i) two or more
capture probes of the sub-sub plurality of capture probes include a
first common sub-sub spatial barcode, and (ii) the first common
sub-sub spatial barcode is associated with the sub-sub region of
interest, and (b) enriching the population of capture probes
including the first sub-sub spatial barcode.
[0046] In some embodiments of methods for determining the location
of an analyte in a biological sample, the first common spatial
barcode is associated with a single cell of the biological
sample.
[0047] In some embodiments of methods for determining the location
of an analyte in a biological sample, the analyte includes an mRNA.
In some embodiments of methods for determining the location of an
analyte in a biological sample, the analyte includes a protein. In
some embodiments of methods for determining the location of an
analyte in a biological sample, the analyte includes an antibody.
In some embodiments of methods for determining the location of an
analyte in a biological sample, the analyte includes DNA. In some
embodiments of methods for determining the location of an analyte
in a biological sample, the DNA encodes a T-cell receptor. In some
embodiments of methods for determining the location of an analyte
in a biological sample, the DNA includes one or more regions of
chromatin in the biological sample.
[0048] In another aspect, provided herein are methods for
generating a spatial array including (a) providing a substrate
including a plurality of first oligonucleotides, where a first
oligonucleotide of the plurality of first oligonucleotides is
attached to a region of the substrate through its 3'-end or its
5'-end, (b) attaching a second oligonucleotide to an unattached end
of the first oligonucleotide, where the second oligonucleotide
includes a spatial barcode.
[0049] In some embodiments, methods for generating a spatial array
further include after (b): (c) attaching a third oligonucleotide
segment to an unattached end of the second oligonucleotide.
[0050] In some embodiments of methods for generating a spatial
array, the first oligonucleotide is attached to the substrate
through its 5'-end and the second oligonucleotide is generated
using spatially-directed in situ synthesis on the 3'-end of the
first oligonucleotide. In some embodiments of methods for
generating a spatial array, the first oligonucleotide is attached
to the substrate through its 3'-end and the second oligonucleotide
is generated using spatially-directed in situ synthesis on the
5'-end of the first oligonucleotide.
[0051] In some embodiments of methods for generating a spatial
array, phosphodiester and exocyclic amine moieties of the first
oligonucleotide are protected.
[0052] In some embodiments of methods for generating a spatial
array, the second oligonucleotide has a length selected from 8
nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12
nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16
nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20
nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24
nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28
nucleotides, 29 nucleotides, or 30 nucleotides.
[0053] In some embodiments of methods for generating a spatial
array, the in situ synthesis is light-directed synthesis.
[0054] In some embodiments of methods for generating a spatial
array, the region of the substrate is less than about 2500
.mu.m.sup.2, less than about 1600 .mu.m.sup.2, less than about 400
.mu.m.sup.2, less than about 100 .mu.m.sup.2, less than about 50
.mu.m.sup.2, less than about 25 .mu.m.sup.2, less than about 15
.mu.m.sup.2, less than about 10 .mu.m.sup.2, or less than about 5
.mu.m.sup.2.
[0055] In some embodiments of methods for generating a spatial
array, two or more second oligonucleotides are attached to two or
more first oligonucleotides, where a member of the two or more
second oligonucleotides differs from another member of the two or
more second oligonucleotides.
[0056] In some embodiments of methods for generating a spatial
array, the first oligonucleotide includes a protected
phosphodiester and exocyclic amine moieties.
[0057] In some embodiments of methods for generating a spatial
array, the substrate further includes a substantially complementary
oligonucleotide hybridized to the first oligonucleotide.
[0058] In some embodiments of methods for generating a spatial
array, the first oligonucleotide has a length of at least about 2
nucleotides, 5 nucleotides, 8 nucleotides, 10 nucleotides, 15
nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, 25
nucleotides, 27 nucleotides, 30 nucleotides, 33 nucleotides, or 35
nucleotides. In some embodiments of methods for generating a
spatial array, the first oligonucleotide has a length of about 2 to
45 nucleotides, 5 to 40 nucleotides, 15 to 40 nucleotides, 25 to 40
nucleotides, 25 to 35 nucleotides, or 27 to 33 nucleotides.
[0059] In some embodiments of methods for generating a spatial
array, the first oligonucleotide is prepared by synthesis off of
the substrate and the first oligonucleotide is subsequently
attached to the substrate.
[0060] In some embodiments of methods for generating a spatial
array, the phosphodiester and exocyclic amine moieties of the first
oligonucleotide are protected prior to attachment of the first
oligonucleotide to the substrate. In some embodiments of methods
for generating a spatial array, the phosphodiester and exocyclic
amine moieties of the first oligonucleotide are protected after
attachment of the first oligonucleotide to the substrate.
[0061] In some embodiments of methods for generating a spatial
array, the first oligonucleotide is prepared by in situ synthesis
on the substrate. In some embodiments of methods for generating a
spatial array, the in situ synthesis is not light-directed
synthesis. In some embodiments of methods for generating a spatial
array, the in situ synthesis is not spatially-directed
synthesis.
[0062] In some embodiments of methods for generating a spatial
array, the first oligonucleotide includes a sequence including a
cleavage site. In some embodiments of methods for generating a
spatial array, the first oligonucleotide is attached to the
substrate through a linker. In some embodiments of methods for
generating a spatial array, a chemical group including: silane,
amine, peptide, polyethylene glycol, biotin, desthiobiotin,
streptavidin, thiol, acrydite, azide, digoxigenin, hexynyl, or a
combination thereof. In some embodiments of methods for generating
a spatial array, the linker is a branched linker or a dendrimeric
linker.
[0063] In some embodiments of methods for generating a spatial
array, the third oligonucleotide is attached to the second
oligonucleotide by ligation.
[0064] In some embodiments of methods for generating a spatial
array, any protected moieties of the first and second
oligonucleotides are deprotected prior to ligating the third
oligonucleotide to the second oligonucleotide.
[0065] In some embodiments of methods for generating a spatial
array, the plurality of third oligonucleotides have a length of at
least about 5 nucleotides, 7 nucleotides, 9 nucleotides, 13
nucleotides, 20 nucleotides, 25 nucleotides, 27 nucleotides, 30
nucleotides, 33 nucleotides, or 35 nucleotides. In some embodiments
of methods for generating a spatial array, the plurality of third
oligonucleotides have a length of about 5 to 40 nucleotides, 5 to
20 nucleotides, 5 to 10 nucleotides, 10 to 35 nucleotides, 15 to 30
nucleotides, 25 to 35 nucleotides, or 27 to 33 nucleotides.
[0066] In some embodiments of methods for generating a spatial
array, the third oligonucleotide includes a capture domain of at
least 6 nucleotides in length at the 3'-end. In some embodiments of
methods for generating a spatial array, the capture domain includes
a poly-T sequence. In some embodiments of methods for generating a
spatial array, the third oligonucleotide includes a random sequence
of 6 to 20 nucleotides in length.
[0067] In another aspect, provided herein are compositions
including a plurality of oligonucleotides attached to a plurality
of regions on a substrate, where an oligonucleotide of the
plurality of oligonucleotides includes: a first oligonucleotide of
2 to 50 nucleotides in length attached to the substrate through its
3'-end or its 5'-end, and a second oligonucleotide of 8 to 30
nucleotides in length that is attached to an unattached end of the
first oligonucleotide, where the second oligonucleotide includes a
spatial barcode.
[0068] In another aspect, provided herein are compositions where
the first oligonucleotide and the second oligonucleotide include
protected phosphodiester and exocyclic amine moieties.
[0069] In another aspect, provided herein are compositions where
the first oligonucleotide is attached to the substrate through its
5'-end and the second oligonucleotide is attached to the 3'-end of
the first oligonucleotide.
[0070] In another aspect, provided herein are compositions where
the first oligonucleotide is attached to the substrate through its
3'-end and the second oligonucleotide is attached to the 5'-end of
the first oligonucleotide.
[0071] In some embodiments, compositions where the first
oligonucleotide is attached to the substrate through its 3'-end and
the second oligonucleotide is attached to the 5'-end of the first
oligonucleotide further include a third oligonucleotide of 5 to 40
nucleotides in length that is attached to an unattached end of the
second oligonucleotide, where the third oligonucleotide includes a
capture domain.
[0072] In some embodiments, compositions where the first
oligonucleotide is attached to the substrate through its 3'-end and
the second oligonucleotide is attached to the 5'-end of the first
oligonucleotide, the second oligonucleotide has a length selected
from 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides,
12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16
nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20
nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24
nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28
nucleotides, 29 nucleotides, or 30 nucleotides.
[0073] In some embodiments, compositions where the first
oligonucleotide is attached to the substrate through its 3'-end and
the second oligonucleotide is attached to the 5'-end of the first
oligonucleotide, the regions are less than about 2500 .mu.m.sup.2,
less than about 1600 .mu.m.sup.2, less than about 400 .mu.m.sup.2,
less than about 100 .mu.m.sup.2, less than about 50 .mu.m.sup.2,
less than about 25 .mu.m.sup.2, less than about 15 .mu.m.sup.2,
less than about 10 .mu.m.sup.2, less than about 5 .mu.m.sup.2, or
less than about 1 .mu.m.sup.2.
[0074] In some embodiments, compositions where the first
oligonucleotide is attached to the substrate through its 3'-end and
the second oligonucleotide is attached to the 5'-end of the first
oligonucleotide, the length of the oligonucleotide in each of the
regions is at least 15 nucleotides, at least 20 nucleotides, at
least 40 nucleotides, at least 50 nucleotides, or at least 60
nucleotides.
[0075] In some embodiments, compositions where the first
oligonucleotide is attached to the substrate through its 3'-end and
the second oligonucleotide is attached to the 5'-end of the first
oligonucleotide, each of the regions is from 15 to 100 nucleotides,
20 to 100 nucleotides, 40 to 100 nucleotides, from 50 to 90
nucleotides, or from 60 to 80 nucleotides.
[0076] In another aspect, provided herein are methods of preparing
a spatial array including (a) providing a substrate, (b)
immobilizing a plurality of sets of primers on the substratet,
where a first set of the plurality of sets of primers includes a
first primer and a second primer, (c) providing a template
oligonucleotide including a reverse complement of a spatial barcode
domain, a reverse complement of a capture domain, a first
hybridization domain capable of hybridizing to the first primer,
and a second hybridization domain, where a reverse complement of
the second hybridization domain is capable of hybridizing to the
second primer, (d) hybridizing the template oligonucleotide to the
first primer, (e) extending the first primer using the template
oligonucleotide as a template to generate a capture probe including
the first primer, the spatial barcode, and the capture domain,
which capture probe is immobilized on the substrate, and (f)
releasing the template oligonucleotide from the capture probe,
thereby producing the spatial array.
[0077] In some embodiments, methods of preparing a spatial array
further include amplifying the capture probe to create a cluster of
one or more capture probes.
[0078] In some embodiments of methods of preparing a spatial array,
amplifying includes (a) hybridizing the capture probe to the second
primer, (b) extending the second primer using the capture probe as
a template to generate a copy of the template oligonucleotide that
is immobilized on the substrate, (c) hybridizing the copy of the
template oligonucleotide to a first primer of a second set of the
plurality of primers, and (d) extending the first primer of the
second set of the plurality of primers using the copy of template
oligonucleotide as a template to generate a copy of the capture
probe that is immobilized on the substrate, where the cluster of
capture probes includes the capture probe and the copy of the
capture probe.
[0079] In some embodiments of methods of preparing a spatial array,
the template oligonucleotide includes a reverse complement of a
unique molecular identifier (UMI), and where the generated capture
probe includes the UMI.
[0080] In some embodiments of methods of preparing a spatial array,
the first primer is affixed to the substrate at a 5' end of the
first primer. In some embodiments of methods of preparing a spatial
array, the first primer includes a sequence that is complementary
to the template oligonucleotide. In some embodiments of methods of
preparing a spatial array, the first primer includes a poly (A)
domain. In some embodiments of methods of preparing a spatial
array, the first primer includes cleavage domain.
[0081] In some embodiments of methods of preparing a spatial array,
the second primer is affixed to the substrate at a 5' end of the
second primer. In some embodiments of methods of preparing a
spatial array, the second primer includes a cleavage domain.
[0082] In some embodiments of methods of preparing a spatial array,
the copy of the template oligonucleotide is released by cleaving
the cleavage domain after the copy of the capture probe is
generated. In some embodiments of methods of preparing a spatial
array, the template oligonucleotide is attached to a bead.
[0083] In some embodiments of methods of preparing a spatial array,
detaching the bead from the template oligonucleotide after the step
of extending the first primer using the template oligonucleotide as
a template to generate a capture probe including the first primer,
the spatial barcode, and the capture domain, which capture probe is
immobilized on the substrate.
[0084] In some embodiments of methods of preparing a spatial array,
the bead is a gel bead. In some embodiments of methods of preparing
a spatial array, the bead is 2 microns or less in diameter. In some
embodiments of methods of preparing a spatial array, the bead is 1
micron or less in diameter. In some embodiments of methods of
preparing a spatial array, the bead is removed prior to the step of
extending the first primer. In some embodiments of methods of
preparing a spatial array, the bead is removed after the step of
extending the first primer.
[0085] In some embodiments of methods of preparing a spatial array,
all or a portion of the sequence of the generated capture probe is
determined. In some embodiments of methods of preparing a spatial
array, all or a portion of the sequence of the generated capture
probe is determined after the template oligonucleotide is
released.
[0086] In some embodiments of methods of preparing a spatial array,
all or a portion of the sequence of the generated capture probe is
determined in situ. In some embodiments of methods of preparing a
spatial array, all or a portion of the sequence of the generated
capture probe is determined by in situ sequencing. In some
embodiments of methods of preparing a spatial array, the in situ
sequencing is performed via sequencing-by-synthesis (SBS),
sequential fluorescence hybridization, sequencing by ligation,
nucleic acid hybridization, or high-throughput digital sequencing
techniques.
[0087] In some embodiments of methods of preparing a spatial array,
the capture domain includes a sequence complementary to an analyte
of interest. In some embodiments of methods of preparing a spatial
array, the capture domain includes a poly(T) domain.
[0088] In some embodiments of methods of preparing a spatial array,
the substrate includes a glass slide. In some embodiments of
methods of preparing a spatial array, the glass slide is a treated
glass slide.
[0089] In another aspect, provided herein are methods for
determining a location of an analyte in a biological sample
including (a) preparing a spatial array using any of the variety of
methods described herein, (b) decoding the one or more capture
probes by determining all or a portion of the sequence of the
spatial barcode of the one or more capture probes, thereby
associating the spatial barcode of the one or more capture probe
with a location on the spatial array, (c) contacting the biological
sample to the spatial array under conditions where the analyte
specifically binds to capture probe(s), (d) extending the one or
more capture probes via a polymerization reaction using the analyte
as a template to generate an extended one or more capture probes
including the one or more capture probes and the reverse complement
of the analyte, and (e) determining the sequence of the extended
one or more capture probes, and associating the spatial barcode of
the one or more capture probes with the analyte, thereby
determining the location of the analyte in the biological
sample.
[0090] In some embodiments of methods for determining a location of
an analyte in a biological sample, one or more capture probes
includes a template oligonucleotide and a bead. In some
embodiments, methods for determining a location of an analyte in a
biological sample further include detaching the bead from the
template oligonucleotide after the step of extending the one or
more capture probes via a polymerization reaction using the analyte
as a template to generate an extended one or more capture probes
including the one or more capture probes and the reverse complement
of the analyte.
[0091] In some embodiments of methods for determining a location of
an analyte in a biological sample, the first primer and the second
primer are at a ratio of between 1:1.5 and 1.5:1. In some
embodiments of methods for determining a location of an analyte in
a biological sample, the first primer and the second primer are
less than 5 .mu.m apart.
[0092] In another aspect, provided herein are spatial arrays
produced by any of the methods described herein.
[0093] In another aspect, provided herein are spatial array
including (a) a substrate, and (b) a plurality of sets of primers
immobilized on the substrate, where a first set of the plurality of
sets of primers includes a first primer and a second primer, and
where the first primer includes a sequence that is complementary to
a capture probe.
[0094] In some embodiments, the spatial array further includes a
template oligonucleotide including a reverse complement of a unique
molecular identifier (UMI), and where the capture probe includes
the UMI.
[0095] In some embodiments of spatial arrays provided herein, the
first primer is affixed to the substrate at a 5' end of the first
primer. In some embodiments of spatial arrays provided herein, the
first primer includes a sequence that is complementary to the
template oligonucleotide. In some embodiments of spatial arrays
provided herein, the first primer includes poly (A) domain. In some
embodiments of spatial arrays provided herein, the first primer
includes a cleavage domain.
[0096] In some embodiments of spatial arrays provided herein, the
second primer is affixed to the substrate at a 5' end of the second
primer. In some embodiments of spatial arrays provided herein, the
second primer includes a cleavage domain.
[0097] In some embodiments of spatial arrays provided herein, the
template oligonucleotide is attached to a bead. In some embodiments
of spatial arrays provided herein, the bead is a gel bead. In some
embodiments of spatial arrays provided herein, the bead is 2
microns or less in diameter. In some embodiments of spatial arrays
provided herein, the bead is 1 micron or less in diameter.
[0098] In some embodiments of spatial arrays provided herein, the
substrate includes a glass slide. In some embodiments of spatial
arrays provided herein, the glass slide is a treated glass
slide.
[0099] In some embodiments of spatial arrays provided herein, the
first primer and the second primer are at a ratio of between 1:1.5
and 1.5:1. In some embodiments of spatial arrays provided herein,
the first primer and the second primer are less than 5 .mu.m
apart.
[0100] In another aspect, provided herein are methods of generating
a spatial array including (a) providing a substrate including an
arrayed plurality of acceptor oligonucleotides, where an acceptor
oligonucleotide of the plurality of acceptor oligonucleotides
includes a spatial barcode and a first ligation handle, and where
the 5' end of the acceptor oligonucleotide is attached to the
substrate, (b) providing a splint oligonucleotide including a
sequence complementary to the first ligation handle and a sequence
complementary to a donor oligonucleotide, and (c) ligating a donor
oligonucleotide including a capture domain to the 3' end the
acceptor oligonucleotide to generate a capture probe, thereby
generating a spatial array.
[0101] In some embodiments of methods of generating a spatial
array, the acceptor oligonucleotides further includes a cleavage
domain, a unique molecular identifier, a functional sequence, or
any combination thereof.
[0102] In some embodiments of methods of generating a spatial
array, the sequence complementary to the capture domain of splint
oligonucleotide is a poly(A) sequence. In some embodiments, the
sequence complementary to the capture domain of the splint
oligonucleotide is a gene-specific sequence. In some embodiments,
the gene-specific sequence is a Penk-specific sequence. In some
embodiments, the sequence complementary to the capture domain of
the splint oligonucleotide is a random sequence. In some
embodiments, the random sequence is a hexamer.
[0103] In some embodiments, methods of generating a spatial array
further include contacting a biological sample to the spatial array
after step (c). In some embodiments, methods of generating a
spatial array further include permeabilizing the biological sample
to allow the analyte in the biological sample to interact with the
capture probe.
[0104] In some embodiments, methods of generating a spatial array
further include migrating the analyte in the biological sample to
the plurality of capture probes. In some embodiments of methods of
generating a spatial array, the migrating includes passive
migration. In some embodiments of methods of generating a spatial
array, the migrating includes active migration.
[0105] In another aspect, provided herein are methods of generating
a spatial array including (a) providing a substrate including an
arrayed plurality of acceptor oligonucleotides, where an acceptor
oligonucleotide of the plurality of acceptor oligonucleotides
includes a spatial barcode and a first ligation handle, and where
the 5' end of the acceptor oligonucleotide is attached to the
substrate, (b) providing a splint oligonucleotide including a
sequence complementary to the first ligation handle and a sequence
complementary to a second ligation handle, and (c) ligating a donor
oligonucleotide including the second ligation handle and a capture
domain to the 3' end the acceptor oligonucleotide to generate a
capture probe, thereby generating a spatial array.
[0106] In some embodiments of methods of generating a spatial
array, the acceptor oligonucleotides further includes a cleavage
domain, a unique molecular identifier, a functional sequence, or
any combination thereof.
[0107] In some embodiments of methods of generating a spatial
array, the capture domain includes a poly(T) sequence. In some
embodiments of methods of generating a spatial array, the capture
domain includes a gene-specific sequence. In some embodiments of
methods of generating a spatial array, the gene-specific sequence
is a Penk-specific sequence. In some embodiments of methods of
generating a spatial array, the capture domain includes a random
sequence. In some embodiments of methods of generating a spatial
array, the random sequence is a hexamer.
[0108] In some embodiments, methods of generating a spatial array
further include contacting a biological sample to the spatial array
after step (c). In some embodiments, methods of generating a
spatial array further include permeabilizing the biological sample
to allow an analyte in the biological sample to interact with the
capture probe.
[0109] In some embodiments, methods of generating a spatial array
further include migrating the analyte in the biological sample to
the capture probe. In some embodiments of methods of generating a
spatial array, the migrating includes passive migration. In some
embodiments of methods of generating a spatial array, the migrating
includes active migration.
[0110] In another aspect, provided herein are methods of generating
a capture probe bound to substrate including dispensing a second
solution onto a portion of a surface of a substrate coated with a
first solution, where after the dispensing, molecules within the
first solution and the second solution do not substantially mix
with each other, where the second solution includes a capture
probe, and the dispensing results in attachment of the capture
probe to the surface of the substrate.
[0111] In some embodiments, methods of generating a capture probe
bound to substrate further include: curing the second solution, and
removing the first solution from the substrate, where the cured
second solution is retained on the surface of the substrate.
[0112] In some embodiments of methods of generating a capture probe
bound to substrate, the surface of the substrate to which the
capture probe is attached has an area that is smaller than a
corresponding area generated by dispensing the second solution onto
the surface of the substrate in the absence of the first solution.
In some embodiments of methods of generating a capture probe bound
to substrate, the surface of the substrate to which the capture
probe is attached has an area that is about two-fold less than the
corresponding area generated by dispensing the second solution onto
the surface of the substrate in the absence of the first
solution.
[0113] In some embodiments, methods of generating a capture probe
bound to substrate further include curing the first and second
solutions on the substrate, thus generating a matrix. In some
embodiments of methods of generating a capture probe bound to
substrate, the first and second solutions are cured by chemical
means. In some embodiments of methods of generating a capture probe
bound to substrate, the first and second solutions are cured by
photo-reactive means.
[0114] In some embodiments, methods of generating a capture probe
bound to substrate further include shrinking the matrix, thus
generating a shrunken matrix having an area to which the capture
probe is attached, where the area in the shrunken matrix is reduced
as compared to a corresponding area in the matrix.
[0115] In some embodiments, methods of generating a capture probe
bound to substrate further include prior to the step of dispensing
the second solution: curing the first solution to form a first
solution matrix, where the first solution matrix can be reversibly
expanded along one or more of its axes. In some embodiments of
methods of generating a capture probe bound to substrate, the step
of dispensing the second solution onto the portion of the substrate
including dispensing the second solution onto a surface of the
first solution matrix in an expanded state. In some embodiments,
methods of generating a capture probe bound to substrate further
includes, after the dispensing step: shrinking the first solution
matrix along the one or more axes of the surface of the
substrate.
[0116] In some embodiments of methods of generating a capture probe
bound to substrate, the first solution has a viscosity that is
about 0.1.times.-fold, 0.2.times.-fold, 0.3.times.-fold,
0.4.times.-fold, 0.5.times.-fold, 0.6.times.-fold, 0.7.times.-fold,
0.8.times.-fold, 0.9.times.-fold, 1.0.times.-fold, 1.1.times.-fold,
1.2.times.-fold, 1.3.times.-fold, 1.4-fold, 1.5.times.-fold,
1.6.times.-fold, 1.7.times.-fold, 1.8.times.-fold, 1.9.times.-fold,
2.0.times.-fold, 2.5.times.-fold, 3.0.times.-fold, 4.0.times.-fold,
5.0.times. fold, 6.0.times.-fold, 7.0.times.-fold, 8.0.times. fold,
9.0.times.-fold or 10.times.-fold greater than the viscosity of the
second solution.
[0117] In some embodiments of methods of generating a capture probe
bound to substrate, the first solution has a lower nucleic acid
diffusivity as compared to the second solution.
[0118] In some embodiments of methods of generating a capture probe
bound to substrate, the capture probe includes a spatial barcode.
In some embodiments, the capture probe includes a unique molecular
identifier. In some embodiments of methods of generating a capture
probe bound to substrate, the capture probe includes a functional
domain. In some embodiments of methods of generating a capture
probe bound to substrate, the capture probe includes a cleavage
domain, a spatial barcode, a unique molecular identifier, a
functional domain, and a capture domain.
[0119] In another aspect, provided herein are methods of generating
an array of capture probes bound to a substrate including
dispensing a droplet of a second solution onto a portion of a
surface of a substrate coated with a first solution, where after
the dispensing, molecules within the first solution and the droplet
of the second solution do not substantially mix with each other,
where the second solution includes a capture probe, and the
dispensing results in attachment of the capture probe to the
surface of the substrate.
[0120] In some embodiments, methods of generating an array of
capture probes bound to a substrate further include: curing the
second solution, and removing the first solution from the
substrate, where the cured second solution is retained on the
surface of the substrate.
[0121] In some embodiments of methods of generating an array of
capture probes bound to a substrate, the surface of the substrate
to which the capture probe is attached has an area that is smaller
than a corresponding area generated by dispensing the second
solution onto the surface of the substrate in the absence of the
first solution. In some embodiments of methods of generating an
array of capture probes bound to a substrate, the surface of the
substrate to which the capture probe is attached has an area that
is about two-fold less than the corresponding area generated by
dispensing the second solution onto the surface of the substrate in
the absence of the first solution.
[0122] In some embodiments, methods of generating an array of
capture probes bound to a substrate further include curing the
first and second solutions on the substrate, thus generating a
matrix. In some embodiments of methods of generating an array of
capture probes bound to a substrate, the first solution and the
second solutions are cured by chemical means. In some embodiments
of methods of generating an array of capture probes bound to a
substrate, the first and second solutions are cured by
photo-reactive means.
[0123] In some embodiments, methods of generating an array of
capture probes bound to a substrate further include shrinking the
matrix, thus generating a shrunken matrix having an area to which
the capture probe is attached, where the area in the shrunken
matrix is reduced as compared to a corresponding area in the
matrix.
[0124] In some embodiments, methods of generating an array of
capture probes bound to a substrate further include, prior to the
step of dispensing the second solution: curing the first solution
to form a first solution matrix, where the first solution matrix
can be reversibly expanded along one or more of its axes. In some
embodiments of methods of generating an array of capture probes
bound to a substrate, the step of dispensing the second solution
onto the portion of the substrate including dispensing the second
solution onto a surface of the first solution matrix in an expanded
state.
[0125] In some embodiments, methods of generating an array of
capture probes bound to a substrate further include after the
dispensing step: shrinking the first solution matrix along the one
or more axes of the surface of the substrate.
[0126] In some embodiments of methods of generating an array of
capture probes bound to a substrate, the first solution has a
viscosity that is about 0.1.times.-fold, 0.2.times.-fold,
0.3.times.-fold, 0.4.times.-fold, 0.5.times.-fold, 0.6.times.-fold,
0.7.times.-fold, 0.8.times.-fold, 0.9.times.-fold, 1.0.times.-fold,
1.1.times.-fold, 1.2.times.-fold, 1.3.times.-fold, 1.4-fold,
1.5.times.-fold, 1.6.times.-fold, 1.7.times.-fold, 1.8.times.-fold,
1.9.times.-fold, 2.0.times.-fold, 2.5.times.-fold, 3.0.times.-fold,
4.0.times.-fold, 5.0.times. fold, 6.0.times.-fold, 7.0.times.-fold,
8.0.times. fold, 9.0.times.-fold or 10.times.-fold greater than the
viscosity of the second solution.
[0127] In some embodiments of methods of generating an array of
capture probes bound to a substrate, the first solution has a lower
nucleic acid diffusivity as compared to the second solution.
[0128] In some embodiments of methods of generating an array of
capture probes bound to a substrate, the capture probe includes a
spatial barcode. In some embodiments of methods of generating an
array of capture probes bound to a substrate, the at least one of
the capture probes includes a unique molecular identifier. In some
embodiments of methods of generating an array of capture probes
bound to a substrate, the capture probe includes a functional
domain. In some embodiments of methods of generating an array of
capture probes bound to a substrate, the capture probe includes a
cleavage domain, a spatial barcode, a unique molecular identifier,
a functional domain, and a capture domain.
[0129] In another aspect, provided herein are methods for spatially
profiling an analyte in a biological sample including (a)
dispensing a droplet of a second solution onto a portion of a
surface of a substrate coated with a the first solution, where
after the dispensing, molecules within the first solution and the
droplet of the second solution do not substantially mix with each
other, where the second solution includes a capture probe, and the
dispensing results in attachment of the capture probe to the
surface of the substrate, (b) contacting the biological sample with
the spatial array such that the analyte present in the biological
sample is captured by the capture probes, thereby determining the
spatial profile of the captured analyte.
[0130] In some embodiments of methods for spatially profiling an
analyte in a biological sample, at least one of the capture probes
includes a cleavage domain, a spatial barcode, a unique molecular
identifier, a functional domain, and a capture domain.
[0131] All publications, patents, patent applications, and
information available on the internet and mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication, patent, patent
application, or item of information was specifically and
individually indicated to be incorporated by reference. To the
extent publications, patents, patent applications, and items of
information incorporated by reference contradict the disclosure
contained in the specification, the specification is intended to
supersede and/or take precedence over any such contradictory
material.
[0132] Where values are described in terms of ranges, it should be
understood that the description includes the disclosure of all
possible sub-ranges within such ranges, as well as specific
numerical values that fall within such ranges irrespective of
whether a specific numerical value or specific sub-range is
expressly stated.
[0133] The term "each," when used in reference to a collection of
items, is intended to identify an individual item in the collection
but does not necessarily refer to every item in the collection,
unless expressly stated otherwise, or unless the context of the
usage clearly indicates otherwise.
[0134] Various embodiments of the features of this disclosure are
described herein. However, it should be understood that such
embodiments are provided merely by way of example, and numerous
variations, changes, and substitutions can occur to those skilled
in the art without departing from the scope of this disclosure. It
should also be understood that various alternatives to the specific
embodiments described herein are also within the scope of this
disclosure.
DESCRIPTION OF DRAWINGS
[0135] The following drawings illustrate certain embodiments of the
features and advantages of this disclosure. These embodiments are
not intended to limit the scope of the appended claims in any
manner. Like reference symbols in the drawings indicate like
elements.
[0136] FIG. 1 shows an exemplary spatial analysis workflow.
[0137] FIG. 2 shows an exemplary spatial analysis workflow.
[0138] FIG. 3 shows an exemplary spatial analysis workflow.
[0139] FIG. 4 shows an exemplary spatial analysis workflow.
[0140] FIG. 5 shows an exemplary spatial analysis workflow.
[0141] FIG. 6 is a schematic diagram showing an example of a
barcoded capture probe, as described herein.
[0142] FIG. 7 is a schematic illustrating a cleavable capture
probe, wherein the cleaved capture probe can enter into a
non-permeabilized cell and bind to target analytes within the
sample.
[0143] FIG. 8 is a schematic diagram of an exemplary multiplexed
spatially-barcoded feature.
[0144] FIG. 9 is a schematic diagram of an exemplary analyte
capture agent.
[0145] FIG. 10 is a schematic diagram depicting an exemplary
interaction between a feature-immobilized capture probe 1024 and an
analyte capture agent 1026.
[0146] FIGS. 11A, 11B, and 11C are schematics illustrating how
streptavidin cell tags can be utilized in an array-based system to
produce a spatially-barcoded cells or cellular contents.
[0147] FIG. 12 is a schematic showing the arrangement of barcoded
features within an array.
[0148] FIG. 13 is a schematic illustrating a side view of a
diffusion-resistant medium, e.g., a lid.
[0149] FIGS. 14A and 14B are schematics illustrating expanded FIG.
14A and side views FIG. 14B of an electrophoretic transfer system
configured to direct transcript analytes toward a
spatially-barcoded capture probe array.
[0150] FIG. 15 is a schematic illustrating an exemplary workflow
protocol utilizing an electrophoretic transfer system.
[0151] FIG. 16 shows an example of a microfluidic channel structure
1600 for partitioning dissociated sample (e.g., biological
particles or individual cells from a sample).
[0152] FIG. 17A shows an example of a microfluidic channel
structure 1700 for delivering spatial barcode carrying beads to
droplets.
[0153] FIG. 17B shows a cross-section view of another example of a
microfluidic channel structure 1750 with a geometric feature for
controlled partitioning.
[0154] FIG. 17C shows an example of a workflow schematic.
[0155] FIG. 18 is a schematic depicting cell tagging using either
covalent conjugation of the analyte binding moiety to the cell
surface or non-covalent interactions with cell membrane
elements.
[0156] FIG. 19 is a schematic depicting cell tagging using either
cell-penetrating peptides or delivery systems.
[0157] FIG. 20A is a workflow schematic illustrating exemplary,
non-limiting, non-exhaustive steps for "pixelating" a sample,
wherein the sample is cut, stamped, microdissected, or transferred
by hollow-needle or microneedle, moving a small portion of the
sample into an individual partition or well.
[0158] FIG. 20B is a schematic depicting multi-needle pixilation,
wherein an array of needles punched through a sample on a scaffold
and into nanowells containing gel beads and reagents below. Once
the needle is in the nanowell, the cell(s) are ejected.
[0159] FIG. 21 shows a workflow schematic illustrating exemplary,
non-limiting, non-exhaustive steps for dissociating a
spatially-barcoded sample for analysis via droplet or flow cell
analysis methods.
[0160] FIG. 22A is a schematic diagram showing an example sample
handling apparatus that can be used to implement various steps and
methods described herein.
[0161] FIG. 22B is a schematic diagram showing an example imaging
apparatus that can be used to obtain images of biological samples,
analytes, and arrays of features.
[0162] FIG. 22C is a schematic diagram of an example of a control
unit of the apparatus of FIGS. 22A and 22B.
[0163] FIG. 23A shows a histological section of an invasive ductal
carcinoma annotated by a pathologist.
[0164] FIG. 23B shows a tissue plot with spots colored by
unsupervised clustering.
[0165] FIG. 23C is a tSNE plot of spots colored by unsupervised
clustering.
[0166] FIG. 23D shows a gene expression heat map of the most
variable genes between 9 clusters.
[0167] FIG. 23E shows the expression levels of genes corresponding
to human epidermal growth factor receptor 2 (Her2), estrogen
receptor (ER), and progesterone receptor (PR) in the tissue
section.
[0168] FIG. 23F shows the expression levels of genes of top
differentially expressed genes from each of the 9 clusters on
individual plots.
[0169] FIG. 23G shows the expression levels of genes of top
differentially expressed genes from each of the 9 clusters on a
single plot.
[0170] FIG. 23H is a plot of the expression levels of the top
differentially expressed genes from each of the 9 clusters in
invasive ductal cell carcinoma (IDC) and normal breast tissue.
[0171] FIG. 23I shows the expression of KRT14 in IDC and match
normal tissue.
[0172] FIG. 23J is a plot of the expression levels of extracellular
matrix genes in IDC and normal tissue.
[0173] FIG. 24 is a schematic diagram showing a method for
generating capture probes of an array for spatial analysis of
analytes in biological sample.
[0174] FIG. 25 is a schematic diagram showing a method of capturing
an analyte with a capture domain and performing reverse
transcription to obtain a complementary cDNA sequence.
[0175] FIG. 26 shows a schematic depicting steps involved in
generating a high-density spatially barcoded array with increased
resolution.
[0176] FIG. 27 is a schematic diagram showing an exemplary two-step
barcode construction process to build an array.
[0177] FIG. 28 is a schematic diagram showing an exemplary two-step
barcode construction process to build an array.
[0178] FIG. 29 is a schematic diagram showing an exemplary spatial
array preparation process.
[0179] FIG. 30 is a schematic diagram showing an exemplary design
for a fluidic isolation chamber.
[0180] FIG. 31 depicts an illustrative embodiment of the methods
for preparing a spatial array of the present disclosure. FIG. 31A
depicts a substrate provided with first oligonucleotides attached
thereto in a 5'-to-3' orientation, wherein the phosphodiester and
exocyclic amine groups are protected. FIG. 31B depicts 5'-to-3
light-directed spatial synthesis of second oligonucleotides to the
free 3'-ends of the first oligonucleotide on the substrate, wherein
the second oligonucleotide have different sequences in the two
different regions. FIG. 31C depicts ligation of a third
oligonucleotide to the free 3'-ends of the two different second
oligonucleotides.
[0181] FIG. 32 depicts an illustrative embodiment of the methods
for preparing a spatial array of the present disclosure. FIG. 32A
depicts ex situ solid phase synthesis of a first oligonucleotide in
3'-to-5' direction followed by cleavage from the feature with the
phosphodiester and exocyclic amine groups protected. FIG. 32B
depicts coupling of the ex situ synthesized first oligonucleotide
to a substrate in a 5'-to-3' orientation. FIG. 32C depicts 5'-to-3'
light-directed spatial synthesis of second oligonucleotides to the
free 3'-ends of the first oligonucleotide on the substrate, wherein
the second oligonucleotides have different sequences in the two
different regions.
[0182] FIG. 33 depicts an illustrative embodiment of the methods
for preparing a spatial array of the present disclosure. FIG. 33A
depicts a first oligonucleotide with phosphodiester and exocyclic
amine protecting groups attached to a substrate in a 5'-to-3'
orientation, wherein the first oligonucleotide has been in situ
synthesized on the surface of the substrate.
[0183] FIG. 33B depicts 5'-to-3 light-directed spatial synthesis of
second oligonucleotides to the free 3'-ends of the first
oligonucleotide (with first segment phosphodiester and exocyclic
amine protecting groups in place) on the substrate, wherein the
second oligonucleotides have different sequences in the two
different regions.
[0184] FIG. 34 depicts an illustrative embodiment of the methods
for preparing a spatial array of the present disclosure. FIG. 34A
depicts a first oligonucleotide phosphodiester and exocyclic amine
protecting groups attached to a substrate in a 5'-to-3'
orientation, wherein the first oligonucleotide has been synthesized
on the surface of the substrate using 5'-to-3' light-directed
spatial synthesis. FIG. 34B depicts 5'-to-3 light-directed spatial
synthesis of second oligonucleotides to the free 3'-ends of the
first oligonucleotide (with first oligonucleotide phosphodiester
and exocyclic amine protecting groups in place) on the substrate,
wherein the second oligonucleotides have different sequences in the
two different regions.
[0185] FIG. 35 depicts an illustrative embodiment of the methods
for preparing a spatial array of the present disclosure. FIG. 35A
depicts coupling of an ex situ synthesized first oligonucleotide in
3'-to-5' direction without phosphodiester and exocyclic amine
groups protected. FIG. 35B depicts in situ protection of the
phosphodiester and exocyclic amine groups of the first
oligonucleotide. FIG. 35C depicts 5'-to-3 light-directed spatial
synthesis of second oligonucleotides to the free 3'-ends of the
first oligonucleotide the substrate, wherein the second
oligonucleotides have different sequences in the two different
regions.
[0186] FIG. 36 depicts an illustrative embodiment of the methods
for preparing a spatial array of the present disclosure. FIG. 36A
depicts coupling of an ex situ synthesized first oligonucleotide in
3'-to-5' direction without phosphodiester and exocyclic amine
groups protected. FIG. 36B depicts in situ hybridization of a
complementary oligonucleotide to the first oligonucleotide, whereby
the phosphodiester and exocyclic amine groups of the first
oligonucleotide are protected. FIG. 36C depicts 5'-to-3
light-directed spatial synthesis of second oligonucleotides to the
free 3'-ends of the first oligonucleotide on the substrate while
the first oligonucleotide is hybridized to a complementary
oligonucleotide, and wherein the synthesized second
oligonucleotides have different sequences in the two different
regions.
[0187] FIG. 37 shows an example of a spatially hierarchical spatial
array, in which one region is divided into nine subregions, which
is further divided into fifteen sub-sub regions. Each spatial
barcode of the 135 sub-sub regions includes a region spatial
barcode, a sub region spatial barcode, and a sub-sub region spatial
barcode.
[0188] FIG. 38 shows an example of a library structure and
amplification scheme including a two level spatial barcode, wherein
the spatial barcode includes a region spatial barcode prefix and a
specific spatial barcode suffix.
[0189] FIG. 39 is a workflow schematic showing exemplary,
non-limiting, non-exhaustive steps for preparing a spatial array
and decoding the clusters on the slide.
[0190] FIG. 40 is a workflow schematic showing oligonucleotide
construction.
[0191] FIG. 41 shows a method for generating a capture probe.
[0192] FIG. 42 is a graph showing detection of cDNA with ligated
and unligated polyT and gene specific, e.g., Penk gene, capture
domains.
[0193] FIG. 43 is a fluorescent image showing detection of Penk
mRNA with a Penk specific capture probe generated by the method
shown in FIG. 41 compared to a control tissue optimization
slide.
[0194] FIG. 44 shows an exemplary ligation-based method for
generating a capture probe using two ligation handles.
DETAILED DESCRIPTION
I. Introduction
[0195] This disclosure describes apparatus, systems, methods, and
compositions for spatial analysis of biological samples. This
section describes certain general terminology, analytes, sample
types, and preparative steps that are referred to in later sections
of the disclosure.
[0196] (a) Spatial Analysis
[0197] Tissues and cells can be obtained from any source. For
example, tissues and cells can be obtained from single-cell or
multicellular organisms (e.g., a mammal). Tissues and cells
obtained from a mammal, e.g., a human, often have varied analyte
levels (e.g., gene and/or protein expression) which can result in
differences in cell morphology and/or function. The position of a
cell within a tissue can affect, e.g., the cell's fate, behavior,
morphology, and signaling and cross-talk with other cells in the
tissue. Information regarding the differences in analyte levels
(gene and/or protein expression) within different cells in a tissue
of a mammal can also help physicians select or administer a
treatment that will be effective in the single-cell or
multicellular organisms (e.g., a mammal) based on the detected
differences in analyte levels within different cells in the tissue.
Differences in analyte levels within different cells in a tissue of
a mammal can also provide information on how tissues (e.g., healthy
and diseased tissues) function and/or develop. Differences in
analyte levels within different cells in a tissue of a mammal can
also provide information of different mechanisms of disease
pathogenesis in a tissue and mechanism of action of a therapeutic
treatment within a tissue. Differences in analyte levels within
different cells in a tissue of a mammal can also provide
information on drug resistance mechanisms and the development of
the same in a tissue of a mammal. Differences in the presence or
absence of analytes within different cells in a tissue of a
multicellular organism (e.g., a mammal) can provide information on
drug resistance mechanisms and the development of the same in a
tissue of a multicellular organism.
[0198] The spatial analysis methodologies herein provide for the
detection of differences in an analyte level (e.g., gene and/or
protein expression) within different cells in a tissue of a mammal
or within a single cell from a mammal. For example, spatial
analysis methodologies can be used to detect the differences in
analyte levels (e.g., gene and/or protein expression) within
different cells in histological slide samples, the data from which
can be reassembled to generate a three-dimensional map of analyte
levels (e.g., gene and/or protein expression) of a tissue sample
obtained from a mammal, e.g., with a degree of spatial resolution
(e.g., single-cell resolution).
[0199] Spatial heterogeneity in developing systems has typically
been studied via RNA hybridization, immunohistochemistry,
fluorescent reporters, or purification or induction of pre-defined
subpopulations and subsequent genomic profiling (e.g., RNA-seq).
Such approaches, however, rely on a relatively small set of
pre-defined markers, therefore introducing selection bias that
limits discovery. These prior approaches also rely on a priori
knowledge. Spatial RNA assays traditionally relied on staining for
a limited number of RNA species. In contrast, single-cell
RNA-sequencing allows for deep profiling of cellular gene
expression (including non-coding RNA), but the established methods
separate cells from their native spatial context.
[0200] Current spatial analysis methodologies provide a vast amount
of analyte level and/or expression data for a variety of multiple
analytes within a sample at high spatial resolution, e.g., while
retaining the native spatial context. Spatial analysis methods
include, e.g., the use of a capture probe including a spatial
barcode (e.g., a nucleic acid sequence that provides information as
to the position of the capture probe within a cell or a tissue
sample (e.g., mammalian cell or a mammalian tissue sample) and a
capture domain that is capable of binding to an analyte (e.g., a
protein and/or nucleic acid) produced by and/or present in a cell.
As described herein, the spatial barcode can be a nucleic acid that
has a unique sequence, a unique fluorophore or a unique combination
of fluorophores, a unique amino acid sequence, a unique heavy metal
or a unique combination of heavy metals, or any other unique
detectable agent. The capture domain can be any agent that is
capable of binding to an analyte produced by and/or present in a
cell (e.g., a nucleic acid that is capable of hybridizing to a
nucleic acid from a cell (e.g., an mRNA, genomic DNA, mitochondrial
DNA, or miRNA), a substrate including an analyte, a binding partner
of an analyte, or an antibody that binds specifically to an
analyte). A capture probe can also include a nucleic acid sequence
that is complementary to a sequence of a universal forward and/or
universal reverse primer. A capture probe can also include a
cleavage site (e.g., a cleavage recognition site of a restriction
endonuclease), a photolabile bond, a thermosensitive bond, or a
chemical-sensitive bond.
[0201] The binding of an analyte to a capture probe can be detected
using a number of different methods, e.g., nucleic acid sequencing,
fluorophore detection, nucleic acid amplification, detection of
nucleic acid ligation, and/or detection of nucleic acid cleavage
products. In some examples, the detection is used to associate a
specific spatial barcode with a specific analyte produced by and/or
present in a cell (e.g., a mammalian cell).
[0202] Capture probes can be, e.g., attached to a surface, e.g., a
solid array, a bead, or a coverslip. In some examples, capture
probes are not attached to a surface. In some examples, capture
probes can be encapsulated within, embedded within, or layered on a
surface of a permeable composition (e.g., any of the substrates
described herein). For example, capture probes can be encapsulated
or disposed within a permeable bead (e.g., a gel bead). In some
examples, capture probes can be encapsulated within, embedded
within, or layered on a surface of a substrate (e.g., any of the
exemplary substrates described herein, such as a hydrogel or a
porous membrane).
[0203] In some examples, a cell or a tissue sample including a cell
are contacted with capture probes attached to a substrate (e.g., a
surface of a substrate), and the cell or tissue sample is
permeabilized to allow analytes to be released from the cell and
bind to the capture probes attached to the substrate. In some
examples, analytes released from a cell can be actively directed to
the capture probes attached to a substrate using a variety of
methods, e.g., electrophoresis, chemical gradient, pressure
gradient, fluid flow, or magnetic field.
[0204] In other examples, a capture probe can be directed to
interact with a cell or a tissue sample using a variety of methods,
e.g., inclusion of a lipid anchoring agent in the capture probe,
inclusion of an agent that binds specifically to, or forms a
covalent bond with a membrane protein in the capture probe, fluid
flow, pressure gradient, chemical gradient, or magnetic field.
[0205] Non-limiting aspects of spatial analysis methodologies are
described in WO 2011/127099, WO 2014/210233, WO 2014/210225, WO
2016/162309, WO 2018/091676, WO 2012/140224, WO 2014/060483, U.S.
Pat. Nos. 10,002,316, 9,727,810, U.S. Patent Application
Publication No. 2017/0016053, Rodrigues et al., Science
363(6434):1463-1467, 2019; WO 2018/045186, Lee et al., Nat. Protoc.
10(3):442-458, 2015; WO 2016/007839, WO 2018/045181, WO
2014/163886, Trejo et al., PLoS ONE 14(2):e0212031, 2019, U.S.
Patent Application Publication No. 2018/0245142, Chen et al.,
Science 348(6233):aaa6090, 2015, Gao et al., BMC Biol. 15:50, 2017,
WO 2017/144338, WO 2018/107054, WO 2017/222453, WO 2019/068880, WO
2011/094669, U.S. Pat. Nos. 7,709,198, 8,604,182, 8,951,726,
9,783,841, 10,041,949, WO 2016/057552, WO 2017/147483, WO
2018/022809, WO 2016/166128, WO 2017/027367, WO 2017/027368, WO
2018/136856, WO 2019/075091, U.S. Pat. No. 10,059,990, WO
2018/057999, WO 2015/161173, and Gupta et al., Nature Biotechnol.
36:1197-1202, 2018, and can be used herein in any combination.
Further non-limiting aspects of spatial analysis methodologies are
described herein.
[0206] (b) General Terminology
[0207] Specific terminology is used throughout this disclosure to
explain various aspects of the apparatus, systems, methods, and
compositions that are described. This sub-section includes
explanations of certain terms that appear in later sections of the
disclosure. To the extent that the descriptions in this section are
in apparent conflict with usage in other sections of this
disclosure, the definitions in this section will control.
[0208] (i) Barcode
[0209] A "barcode" is a label, or identifier, that conveys or is
capable of conveying information (e.g., information about an
analyte in a sample, a bead, and/or a capture probe). A barcode can
be part of an analyte, or independent of an analyte. A barcode can
be attached to an analyte. A particular barcode can be unique
relative to other barcodes.
[0210] Barcodes can have a variety of different formats. For
example, barcodes can include polynucleotide barcodes, random
nucleic acid and/or amino acid sequences, and synthetic nucleic
acid and/or amino acid sequences. A barcode can be attached to an
analyte or to another moiety or structure in a reversible or
irreversible manner. A barcode can be added to, for example, a
fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
sample before or during sequencing of the sample. Barcodes can
allow for identification and/or quantification of individual
sequencing-reads (e.g., a barcode can be or can include a unique
molecular identifier or "UMI").
[0211] Barcodes can spatially-resolve molecular components found in
biological samples, for example, at single-cell resolution (e.g., a
barcode can be or can include a "spatial barcode"). In some
embodiments, a barcode includes both a UMI and a spatial barcode.
In some embodiments, a barcode includes two or more sub-barcodes
that together function as a single barcode. For example, a
polynucleotide barcode can include two or more polynucleotide
sequences (e.g., sub-barcodes) that are separated by one or more
non-barcode sequences.
[0212] (ii) Nucleic Acid and Nucleotide
[0213] The terms "nucleic acid" and "nucleotide" are intended to be
consistent with their use in the art and to include
naturally-occurring species or functional analogs thereof.
Particularly useful functional analogs of nucleic acids are capable
of hybridizing to a nucleic acid in a sequence-specific fashion
(e.g., capable of hybridizing to two nucleic acids such that
ligation can occur between the two hybridized nucleic acids) or are
capable of being used as a template for replication of a particular
nucleotide sequence. Naturally-occurring nucleic acids generally
have a backbone containing phosphodiester bonds. An analog
structure can have an alternate backbone linkage including any of a
variety of those known in the art. Naturally-occurring nucleic
acids generally have a deoxyribose sugar (e.g., found in
deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in
ribonucleic acid (RNA)).
[0214] A nucleic acid can contain nucleotides having any of a
variety of analogs of these sugar moieties that are known in the
art. A nucleic acid can include native or non-native nucleotides.
In this regard, a native deoxyribonucleic acid can have one or more
bases selected from the group consisting of adenine (A), thymine
(T), cytosine (C), or guanine (G), and a ribonucleic acid can have
one or more bases selected from the group consisting of uracil (U),
adenine (A), cytosine (C), or guanine (G). Useful non-native bases
that can be included in a nucleic acid or nucleotide are known in
the art.
[0215] (iii) Probe and Target
[0216] A "probe" or a "target," when used in reference to a nucleic
acid or sequence of a nucleic acids, is intended as a semantic
identifier for the nucleic acid or sequence in the context of a
method or composition, and does not limit the structure or function
of the nucleic acid or sequence beyond what is expressly
indicated.
[0217] (iv) Oligonucleotide and Polynucleotide
[0218] The terms "oligonucleotide" and "polynucleotide" are used
interchangeably to refer to a single-stranded multimer of
nucleotides from about 2 to about 500 nucleotides in length.
Oligonucleotides can be synthetic, made enzymatically (e.g., via
polymerization), or using a "split-pool" method. Oligonucleotides
can include ribonucleotide monomers (i.e., can be
oligoribonucleotides) and/or deoxyribonucleotide monomers (i.e.,
oligodeoxyribonucleotides). In some examples, oligonucleotides can
include a combination of both deoxyribonucleotide monomers and
ribonucleotide monomers in the oligonucleotide (e.g., random or
ordered combination of deoxyribonucleotide monomers and
ribonucleotide monomers). An oligonucleotide can be 4 to 10, 10 to
20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80
to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350,
350 to 400, or 400-500 nucleotides in length, for example.
Oligonucleotides can include one or more functional moieties that
are attached (e.g., covalently or non-covalently) to the multimer
structure. For example, an oligonucleotide can include one or more
detectable labels (e.g., a radioisotope or fluorophore).
[0219] (v) Subject
[0220] A "subject" is an animal, such as a mammal (e.g., human or a
non-human simian), or avian (e.g., bird), or other organism, such
as a plant. Examples of subjects include, but are not limited to, a
mammal such as a rodent, mouse, rat, rabbit, guinea pig, ungulate,
horse, sheep, pig, goat, cow, cat, dog, primate (i.e. human or
non-human primate); a plant such as Arabidopsis thaliana, corn,
sorghum, oat, wheat, rice, canola, or soybean; an algae such as
Chlamydomonas reinhardtii; a nematode such as Caenorhabditis
elegans; an insect such as Drosophila melanogaster, mosquito, fruit
fly, or honey bee; an arachnid such as a spider; a fish such as
zebrafish; a reptile; an amphibian such as a frog or Xenopus
laevis; a Dictyostelium discoideum; a fungi such as Pneumocystis
carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or
Schizosaccharomyces pombe; or a Plasmodium falciparum.
[0221] (vi) Genome
[0222] A "genome" generally refers to genomic information from a
subject, which can be, for example, at least a portion of, or the
entirety of, the subject's gene-encoded hereditary information. A
genome can include coding regions (e.g., that code for proteins) as
well as non-coding regions. A genome can include the sequences of
some or all of the subject's chromosomes. For example, the human
genome ordinarily has a total of 46 chromosomes. The sequences of
some or all of these can constitute the genome.
[0223] (vii) Adaptor, Adapter, and Tag
[0224] An "adaptor," an "adapter," and a "tag" are terms that are
used interchangeably in this disclosure, and refer to species that
can be coupled to a polynucleotide sequence (in a process referred
to as "tagging") using any one of many different techniques
including (but not limited to) ligation, hybridization, and
tagmentation. Adaptors can also be nucleic acid sequences that add
a function, e.g., spacer sequences, primer sequences/sites, barcode
sequences, unique molecular identifier sequences.
[0225] (viii) Hybridizing, Hybridize, Annealing, and Anneal
[0226] The terms "hybridizing," "hybridize," "annealing," and
"anneal" are used interchangeably in this disclosure, and refer to
the pairing of substantially complementary or complementary nucleic
acid sequences within two different molecules. Pairing can be
achieved by any process in which a nucleic acid sequence joins with
a substantially or fully complementary sequence through base
pairing to form a hybridization complex. For purposes of
hybridization, two nucleic acid sequences are "substantially
complementary" if at least 60% (e.g., at least 70%, at least 80%,
or at least 90%) of their individual bases are complementary to one
another.
[0227] (ix) Primer
[0228] A "primer" is a single-stranded nucleic acid sequence having
a 3' end that can be used as a chemical substrate for a nucleic
acid polymerase in a nucleic acid extension reaction. RNA primers
are formed of RNA nucleotides, and are used in RNA synthesis, while
DNA primers are formed of DNA nucleotides and used in DNA
synthesis. Primers can also include both RNA nucleotides and DNA
nucleotides (e.g., in a random or designed pattern). Primers can
also include other natural or synthetic nucleotides described
herein that can have additional functionality. In some examples,
DNA primers can be used to prime RNA synthesis and vice versa
(e.g., RNA primers can be used to prime DNA synthesis). Primers can
vary in length. For example, primers can be about 6 bases to about
120 bases. For example, primers can include up to about 25
bases.
[0229] (x) Primer Extension
[0230] A "primer extension" refers to any method where two nucleic
acid sequences (e.g., a constant region from each of two distinct
capture probes) become linked (e.g., hybridized) by an overlap of
their respective terminal complementary nucleic acid sequences
(i.e., for example, 3' termini). Such linking can be followed by
nucleic acid extension (e.g., an enzymatic extension) of one, or
both termini using the other nucleic acid sequence as a template
for extension. Enzymatic extension can be performed by an enzyme
including, but not limited to, a polymerase and/or a reverse
transcriptase.
[0231] (xi) Proximity Ligation
[0232] A "proximity ligation" is a method of ligating two (or more)
nucleic acid sequences that are in proximity with each other
through enzymatic means (e.g., a ligase). In some embodiments,
proximity ligation can include a "gap-filling" step that involves
incorporation of one or more nucleic acids by a polymerase, based
on the nucleic acid sequence of a template nucleic acid molecule,
spanning a distance between the two nucleic acid molecules of
interest (see, e.g., U.S. Pat. No. 7,264,929, the entire contents
of which are incorporated herein by reference).
[0233] A wide variety of different methods can be used for
proximity ligating nucleic acid molecules, including (but not
limited to) "sticky-end" and "blunt-end" ligations. Additionally,
single-stranded ligation can be used to perform proximity ligation
on a single-stranded nucleic acid molecule. Sticky-end proximity
ligations involve the hybridization of complementary
single-stranded sequences between the two nucleic acid molecules to
be joined, prior to the ligation event itself. Blunt-end proximity
ligations generally do not include hybridization of complementary
regions from each nucleic acid molecule because both nucleic acid
molecules lack a single-stranded overhang at the site of
ligation.
[0234] (xii) Nucleic Acid Extension
[0235] A "nucleic acid extension" generally involves incorporation
of one or more nucleic acids (e.g., A, G, C, T, U, nucleotide
analogs, or derivatives thereof) into a molecule (such as, but not
limited to, a nucleic acid sequence) in a template-dependent
manner, such that consecutive nucleic acids are incorporated by an
enzyme (such as a polymerase or reverse transcriptase), thereby
generating a newly synthesized nucleic acid molecule. For example,
a primer that hybridizes to a complementary nucleic acid sequence
can be used to synthesize a new nucleic acid molecule by using the
complementary nucleic acid sequence as a template for nucleic acid
synthesis. Similarly, a 3' polyadenylated tail of an mRNA
transcript that hybridizes to a poly (dT) sequence (e.g., capture
domain) can be used as a template for single-strand synthesis of a
corresponding cDNA molecule.
[0236] (xiii) PCR Amplification
[0237] A "PCR amplification" refers to the use of a polymerase
chain reaction (PCR) to generate copies of genetic material,
including DNA and RNA sequences. Suitable reagents and conditions
for implementing PCR are described, for example, in U.S. Pat. Nos.
4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the
entire contents of each of which are incorporated herein by
reference. In a typical PCR amplification, the reaction mixture
includes the genetic material to be amplified, an enzyme, one or
more primers that are employed in a primer extension reaction, and
reagents for the reaction. The oligonucleotide primers are of
sufficient length to provide for hybridization to complementary
genetic material under annealing conditions. The length of the
primers generally depends on the length of the amplification
domains, but will typically be at least 4 bases, at least 5 bases,
at least 6 bases, at least 8 bases, at least 9 bases, at least 10
base pairs (bp), at least 11 bp, at least 12 bp, at least 13 bp, at
least 14 bp, at least 15 bp, at least 16 bp, at least 17 bp, at
least 18 bp, at least 19 bp, at least 20 bp, at least 25 bp, at
least 30 bp, at least 35 bp, and can be as long as 40 bp or longer,
where the length of the primers will generally range from 18 to 50
bp. The genetic material can be contacted with a single primer or a
set of two primers (forward and reverse primers), depending upon
whether primer extension, linear or exponential amplification of
the genetic material is desired.
[0238] In some embodiments, the PCR amplification process uses a
DNA polymerase enzyme. The DNA polymerase activity can be provided
by one or more distinct DNA polymerase enzymes. In certain
embodiments, the DNA polymerase enzyme is from a bacterium, e.g.,
the DNA polymerase enzyme is a bacterial DNA polymerase enzyme. For
instance, the DNA polymerase can be from a bacterium of the genus
Escherichia, Bacillus, Thermophilus, or Pyrococcus.
[0239] Suitable examples of DNA polymerases that can be used
include, but are not limited to: E. coli DNA polymerase I, Bsu DNA
polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT.TM. DNA
polymerase, DEEPVENT.TM. DNA polymerase, LongAmp.RTM. Taq DNA
polymerase, LongAmp.RTM. Hot Start Taq DNA polymerase, Crimson
LongAmp.RTM. Taq DNA polymerase, Crimson Taq DNA polymerase,
OneTaq.RTM. DNA polymerase, OneTaq.RTM. Quick-Load.RTM. DNA
polymerase, Hemo KlenTaq.RTM. DNA polymerase, REDTaq.RTM. DNA
polymerase, Phusion.RTM. DNA polymerase, Phusion.RTM. High-Fidelity
DNA polymerase, Platinum Pfx DNA polymerase, AccuPrime Pfx DNA
polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA
polymerase, Pfu DNA polymerase, T4 DNA polymerase and T7 DNA
polymerase enzymes.
[0240] The term "DNA polymerase" includes not only
naturally-occurring enzymes but also all modified derivatives
thereof, including also derivatives of naturally-occurring DNA
polymerase enzymes. For instance, in some embodiments, the DNA
polymerase can have been modified to remove 5'-3' exonuclease
activity. Sequence-modified derivatives or mutants of DNA
polymerase enzymes that can be used include, but are not limited
to, mutants that retain at least some of the functional, e.g., DNA
polymerase activity of the wild-type sequence. Mutations can affect
the activity profile of the enzymes, e.g., enhance or reduce the
rate of polymerization, under different reaction conditions, e.g.,
temperature, template concentration, primer concentration, etc.
Mutations or sequence-modifications can also affect the exonuclease
activity and/or thermostability of the enzyme.
[0241] In some embodiments, PCR amplification can include reactions
such as, but not limited to, a strand-displacement amplification
reaction, a rolling circle amplification reaction, a ligase chain
reaction, a transcription-mediated amplification reaction, an
isothermal amplification reaction, and/or a loop-mediated
amplification reaction.
[0242] In some embodiments, PCR amplification uses a single primer
that is complementary to the 3' tag of target DNA fragments. In
some embodiments, PCR amplification uses a first and a second
primer, where at least a 3' end portion of the first primer is
complementary to at least a portion of the 3' tag of the target
nucleic acid fragments, and where at least a 3' end portion of the
second primer exhibits the sequence of at least a portion of the 5'
tag of the target nucleic acid fragments. In some embodiments, a 5'
end portion of the first primer is non-complementary to the 3' tag
of the target nucleic acid fragments, and a 5' end portion of the
second primer does not exhibit the sequence of at least a portion
of the 5' tag of the target nucleic acid fragments. In some
embodiments, the first primer includes a first universal sequence
and/or the second primer includes a second universal sequence.
[0243] In some embodiments (e.g., when the PCR amplification
amplifies captured DNA), the PCR amplification products can be
ligated to additional sequences using a DNA ligase enzyme. The DNA
ligase activity can be provided by one or more distinct DNA ligase
enzymes. In some embodiments, the DNA ligase enzyme is from a
bacterium, e.g., the DNA ligase enzyme is a bacterial DNA ligase
enzyme. In some embodiments, the DNA ligase enzyme is from a virus
(e.g., a bacteriophage). For instance, the DNA ligase can be T4 DNA
ligase. Other enzymes appropriate for the ligation step include,
but are not limited to, Tth DNA ligase, Taq DNA ligase,
Thermococcus sp. (strain 9oN) DNA ligase (9oN.TM. DNA ligase,
available from New England Biolabs, Ipswich, Mass.), and
Ampligase.TM. (available from Epicentre Biotechnologies, Madison,
Wis.). Derivatives, e.g., sequence-modified derivatives, and/or
mutants thereof, can also be used.
[0244] In some embodiments, genetic material is amplified by
reverse transcription polymerase chain reaction (RT-PCR). The
desired reverse transcriptase activity can be provided by one or
more distinct reverse transcriptase enzymes, suitable examples of
which include, but are not limited to: M-MLV, MuLV, AMV, HIV,
ArrayScript.TM., MultiScribe.TM. ThermoScript.TM., and
SuperScript.RTM. I, II, III, and IV enzymes. "Reverse
transcriptase" includes not only naturally occurring enzymes, but
all such modified derivatives thereof, including also derivatives
of naturally-occurring reverse transcriptase enzymes.
[0245] In addition, reverse transcription can be performed using
sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and
HIV reverse transcriptase enzymes, including mutants that retain at
least some of the functional, e.g., reverse transcriptase, activity
of the wild-type sequence. The reverse transcriptase enzyme can be
provided as part of a composition that includes other components,
e.g., stabilizing components that enhance or improve the activity
of the reverse transcriptase enzyme, such as RNase inhibitor(s),
inhibitors of DNA-dependent DNA synthesis, e.g., actinomycin D.
Many sequence-modified derivative or mutants of reverse
transcriptase enzymes, e.g., M-MLV, and compositions including
unmodified and modified enzymes are commercially available, e.g.,
ArrayScript.TM. MultiScribe.TM., ThermoScript.TM., and
SuperScript.RTM. I, II, III, and IV enzymes.
[0246] Certain reverse transcriptase enzymes (e.g., Avian
Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine
Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase) can synthesize
a complementary DNA strand using both RNA (cDNA synthesis) and
single-stranded DNA (ssDNA) as a template. Thus, in some
embodiments, the reverse transcription reaction can use an enzyme
(reverse transcriptase) that is capable of using both RNA and ssDNA
as the template for an extension reaction, e.g., an AMV or MMLV
reverse transcriptase.
[0247] In some embodiments, the quantification of RNA and/or DNA is
carried out by real-time PCR (also known as quantitative PCR or
qPCR), using techniques well known in the art, such as but not
limited to "TAQMAN.TM." or "SYBR.RTM.", or on capillaries
("LightCycler.RTM. Capillaries"). In some embodiments, the
quantification of genetic material is determined by optical
absorbance and with real-time PCR. In some embodiments, the
quantification of genetic material is determined by digital PCR. In
some embodiments, the genes analyzed can be compared to a reference
nucleic acid extract (DNA and RNA) corresponding to the expression
(mRNA) and quantity (DNA) in order to compare expression levels of
the target nucleic acids.
[0248] (xiv) Antibody
[0249] An "antibody" is a polypeptide molecule that recognizes and
binds to a complementary target antigen. Antibodies typically have
a molecular structure shape that resembles a Y shape.
Naturally-occurring antibodies, referred to as immunoglobulins,
belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD, and
IgE. Antibodies can also be produced synthetically. For example,
recombinant antibodies, which are monoclonal antibodies, can be
synthesized using synthetic genes by recovering the antibody genes
from source cells, amplifying into an appropriate vector, and
introducing the vector into a host to cause the host to express the
recombinant antibody. In general, recombinant antibodies can be
cloned from any species of antibody-producing animal using suitable
oligonucleotide primers and/or hybridization probes. Recombinant
techniques can be used to generate antibodies and antibody
fragments, including non-endogenous species.
[0250] Synthetic antibodies can be derived from non-immunoglobulin
sources. For example, antibodies can be generated from nucleic
acids (e.g., aptamers), and from non-immunoglobulin protein
scaffolds (such as peptide aptamers) into which hypervariable loops
are inserted to form antigen binding sites. Synthetic antibodies
based on nucleic acids or peptide structures can be smaller than
immunoglobulin-derived antibodies, leading to greater tissue
penetration.
[0251] Antibodies can also include affimer proteins, which are
affinity reagents that typically have a molecular weight of about
12-14 kDa. Affimer proteins generally bind to a target (e.g., a
target protein) with both high affinity and specificity. Examples
of such targets include, but are not limited to, ubiquitin chains,
immunoglobulins, and C-reactive protein. In some embodiments,
affimer proteins are derived from cysteine protease inhibitors, and
include peptide loops and a variable N-terminal sequence that
provides the binding site.
[0252] Antibodies can also include single domain antibodies (VHH
domains and VNAR domains), scFvs, and Fab fragments.
[0253] (xv) Affinity Group
[0254] An "affinity group" is a molecule or molecular moiety which
has a high affinity or preference for associating or binding with
another specific or particular molecule or moiety. The association
or binding with another specific or particular molecule or moiety
can be via a non-covalent interaction, such as hydrogen bonding,
ionic forces, and van der Waals interactions. An affinity group
can, for example, be biotin, which has a high affinity or
preference to associate or bind to the protein avidin or
streptavidin. An affinity group, for example, can also refer to
avidin or streptavidin which has an affinity to biotin. Other
examples of an affinity group and specific or particular molecule
or moiety to which it binds or associates with include, but are not
limited to, antibodies or antibody fragments and their respective
antigens, such as digoxigenin and anti-digoxigenin antibodies,
lectin, and carbohydrates (e.g., a sugar, a monosaccharide, a
disaccharide, or a polysaccharide), and receptors and receptor
ligands.
[0255] Any pair of affinity group and its specific or particular
molecule or moiety to which it binds or associates with can have
their roles reversed, for example, such that between a first
molecule and a second molecule, in a first instance the first
molecule is characterized as an affinity group for the second
molecule, and in a second instance the second molecule is
characterized as an affinity group for the first molecule.
[0256] (xvi) Label, Detectable Label, and Optical Label
[0257] The terms "detectable label," "optical label," and "label"
are used interchangeably herein to refer to a directly or
indirectly detectable moiety that is associated with (e.g.,
conjugated to) a molecule to be detected, e.g., a capture probe or
analyte. The detectable label can be directly detectable by itself
(e.g., radioisotope labels or fluorescent labels) or, in the case
of an enzymatic label, can be indirectly detectable, e.g., by
catalyzing chemical alterations of a chemical substrate compound or
composition, which chemical substrate compound or composition is
directly detectable. Detectable labels can be suitable for small
scale detection and/or suitable for high-throughput screening. As
such, suitable detectable labels include, but are not limited to,
radioisotopes, fluorophores, chemiluminescent compounds,
bioluminescent compounds, and dyes.
[0258] The detectable label can be qualitatively detected (e.g.,
optically or spectrally), or it can be quantified. Qualitative
detection generally includes a detection method in which the
existence or presence of the detectable label is confirmed, whereas
quantifiable detection generally includes a detection method having
a quantifiable (e.g., numerically reportable) value such as an
intensity, duration, polarization, and/or other properties. In some
embodiments, the detectable label is bound to a feature or to a
capture probe associated with a feature. For example, detectably
labeled features can include a fluorescent, a colorimetric, or a
chemiluminescent label attached to a bead (see, for example,
Rajeswari et al., J. Microbiol Methods 139:22-28, 2017, and
Forcucci et al., J. Biomed Opt. 10:105010, 2015, the entire
contents of each of which are incorporated herein by
reference).
[0259] In some embodiments, a plurality of detectable labels can be
attached to a feature, capture probe, or composition to be
detected. For example, detectable labels can be incorporated during
nucleic acid polymerization or amplification (e.g.,
Cy5.RTM.-labelled nucleotides, such as Cy5.RTM.-dCTP). Any suitable
detectable label can be used. In some embodiments, the detectable
label is a fluorophore. For example, the fluorophore can be from a
group that includes: 7-AAD (7-Aminoactinomycin D), Acridine Orange
(+DNA), Acridine Orange (+RNA), Alexa Fluor.RTM. 350, Alexa
Fluor.RTM. 430, Alexa Fluor.RTM. 488, Alexa Fluor.RTM. 532, Alexa
Fluor.RTM. 546, Alexa Fluor.RTM. 555, Alexa Fluor.RTM. 568, Alexa
Fluor.RTM. 594, Alexa Fluor.RTM. 633, Alexa Fluor.RTM. 647, Alexa
Fluor.RTM. 660, Alexa Fluor.RTM. 680, Alexa Fluor.RTM. 700, Alexa
Fluor.RTM. 750, Allophycocyanin (APC), AMCA/AMCA-X,
7-Aminoactinomycin D (7-AAD), 7-Amino-4-methylcoumarin,
6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG.TM. CBQCA,
ATTO-TAG.TM. FQ, Auramine O-Feulgen, BCECF (high pH), BFP (Blue
Fluorescent Protein), BFP/GFP FRET, BOBO.TM.-1/BO-PRO.TM.-1,
BOBO.TM.-3/BO-PRO.TM.-3, BODIPY.RTM. FL, BODIPY.RTM. TMR,
BODIPY.RTM. TR-X, BODIPY.RTM. 530/550, BODIPY.RTM. 558/568,
BODIPY.RTM. 564/570, BODIPY.RTM. 581/591, BODIPY.RTM. 630/650-X,
BODIPY.RTM. 650-665-X, BTC, Calcein, Calcein Blue, Calcium
Crimson.TM., Calcium Green-1.TM., Calcium Orange.TM.,
Calcofluor.RTM. White, 5-Carboxyfluoroscein (5-FAM),
5-Carboxynaphthofluoroscein, 6-Carboxyrhodamine 6G,
5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine
(5-ROX), Cascade Blue.RTM., Cascade Yellow.TM., CCF2
(GeneBLAzer.TM.), CFP (Cyan Fluorescent Protein), CFP/YFP FRET,
Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC Formazan,
Cy2.RTM., Cy3.RTM., Cy3.5.RTM., Cy5.RTM., Cy5.5.RTM., Cy7.RTM.,
Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansylchloride,
DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS,
Dil (DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4 ANEPPS,
Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (Red Fluorescent Protein),
EBFP, ECFP, EGFP, ELF.RTM.-97 alcohol, Eosin, Erythrosin, Ethidium
bromide, Ethidium homodimer-1 (EthD-1), Europium (III) Chloride,
5-FAM (5-Carboxyfluorescein), Fast Blue, Fluorescein-dT
phosphoramidite, FITC, Fluo-3, Fluo-4, FluorX.RTM., Fluoro-Gold.TM.
(high pH), Fluoro-Gold.TM. (low pH), Fluoro-Jade, FM.RTM. 1-43,
Fura-2 (high calcium), Fura-2/BCECF, Fura Red.TM. (high calcium),
Fura Red.TM./Fluo-3, GeneBLAzer.TM. (CCF2), GFP Red Shifted
(rsGFP), GFP Wild Type, GFP/BFP FRET, GFP/DsRed FRET, Hoechst 33342
& 33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1
(high calcium), Indo-1 (low calcium), Indodicarbocyanine,
Indotricarbocyanine, JC-1, 6-JOE, JOJO.TM.-1/JO-PRO.TM.-1, LDS 751
(+DNA), LDS 751 (+RNA), LOLO.TM.-1/LO-PRO.TM.-1, Lucifer Yellow,
LysoSensor.TM. Blue (pH 5), LysoSensor.TM. Green (pH 5),
LysoSensor.TM. Yellow/Blue (pH 4.2), LysoTracker.RTM. Green,
LysoTracker.RTM. Red, LysoTracker.RTM. Yellow, Mag-Fura-2,
Mag-Indo-1, Magnesium Green.TM. Marina Blue.RTM.,
4-Methylumbelliferone, Mithramycin, MitoTracker.RTM. Green,
MitoTracker.RTM. Orange, MitoTracker.RTM. Red, NBD (amine), Nile
Red, Oregon Green.RTM. 488, Oregon Green.RTM. 500, Oregon
Green.RTM. 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5,
PE-Cy7, PE-Texas Red, PerCP (Peridinin chlorphyll protein),
PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin,
R-phycocyanin, R-phycoerythrin (PE), PI (Propidium Iodide), PKH26,
PKH67, POPO.TM.-1/PO-PRO.TM.-1, POPO.TM.-3/PO-PRO.TM.-3, Propidium
Iodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red (PE-Cy5),
Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red
Fluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine
B, Rhodamine Green.TM., Rhodamine Red.TM., Rhodamine Phalloidin,
Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A,
S65C, S65L, S65T, SBFI, SITS, SNAFL.RTM.-1 (high pH), SNAFL.RTM.-2,
SNARF.RTM.-1 (high pH), SNARF.RTM.-1 (low pH), Sodium Green.TM.,
SpectrumAqua.RTM., SpectrumGreen.RTM. #1, SpectrumGreen.RTM. #2,
SpectrumOrange.RTM., SpectrumRed.RTM., SYTO.RTM. 11, SYTO.RTM. 13,
SYTO.RTM. 17, SYTO.RTM. 45, SYTOX.RTM. Blue, SYTOX.RTM. Green,
SYTOX.RTM. Orange, 5-TAMRA (5-Carboxytetramethylrhodamine),
Tetramethylrhodamine (TRITC), Texas Red.RTM./Texas Red.RTM.-X,
Texas Red.RTM.-X (NHS Ester), Thiadicarbocyanine, Thiazole Orange,
TOTO.RTM.-1/TO-PRO.RTM.-1, TOTO.RTM.-3/TO-PRO.RTM.-3,
TO-PRO.RTM.-5, Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine),
TruRed (PerCP-Cy5.5), WW 781, X-Rhodamine (XRITC), Y66F, Y66H, Y66
W, YFP (Yellow Fluorescent Protein), YOYO.RTM.-1/YO-PRO.RTM.-1,
YOYO.RTM.-3/YO-PRO.RTM.-3, 6-FAM (Fluorescein), 6-FAM (NHS Ester),
6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow, MAX, TET,
TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101, ATTO
590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5' IRDye.RTM.
700, 5' IRDye.RTM. 800, 5' IRDye.RTM. 800CW (NHS Ester), WellRED D4
Dye, WellRED D3 Dye, WellRED D2 Dye, Lightcycler.RTM. 640 (NHS
Ester), and Dy 750 (NHS Ester).
[0260] As mentioned above, in some embodiments, a detectable label
is or includes a luminescent or chemiluminescent moiety. Common
luminescent/chemiluminescent moieties include, but are not limited
to, peroxidases such as horseradish peroxidase (HRP), soybean
peroxidase (SP), alkaline phosphatase, and luciferase. These
protein moieties can catalyze chemiluminescent reactions given the
appropriate chemical substrates (e.g., an oxidizing reagent plus a
chemiluminescent compound). A number of compound families are known
to provide chemiluminescence under a variety of conditions.
Non-limiting examples of chemiluminescent compound families include
2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy-
and the dimethylamino[ca]benz analog. These compounds can luminesce
in the presence of alkaline hydrogen peroxide or calcium
hypochlorite and base. Other examples of chemiluminescent compound
families include, e.g., 2,4,5-triphenylimidazoles,
para-dimethylamino and--methoxy substituents, oxalates such as
oxalyl active esters, p-nitrophenyl, N-alkyl acridinum esters,
luciferins, lucigenins, or acridinium esters.
[0261] (xvii) Template Switching Oligonucleotide
[0262] A "template switching oligonucleotide" is an oligonucleotide
that hybridizes to untemplated nucleotides added by a reverse
transcriptase (e.g., enzyme with terminal transferase activity)
during reverse transcription. In some embodiments, a template
switching oligonucleotide hybridizes to untemplated poly(C)
nucleotides added by a reverse transcriptase. In some embodiments,
the template switching oligonucleotide adds a common 5' sequence to
full-length cDNA that is used for cDNA amplification.
[0263] In some embodiments, the template switching oligonucleotide
adds a common sequence onto the 5' end of the RNA being reverse
transcribed. For example, a template switching oligonucleotide can
hybridize to untemplated poly(C) nucleotides added onto the end of
a cDNA molecule and provide a template for the reverse
transcriptase to continue replication to the 5' end of the template
switching oligonucleotide, thereby generating full-length cDNA
ready for further amplification. In some embodiments, once a
full-length cDNA molecule is generated, the template switching
oligonucleotide can serve as a primer in a cDNA amplification
reaction.
[0264] In some embodiments, a template switching oligonucleotide is
added before, contemporaneously with, or after a reverse
transcription, or other terminal transferase-based reaction. In
some embodiments, a template switching oligonucleotide is included
in the capture probe. In certain embodiments, methods of sample
analysis using template switching oligonucleotides can involve the
generation of nucleic acid products from analytes of the tissue
sample, followed by further processing of the nucleic acid products
with the template switching oligonucleotide.
[0265] Template switching oligonucleotides can include a
hybridization region and a template region. The hybridization
region can include any sequence capable of hybridizing to the
target. In some embodiments, the hybridization region can, e.g.,
include a series of G bases to complement the overhanging C bases
at the 3' end of a cDNA molecule. The series of G bases can include
1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than
5 G bases. The template sequence can include any sequence to be
incorporated into the cDNA. In other embodiments, the hybridization
region can include at least one base in addition to at least one G
base. In other embodiments, the hybridization can include bases
that are not a G base. In some embodiments, the template region
includes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag
sequences and/or functional sequences. In some embodiments, the
template region and hybridization region are separated by a
spacer.
[0266] In some embodiments, the template regions include a barcode
sequence. The barcode sequence can act as a spatial barcode and/or
as a unique molecular identifier. Template switching
oligonucleotides can include deoxyribonucleic acids; ribonucleic
acids; modified nucleic acids including 2-aminopurine,
2,6-diaminopurine (2-amino-dA), inverted dT, 5-methyl dC,
2'-deoxylnosine, Super T (5-hydroxybutynl-2'-deoxyuridine), Super G
(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked
nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG,
Iso-dC, 2' fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and
Fluoro G), or any combination of the foregoing.
[0267] In some embodiments, the length of a template switching
oligonucleotide can be at least about 1, 2, 10, 20, 50, 75, 100,
150, 200, or 250 nucleotides or longer. In some embodiments, the
length of a template switching oligonucleotide can be at most about
2, 10, 20, 50, 100, 150, 200, or 250 nucleotides or longer.
[0268] (xviii) Splint Oligonucleotide
[0269] A "splint oligonucleotide" is an oligonucleotide that, when
hybridized to other polynucleotides, acts as a "splint" to position
the polynucleotides next to one another so that they can be ligated
together. In some embodiments, the splint oligonucleotide is DNA or
RNA. The splint oligonucleotide can include a nucleotide sequence
that is partially complimentary to nucleotide sequences from two or
more different oligonucleotides. In some embodiments, the splint
oligonucleotide assists in ligating a "donor" oligonucleotide and
an "acceptor" oligonucleotide. In general, an RNA ligase, a DNA
ligase, or another other variety of ligase is used to ligate two
nucleotide sequences together
[0270] In some embodiments, the splint oligonucleotide is between
10 and 50 oligonucleotides in length, e.g., between 10 and 45, 10
and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20
oligonucleotides in length. In some embodiments, the splint
oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and
35, 15 and 30, 15 and 30, or 15 and 25 nucleotides in length.
[0271] (c) Analytes
[0272] The apparatus, systems, methods, and compositions described
in this disclosure can be used to detect and analyze a wide variety
of different analytes. For the purpose of this disclosure, an
"analyte" can include any biological substance, structure, moiety,
or component to be analyzed. The term "target" can similarly refer
to an analyte of interest.
[0273] Analytes can be broadly classified into one of two groups:
nucleic acid analytes, and non-nucleic acid analytes. Examples of
non-nucleic acid analytes include, but are not limited to, lipids,
carbohydrates, peptides, proteins, glycoproteins (N-linked or
O-linked), lipoproteins, phosphoproteins, specific phosphorylated
or acetylated variants of proteins, amidation variants of proteins,
hydroxylation variants of proteins, methylation variants of
proteins, ubiquitylation variants of proteins, sulfation variants
of proteins, viral coat proteins, extracellular and intracellular
proteins, antibodies, and antigen binding fragments. In some
embodiments, the analyte can be an organelle (e.g., nuclei or
mitochondria).
[0274] Cell surface features corresponding to analytes can include,
but are not limited to, a receptor, an antigen, a surface protein,
a transmembrane protein, a cluster of differentiation protein, a
protein channel, a protein pump, a carrier protein, a phospholipid,
a glycoprotein, a glycolipid, a cell-cell interaction protein
complex, an antigen-presenting complex, a major histocompatibility
complex, an engineered T-cell receptor, a T-cell receptor, a B-cell
receptor, a chimeric antigen receptor, an extracellular matrix
protein, a posttranslational modification (e.g., phosphorylation,
glycosylation, ubiquitination, nitrosylation, methylation,
acetylation or lipidation) state of a cell surface protein, a gap
junction, and an adherens junction.
[0275] Analytes can be derived from a specific type of cell and/or
a specific sub-cellular region. For example, analytes can be
derived from cytosol, from cell nuclei, from mitochondria, from
microsomes, and more generally, from any other compartment,
organelle, or portion of a cell. Permeabilizing agents that
specifically target certain cell compartments and organelles can be
used to selectively release analytes from cells for analysis.
[0276] Examples of nucleic acid analytes include DNA analytes such
as genomic DNA, methylated DNA, specific methylated DNA sequences,
fragmented DNA, mitochondrial DNA, in situ synthesized PCR
products, and RNA/DNA hybrids.
[0277] Examples of nucleic acid analytes also include RNA analytes
such as various types of coding and non-coding RNA. Examples of the
different types of RNA analytes include messenger RNA (mRNA),
ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and
viral RNA. The RNA can be a transcript (e.g., present in a tissue
section). The RNA can be small (e.g., less than 200 nucleic acid
bases in length) or large (e.g., RNA greater than 200 nucleic acid
bases in length). Small RNAs mainly include 5.8S ribosomal RNA
(rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small
interfering RNA (siRNA), small nucleolar RNA (snoRNAs),
Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and
small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA
or single-stranded RNA. The RNA can be circular RNA. The RNA can be
a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).
[0278] Additional examples of analytes include mRNA and cell
surface features (e.g., using the labelling agents described
herein), mRNA and intracellular proteins (e.g., transcription
factors), mRNA and cell methylation status, mRNA and accessible
chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq), mRNA and
metabolites (e.g., using the labelling agents described herein), a
barcoded labelling agent (e.g., the oligonucleotide tagged
antibodies described herein) and a V(D)J sequence of an immune cell
receptor (e.g., T-cell receptor), mRNA and a perturbation agent
(e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or
antisense oligonucleotide as described herein). In some
embodiments, a perturbation agent can be a small molecule, an
antibody, a drug, an aptamer, a miRNA, a physical environmental
(e.g., temperature change), or any other known perturbation
agents.
[0279] Analytes can include a nucleic acid molecule with a nucleic
acid sequence encoding at least a portion of a V(D)J sequence of an
immune cell receptor (e.g., a TCR or BCR). In some embodiments, the
nucleic acid molecule is cDNA first generated from reverse
transcription of the corresponding mRNA, using a poly(T) containing
primer. The generated cDNA can then be barcoded using a capture
probe, featuring a barcode sequence (and optionally, a UMI
sequence) that hybridizes with at least a portion of the generated
cDNA. In some embodiments, a template switching oligonucleotide
hybridizes to a poly(C) tail added to a 3'end of the cDNA by a
reverse transcriptase enzyme. The original mRNA template and
template switching oligonucleotide can then be denatured from the
cDNA and the barcoded capture probe can then hybridize with the
cDNA and a complement of the cDNA generated. Additional methods and
compositions suitable for barcoding cDNA generated from mRNA
transcripts including those encoding V(D)J regions of an immune
cell receptor and/or barcoding methods and composition including a
template switch oligonucleotide are described in PCT Patent
Application PCT/US2017/057269, filed Oct. 18, 2017, and U.S. patent
application Ser. No. 15/825,740, filed Nov. 29, 2017, both of which
are incorporated herein by reference in their entireties. V(D)J
analysis can also be completed with the use of one or more
labelling agents that bind to particular surface features of immune
cells and associated with barcode sequences. The one or more
labelling agents can include an MHC or MHC multimer.
[0280] As described above, the analyte can include a nucleic acid
capable of functioning as a component of a gene editing reaction,
such as, for example, clustered regularly interspaced short
palindromic repeats (CRISPR)-based gene editing. Accordingly, the
capture probe can include a nucleic acid sequence that is
complementary to the analyte (e.g., a sequence that can hybridize
to the CRISPR RNA (crRNA), single guide RNA (sgRNA), or an adapter
sequence engineered into a crRNA or sgRNA).
[0281] In certain embodiments, an analyte can be extracted from a
live cell. Processing conditions can be adjusted to ensure that a
biological sample remains live during analysis, and analytes are
extracted from (or released from) live cells of the sample. Live
cell-derived analytes can be obtained only once from the sample, or
can be obtained at intervals from a sample that continues to remain
in viable condition.
[0282] In general, the systems, apparatus, methods, and
compositions can be used to analyze any number of analytes. For
example, the number of analytes that are analyzed can be at least
about 2, at least about 3, at least about 4, at least about 5, at
least about 6, at least about 7, at least about 8, at least about
9, at least about 10, at least about 11, at least about 12, at
least about 13, at least about 14, at least about 15, at least
about 20, at least about 25, at least about 30, at least about 40,
at least about 50, at least about 100, at least about 1,000, at
least about 10,000, at least about 100,000 or more different
analytes present in a region of the sample or within an individual
feature of the substrate. Methods for performing multiplexed assays
to analyze two or more different analytes will be discussed in a
subsequent section of this disclosure.
[0283] (d) Biological Samples
[0284] (i) Types of Biological Samples
[0285] A "biological sample" is obtained from the subject for
analysis using any of a variety of techniques including, but not
limited to, biopsy, surgery, and laser capture microscopy (LCM),
and generally includes cells and/or other biological material from
the subject. In addition to the subjects described above, a
biological sample can be obtained from non-mammalian organisms
(e.g., a plants, an insect, an arachnid, a nematode (e.g.,
Caenorhabditis elegans), a fungi, an amphibian, or a fish (e.g.,
zebrafish)). A biological sample can be obtained from a prokaryote
such as a bacterium, e.g., Escherichia coli, Staphylococci or
Mycoplasma pneumoniae; an archaea; a virus such as Hepatitis C
virus or human immunodeficiency virus; or a viroid. A biological
sample can be obtained from a eukaryote, such as a patient derived
organoid (PDO) or patient derived xenograft (PDX). The biological
sample can include organoids, a miniaturized and simplified version
of an organ produced in vitro in three dimensions that shows
realistic micro-anatomy. Organoids can be generated from one or
more cells from a tissue, embryonic stem cells, and/or induced
pluripotent stem cells, which can self-organize in
three-dimensional culture owing to their self-renewal and
differentiation capacities. In some embodiments, an organoid is a
cerebral organoid, an intestinal organoid, a stomach organoid, a
lingual organoid, a thyroid organoid, a thymic organoid, a
testicular organoid, a hepatic organoid, a pancreatic organoid, an
epithelial organoid, a lung organoid, a kidney organoid, a
gastruloid, a cardiac organoid, or a retinal organoid. Subjects
from which biological samples can be obtained can be healthy or
asymptomatic individuals, individuals that have or are suspected of
having a disease (e.g., cancer) or a pre-disposition to a disease,
and/or individuals that are in need of therapy or suspected of
needing therapy.
[0286] Biological samples can be derived from a homogeneous culture
or population of the subjects or organisms mentioned herein or
alternatively from a collection of several different organisms, for
example, in a community or ecosystem.
[0287] Biological samples can include one or more diseased cells. A
diseased cell can have altered metabolic properties, gene
expression, protein expression, and/or morphologic features.
Examples of diseases include inflammatory disorders, metabolic
disorders, nervous system disorders, and cancer. Cancer cells can
be derived from solid tumors, hematological malignancies, cell
lines, or obtained as circulating tumor cells.
[0288] Biological samples can also include fetal cells. For
example, a procedure such as amniocentesis can be performed to
obtain a fetal cell sample from maternal circulation. Sequencing of
fetal cells can be used to identify any of a number of genetic
disorders, including, e.g., aneuploidy such as Down's syndrome,
Edwards syndrome, and Patau syndrome. Further, cell surface
features of fetal cells can be used to identify any of a number of
disorders or diseases.
[0289] Biological samples can also include immune cells. Sequence
analysis of the immune repertoire of such cells, including genomic,
proteomic, and cell surface features, can provide a wealth of
information to facilitate an understanding the status and function
of the immune system. By way of example, determining the status
(e.g., negative or positive) of minimal residue disease (MRD) in a
multiple myeloma (MM) patient following autologous stem cell
transplantation is considered a predictor of MRD in the MM patient
(see, e.g., U.S. Patent Application Publication No. 2018/0156784,
the entire contents of which are incorporated herein by
reference).
[0290] Examples of immune cells in a biological sample include, but
are not limited to, B cells, T cells (e.g., cytotoxic T cells,
natural killer T cells, regulatory T cells, and T helper cells),
natural killer cells, cytokine induced killer (CIK) cells, myeloid
cells, such as granulocytes (basophil granulocytes, eosinophil
granulocytes, neutrophil granulocytes/hypersegmented neutrophils),
monocytes/macrophages, mast cells, thrombocytes/megakaryocytes, and
dendritic cells.
[0291] The biological sample can include any number of
macromolecules, for example, cellular macromolecules and organelles
(e.g., mitochondria and nuclei). The biological sample can be a
nucleic acid sample and/or protein sample. The biological sample
can be a carbohydrate sample or a lipid sample. The biological
sample can be obtained as a tissue sample, such as a tissue
section, biopsy, a core biopsy, needle aspirate, or fine needle
aspirate. The sample can be a fluid sample, such as a blood sample,
urine sample, or saliva sample. The sample can be a skin sample, a
colon sample, a cheek swab, a histology sample, a histopathology
sample, a plasma or serum sample, a tumor sample, living cells,
cultured cells, a clinical sample such as, for example, whole blood
or blood-derived products, blood cells, or cultured tissues or
cells, including cell suspensions.
[0292] Cell-free biological samples can include extracellular
polynucleotides. Extracellular polynucleotides can be isolated from
a bodily sample, e.g., blood, plasma, serum, urine, saliva, mucosal
excretions, sputum, stool, and tears.
[0293] As discussed above, a biological sample can include a single
analyte of interest, or more than one analyte of interest. Methods
for performing multiplexed assays to analyze two or more different
analytes in a single biological sample is discussed in a subsequent
section of this disclosure.
[0294] (ii) Preparation of Biological Samples
[0295] A variety of steps can be performed to prepare a biological
sample for analysis. Except where indicated otherwise, the
preparative steps described below can generally be combined in any
manner to appropriately prepare a particular sample for
analysis.
[0296] (1) Tissue Sectioning
[0297] A biological sample can be harvested from a subject (e.g.,
via surgical biopsy, whole subject sectioning), grown in vitro on a
growth substrate or culture dish as a population of cells, or
prepared as a tissue slice or tissue section. Grown samples may be
sufficiently thin for analysis without further processing steps.
Alternatively, grown samples, and samples obtained via biopsy or
sectioning, can be prepared as thin tissue sections using a
mechanical cutting apparatus such as a vibrating blade microtome.
As another alternative, in some embodiments, a thin tissue section
can be prepared by applying a touch imprint of a biological sample
to a suitable substrate material.
[0298] The thickness of the tissue section can be a fraction of
(e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1)
the maximum cross-sectional dimension of a cell. However, tissue
sections having a thickness that is larger than the maximum
cross-section cell dimension can also be used. For example,
cryostat sections can be used, which can be, e.g., 10-20
micrometers thick.
[0299] More generally, the thickness of a tissue section typically
depends on the method used to prepare the section and the physical
characteristics of the tissue, and therefore sections having a wide
variety of different thicknesses can be prepared and used. For
example, the thickness of the tissue section can be at least 0.1,
0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,
13, 14, 15, 20, 30, 40, or 50 micrometers. Thicker sections can
also be used if desired or convenient, e.g., at least 70, 80, 90,
or 100 micrometers or more. Typically, the thickness of a tissue
section is between 1-100 micrometers, 1-50 micrometers, 1-30
micrometers, 1-25 micrometers, 1-20 micrometers, 1-15 micrometers,
1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6
micrometers, but as mentioned above, sections with thicknesses
larger or smaller than these ranges can also be analysed.
[0300] Multiple sections can also be obtained from a single
biological sample. For example, multiple tissue sections can be
obtained from a surgical biopsy sample by performing serial
sectioning of the biopsy sample using a sectioning blade. Spatial
information among the serial sections can be preserved in this
manner, and the sections can be analysed successively to obtain
three-dimensional information about the biological sample.
[0301] (2) Freezing
[0302] In some embodiments, the biological sample (e.g., a tissue
section as described above) can be prepared by deep freezing at a
temperature suitable to maintain or preserve the integrity (e.g.,
the physical characteristics) of the tissue structure. Such a
temperature can be, e.g., less than -20.degree. C., or less than
-25.degree. C., -30.degree. C., -40.degree. C., -50.degree. C.,
-60.degree. C., -70.degree. C., -80.degree. C. -90.degree. C.,
-100.degree. C., -110.degree. C., -120.degree. C., -130.degree. C.,
-140.degree. C., -150.degree. C., -160.degree. C., -170.degree. C.,
-180.degree. C., -190.degree. C., or -200.degree. C. The frozen
tissue sample can be sectioned, e.g., thinly sliced, onto a
substrate surface using any number of suitable methods. For
example, a tissue sample can be prepared using a chilled microtome
(e.g., a cryostat) set at a temperature suitable to maintain both
the structural integrity of the tissue sample and the chemical
properties of the nucleic acids in the sample. Such a temperature
can be, e.g., less than -15.degree. C., less than -20.degree. C.,
or less than -25.degree. C. A sample can be snap frozen in
isopentane and liquid nitrogen. Frozen samples can be stored in a
sealed container prior to embedding.
[0303] (3) Formalin Fixation and Paraffin Embedding
[0304] In some embodiments, the biological sample can be prepared
using formalin-fixation and paraffin-embedding (FFPE), which are
established methods. In some embodiments, cell suspensions and
other non-tissue samples can be prepared using formalin-fixation
and paraffin-embedding. Following fixation of the sample and
embedding in a paraffin or resin block, the sample can be sectioned
as described above. Prior to analysis, the paraffin-embedding
material can be removed from the tissue section (e.g.,
deparaffinization) by incubating the tissue section in an
appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5%
ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol
for 2 minutes).
[0305] (4) Fixation
[0306] As an alternative to formalin fixation described above, a
biological sample can be fixed in any of a variety of other
fixatives to preserve the biological structure of the sample prior
to analysis. For example, a sample can be fixed via immersion in
ethanol, methanol, acetone, formaldehyde (e.g., 2% formaldehyde),
paraformaldehyde-Triton, glutaraldehyde, or combinations
thereof.
[0307] In some embodiments, acetone fixation is used with fresh
frozen samples, which can include, but are not limited to, cortex
tissue, mouse olfactory bulb, human brain tumor, human post-mortem
brain, and breast cancer samples. In some embodiments, a compatible
fixation method is chosen and/or optimized based on a desired
workflow. For example, formaldehyde fixation may be chosen as
compatible for workflows using IHC/IF protocols for protein
visualization. As another example, methanol fixation may be chosen
for workflows emphasizing RNA/DNA library quality. Acetone fixation
may be chosen in some applications to permeabilize the tissue. When
acetone fixation is performed, pre-permeabilization steps
(described below) may not be performed. Alternatively, acetone
fixation can be performed in conjunction with permeabilization
steps.
[0308] (5) Embedding
[0309] As an alternative to paraffin embedding described above, a
biological sample can be embedded in any of a variety of other
embedding materials to provide a substrate to the sample prior to
sectioning and other handling steps. In general, the embedding
material is removed prior to analysis of tissue sections obtained
from the sample. Suitable embedding materials include, but are not
limited to, waxes, resins (e.g., methacrylate resins), epoxies, and
agar.
[0310] (6) Staining
[0311] To facilitate visualization, biological samples can be
stained using a wide variety of stains and staining techniques. In
some embodiments, a sample can be stained using any number of
biological stains, including but not limited to, acridine orange,
Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI,
eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst
stains, iodine, methyl green, methylene blue, neutral red, Nile
blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or
safranin.
[0312] The sample can be stained using known staining techniques,
including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E),
Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky,
silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining
techniques. PAS staining is typically performed after formalin or
acetone fixation.
[0313] In some embodiments, the biological sample can be stained
using a detectable label (e.g., radioisotopes, fluorophores,
chemiluminescent compounds, bioluminescent compounds, and dyes) as
described elsewhere herein. In some embodiments, a biological
sample is stained using only one type of stain or one technique. In
some embodiments, staining includes biological staining techniques
such as H&E staining. In some embodiments, staining includes
identifying analytes using fluorescently-conjugated antibodies. In
some embodiments, a biological sample is stained using two or more
different types of stains, or two or more different staining
techniques. For example, a biological sample can be prepared by
staining and imaging using one technique (e.g., H&E staining
and brightfield imaging), followed by staining and imaging using
another technique (e.g., IHC/IF staining and fluorescence
microscopy) for the same biological sample.
[0314] In some embodiments, biological samples can be destained.
Methods of destaining or discoloring a biological sample are known
in the art, and generally depend on the nature of the stain(s)
applied to the sample. For example, H&E staining can be
destined by washing the sample in HCl. In some embodiments,
destaining can include 1, 2, 3, or more washes in HCl. In some
embodiments, destaining can include adding HCl to a downstream
solution (e.g., permeabilization solution). As another example, in
some embodiments, one or more immunofluorescence stains are applied
to the sample via antibody coupling. Such stains can be removed
using techniques such as cleavage of disulfide linkages via
treatment with a reducing agent and detergent washing, chaotropic
salt treatment, treatment with antigen retrieval solution, and
treatment with an acidic glycine buffer. Methods for multiplexed
staining and destaining are described, for example, in Bolognesi et
al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat
Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009;
57:567-75, and Glass et al., J. Histochem. Cytochem. 2009;
57:899-905, the entire contents of each of which are incorporated
herein by reference.
[0315] (7) Hydrogel Embedding
[0316] In some embodiments, hydrogel formation occurs within a
biological sample. In some embodiments, a biological sample (e.g.,
tissue section) is embedded in a hydrogel. In some embodiments,
hydrogel subunits are infused into the biological sample, and
polymerization of the hydrogel is initiated by an external or
internal stimulus. A "hydrogel" as described herein can include a
cross-linked 3D network of hydrophilic polymer chains. A "hydrogel
subunit" can be a hydrophilic monomer, a molecular precursor, or a
polymer that can be polymerized (e.g., cross-linked) to form a
three-dimensional (3D) hydrogel network.
[0317] A hydrogel can swell in the presence of water. In some
embodiments, a hydrogel comprises a natural material. In some
embodiments, a hydrogel includes a synthetic material. In some
embodiments, a hydrogel includes a hybrid material, e.g., the
hydrogel material comprises elements of both synthetic and natural
polymers. Any of the materials used in hydrogels or hydrogels
comprising a polypeptide-based material described herein can be
used. Embedding the sample in this manner typically involves
contacting the biological sample with a hydrogel such that the
biological sample becomes surrounded by the hydrogel. For example,
the sample can be embedded by contacting the sample with a suitable
polymer material, and activating the polymer material to form a
hydrogel. In some embodiments, the hydrogel is formed such that the
hydrogel is internalized within the biological sample.
[0318] In some embodiments, the biological sample is immobilized in
the hydrogel via cross-linking of the polymer material that forms
the hydrogel. Cross-linking can be performed chemically and/or
photochemically, or alternatively by any other hydrogel-formation
method known in the art. For example, the biological sample can be
immobilized in the hydrogel by polyacrylamide crosslinking.
Further, analytes of a biological sample can be immobilized in a
hydrogel by crosslinking (e.g., polyacrylamide crosslinking).
[0319] The composition and application of the hydrogel to a
biological sample typically depends on the nature and preparation
of the biological sample (e.g., sectioned, non-sectioned,
fresh-frozen tissue, type of fixation). A hydrogel can be any
appropriate hydrogel where upon formation of the hydrogel on the
biological sample the biological sample becomes anchored to or
embedded in the hydrogel. Non-limiting examples of hydrogels are
described herein or are known in the art. As one example, where the
biological sample is a tissue section, the hydrogel can include a
monomer solution and an ammonium persulfate (APS)
initiator/tetramethylethylenediamine (TEMED) accelerator solution.
As another example, where the biological sample consists of cells
(e.g., cultured cells or cells disassociated from a tissue sample),
the cells can be incubated with the monomer solution and APS/TEMED
solutions. For cells, hydrogel are formed in compartments,
including but not limited to devices used to culture, maintain, or
transport the cells. For example, hydrogels can be formed with
monomer solution plus APS/TEMED added to the compartment to a depth
ranging from about 0.1 .mu.m to about 5 mm.
[0320] In some embodiments, a hydrogel includes a linker that
allows anchoring of the biological sample to the hydrogel. In some
embodiments, a hydrogel includes linkers that allow anchoring of
biological analytes to the hydrogel. In such cases, the linker can
be added to the hydrogel before, contemporaneously with, or after
hydrogel formation. Non-limiting examples of linkers that anchor
nucleic acids to the hydrogel can include 6-((Acryloyl)amino)
hexanoic acid (Acryloyl-X SE) (available from ThermoFisher,
Waltham, Mass.), Label-IT Amine (available from MirusBio, Madison,
Wis.) and Label X).
[0321] In some embodiments, functionalization chemistry can be
used. In some embodiments, functionalization chemistry includes
hydrogel-tissue chemistry (HTC). Any hydrogel-tissue backbone
(e.g., synthetic or native) suitable for HTC can be used for
anchoring biological macromolecules and modulating
functionalization. Non-limiting examples of methods using HTC
backbone variants include CLARITY, PACT, ExM, SWITCH and ePACT. In
some embodiments, hydrogel formation within a biological sample is
permanent. For example, biological macromolecules can permanently
adhere to the hydrogel allowing multiple rounds of interrogation.
In some embodiments, hydrogel formation within a biological sample
is reversible.
[0322] In some embodiments, additional reagents are added to the
hydrogel subunits before, contemporaneously with, and/or after
polymerization. For example, additional reagents can include but
are not limited to oligonucleotides (e.g., capture probes),
endonucleases to fragment DNA, fragmentation buffer for DNA, DNA
polymerase enzymes, dNTPs used to amplify the nucleic acid and to
attach the barcode to the amplified fragments. Other enzymes can be
used, including without limitation, RNA polymerase, transposase,
ligase, proteinase K, and DNAse. Additional reagents can also
include reverse transcriptase enzymes, including enzymes with
terminal transferase activity, primers, and switch
oligonucleotides. In some embodiments, optical labels are added to
the hydrogel subunits before, contemporaneously with, and/or after
polymerization.
[0323] In some embodiments, HTC reagents are added to the hydrogel
before, contemporaneously with, and/or after polymerization. In
some embodiments, an analyte capture agent is added to the hydrogel
before, contemporaneously with, and/or after polymerization. In
some embodiments, a cell-penetrating agent is added to the hydrogel
before, contemporaneously with, and/or after polymerization.
[0324] In some embodiments, a biological sample is embedded in a
hydrogel to facilitate sample transfer to another location (e.g.,
to an array). For example, archived biological samples (e.g., FFPE
tissue sections) can be transferred from storage to a spatial array
to perform spatial analysis. In some embodiments, a biological
sample on a substrate can be covered with any of the prepolymer
solutions described herein. In some embodiments, the prepolymer
solution can be polymerized such that a hydrogel is formed on top
of and/or around the biological sample. Hydrogel formation can
occur in a manner sufficient to anchor (e.g., embed) the biological
sample to the hydrogel. After hydrogel formation, the biological
sample is anchored to (e.g., embedded in) the hydrogel wherein
separating the hydrogel from the substrate (e.g., glass slide)
results in the biological sample separating from the substrate
along with the hydrogel. The biological sample contained in the
hydrogel can then be contacted with a spatial array, and spatial
analysis can be performed on the biological sample.
[0325] Any variety of characteristics can determine the transfer
conditions required for a given biological sample. Non-limiting
examples of characteristics likely to impact transfer conditions
include the sample (e.g., thickness, fixation, and cross-linking)
and/or the analyte of interest (different conditions to preserve
and/or transfer different analytes (e.g., DNA, RNA, and
protein)).
[0326] In some embodiments, the hydrogel is removed after
contacting the biological sample with the spatial array. For
example, methods described herein can include an event-dependent
(e.g., light or chemical) depolymerizing hydrogel, wherein upon
application of the event (e.g., external stimuli) the hydrogel
depolymerizes. In one example, a biological sample can be anchored
to a DTT-sensitive hydrogel, where addition of DTT can cause the
hydrogel to depolymerize and release the anchored biological
sample.
[0327] Hydrogels embedded within biological samples can be cleared
using any suitable method. For example, electrophoretic tissue
clearing methods can be used to remove biological macromolecules
from the hydrogel-embedded sample. In some embodiments, a
hydrogel-embedded sample is stored in a medium before or after
clearing of hydrogel (e.g., a mounting medium, methylcellulose, or
other semi-solid mediums).
[0328] In some embodiments, the hydrogel chemistry can be tuned to
specifically bind (e.g., retain) particular species of analytes
(e.g., RNA, DNA, protein, etc.). In some embodiments, a hydrogel
includes a linker that allows anchoring of the biological sample to
the hydrogel. In some embodiments, a hydrogel includes linkers that
allow anchoring of biological analytes to the hydrogel. In such
cases, the linker can be added to the hydrogel before,
contemporaneously with, or after hydrogel formation. Non-limiting
examples of linkers that anchor nucleic acids to the hydrogel can
include 6-((Acryloyl)amino) hexanoic acid (Acryloyl-X SE), Label-IT
Amine and Label X. Non-limiting examples of characteristics likely
to impact transfer conditions include the sample (e.g., thickness,
fixation, and cross-linking) and/or the analyte of interest
(different conditions to preserve and/or transfer different
analytes (e.g., DNA, RNA, and protein)).
[0329] Additional methods and aspects of hydrogel embedding of
biological samples are described for example in Chen et al.,
Science 347(6221):543-548, 2015, the entire contents of which are
incorporated herein by reference.
[0330] (8) Biological Sample Transfer
[0331] In some embodiments, a biological sample immobilized on a
substrate (e.g., a biological sample prepared using methanol
fixation or formalin-fixation and paraffin-embedding (FFPE)) is
transferred to a spatial array using a hydrogel. In some
embodiments, a hydrogel is formed on top of a biological sample on
a substrate (e.g., glass slide). For example, hydrogel formation
can occur in a manner sufficient to anchor (e.g., embed) the
biological sample to the hydrogel. After hydrogel formation, the
biological sample is anchored to (e.g., embedded in) the hydrogel
wherein separating the hydrogel from the substrate results in the
biological sample separating from the substrate along with the
hydrogel. The biological sample can then be contacted with a
spatial array, thereby allowing spatial profiling of the biological
sample. In some embodiments, the hydrogel is removed after
contacting the biological sample with the spatial array. For
example, methods described herein can include an event-dependent
(e.g., light or chemical) depolymerizing hydrogel, wherein upon
application of the event (e.g., external stimuli) the hydrogel
depolymerizes. In one example, a biological sample can be anchored
to a DTT-sensitive hydrogel, where addition of DTT can cause the
hydrogel to depolymerize and release the anchored biological
sample. A hydrogel can be any appropriate hydrogel where upon
formation of the hydrogel on the biological sample the biological
sample becomes anchored to or embedded in the hydrogel.
Non-limiting examples of hydrogels are described herein or are
known in the art. In some embodiments, a hydrogel includes a linker
that allows anchoring of the biological sample to the hydrogel. In
some embodiments, a hydrogel includes linkers that allow anchoring
of biological analytes to the hydrogel. In such cases, the linker
can be added to the hydrogel before, contemporaneously with, or
after hydrogel formation. Non-limiting examples of linkers that
anchor nucleic acids to the hydrogel can include
6-((Acryloyl)amino) hexanoic acid (Acryloyl-X SE) (available from
ThermoFisher, Waltham, Mass.), Label-IT Amine (available from
MirusBio, Madison, Wis.) and Label X). Any variety of
characteristics can determine the transfer conditions required for
a given biological sample. Non-limiting examples of characteristics
likely to impact transfer conditions include the sample (e.g.,
thickness, fixation, and cross-linking) and/or the analyte of
interest (different conditions to preserve and/or transfer
different analytes (e.g., DNA, RNA, and protein)). In some
embodiments, hydrogel formation can occur in a manner sufficient to
anchor the analytes (e.g., embed) in the biological sample to the
hydrogel. In some embodiments, the hydrogel can be imploded (e.g.,
shrunk) with the anchored analytes (e.g., embedded in the hydrogel)
present in the biological sample. In some embodiments, the hydrogel
can be expanded (e.g., isometric expansion) with the anchored
analytes (e.g., embedded in the hydrogel) present in the biological
sample. In some embodiments, the hydrogel can be imploded (e.g.,
shrunk) and subsequently expanded with anchored analytes (e.g.,
embedded in the hydrogel) present in the biological sample.
[0332] (9) Isometric Expansion
[0333] In some embodiments, a biological sample embedded in a
hydrogel can be isometrically expanded. Isometric expansion methods
that can be used include hydration, a preparative step in expansion
microscopy, as described in Chen et al., Science 347(6221):543-548,
2015; Asano et al. Current Protocols. 2018, 80:1,
doi:10.1002/cpcb.56 and Gao et al. BMC Biology. 2017, 15:50,
doi:10.1186/s12915-017-0393-3, Wassie, A. T., et al, Expansion
microscopy: principles and uses in biological research, Nature
Methods, 16(1): 33-41 (2018), each of which is incorporated by
reference in its entirety.
[0334] In general, the steps used to perform isometric expansion of
the biological sample can depend on the characteristics of the
sample (e.g., thickness of tissue section, fixation,
cross-linking), and/or the analyte of interest (e.g., different
conditions to anchor RNA, DNA, and protein to a gel).
[0335] Isometric expansion can be performed by anchoring one or
more components of a biological sample to a gel, followed by gel
formation, proteolysis, and swelling. Isometric expansion of the
biological sample can occur prior to immobilization of the
biological sample on a substrate, or after the biological sample is
immobilized to a substrate. In some embodiments, the isometrically
expanded biological sample can be removed from the substrate prior
to contacting the expanded biological sample with a spatially
barcoded array (e.g., spatially barcoded capture probes on a
substrate).
[0336] In some embodiments, proteins in the biological sample are
anchored to a swellable gel such as a polyelectrolyte gel. An
antibody can be directed to the protein before, after, or in
conjunction with being anchored to the swellable gel. DNA and/or
RNA in a biological sample can also be anchored to the swellable
gel via a suitable linker. Examples of such linkers include, but
are not limited to, 6-((Acryloyl)amino) hexanoic acid (Acryloyl-X
SE) (available from ThermoFisher, Waltham, Mass.), Label-IT Amine
(available from MirusBio, Madison, Wis.) and Label X (described for
example in Chen et al., Nat. Methods 13:679-684, 2016, the entire
contents of which are incorporated herein by reference).
[0337] Isometric expansion of the sample can increase the spatial
resolution of the subsequent analysis of the sample. For example,
isometric expansion of the biological sample can result in
increased resolution in spatial profiling (e.g., single-cell
profiling). The increased resolution in spatial profiling can be
determined by comparison of an isometrically expanded sample with a
sample that has not been isometrically expanded.
[0338] In some embodiments, a biological sample is isometrically
expanded to a volume at least 2.times., 2.1.times., 2.2.times.,
2.3.times., 2.4.times., 2.5.times., 2.6.times., 2.7.times.,
2.8.times., 2.9.times., 3.times., 3.1.times., 3.2.times.,
3.3.times., 3.4.times., 3.5.times., 3.6.times., 3.7.times.,
3.8.times., 3.9.times., 4.times., 4.1.times., 4.2.times.,
4.3.times., 4.4.times., 4.5.times., 4.6.times., 4.7.times.,
4.8.times., or 4.9.times. its non-expanded volume. In some
embodiments, the sample is isometrically expanded to at least
2.times. and less than 20.times. of its non-expanded volume.
[0339] In some embodiments, a biological sample embedded in a
hydrogel is isometrically expanded to a volume at least 2.times.,
2.1.times., 2.2.times., 2.3.times., 2.4.times., 2.5.times.,
2.6.times., 2.7.times., 2.8.times., 2.9.times., 3.times.,
3.1.times., 3.2.times., 3.3.times., 3.4.times., 3.5.times.,
3.6.times., 3.7.times., 3.8.times., 3.9.times., 4.times.,
4.1.times., 4.2.times., 4.3.times., 4.4.times., 4.5.times.,
4.6.times., 4.7.times., 4.8.times., or 4.9.times. its non-expanded
volume. In some embodiments, the biological sample embedded in a
hydrogel is isometrically expanded to at least 2.times. and less
than 20.times. of its non-expanded volume.
[0340] (10) Substrate Attachment
[0341] In some embodiments, the biological sample can be attached
to a substrate. Examples of substrates suitable for this purpose
are described in detail below. Attachment of the biological sample
can be irreversible or reversible, depending upon the nature of the
sample and subsequent steps in the analytical method.
[0342] In certain embodiments, the sample can be attached to the
substrate reversibly by applying a suitable polymer coating to the
substrate, and contacting the sample to the polymer coating. The
sample can then be detached from the substrate using an organic
solvent that at least partially dissolves the polymer coating.
Hydrogels are examples of polymers that are suitable for this
purpose.
[0343] More generally, in some embodiments, the substrate can be
coated or functionalized with one or more substances to facilitate
attachment of the sample to the substrate. Suitable substances that
can be used to coat or functionalize the substrate include, but are
not limited to, lectins, poly-lysine, antibodies, and
polysaccharides.
[0344] (11) Unaggregated Cells
[0345] In some embodiments, the biological sample corresponds to
cells (e.g., derived from a cell culture or a tissue sample). In a
cell sample with a plurality of cells, individual cells can be
naturally unaggregated. For example, cells can be derived from a
suspension of cells and/or disassociated or disaggregated cells
from a tissue or tissue section.
[0346] Alternatively, the cells in the sample may be aggregated,
and may be disaggregated into individual cells using, for example,
enzymatic or mechanical techniques. Examples of enzymes used in
enzymatic disaggregation include, but are not limited to, dispase,
collagenase, trypsin, or combinations thereof. Mechanical
disaggregation can be performed, for example, using a tissue
homogenizer.
[0347] In some embodiments of unaggregated cells or disaggregated
cells, the cells are distributed onto the substrate such that at
least one cell occupies a distinct spatial feature on the
substrate. The cells can be immobilized on the substrate (e.g., to
prevent lateral diffusion of the cells). In some embodiments, a
cell immobilization agent can be used to immobilize a
non-aggregated or disaggregated sample on a spatially-barcoded
array prior to analyte capture. A "cell immobilization agent" can
refer to an antibody, attached to a substrate, which can bind to a
cell surface marker. In some embodiments, the distribution of the
plurality of cells on the substrate follows Poisson statistics.
[0348] In some embodiments, cells from a plurality of cells are
immobilized on a substrate. In some embodiments, the cells are
immobilized to prevent lateral diffusion, for example, by adding a
hydrogel and/or by the application of an electric field.
[0349] (12) Suspended and Adherent Cells
[0350] In some embodiments, the biological sample can be derived
from a cell culture grown in vitro. Samples derived from a cell
culture can include one or more suspension cells which are
anchorage-independent within the cell culture. Examples of such
cells include, but are not limited to, cell lines derived from
hematopoietic cells, and from the following cell lines: Colo205,
CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92, NCI-H322M,
and MALME-3M.
[0351] Samples derived from a cell culture can include one or more
adherent cells which grow on the surface of the vessel that
contains the culture medium. Non-limiting examples of adherent
cells include DU145 (prostate cancer) cells, H295R (adrenocortical
cancer) cells, HeLa (cervical cancer) cells, KBM-7 (chronic
myelogenous leukemia) cells, LNCaP (prostate cancer) cells, MCF-7
(breast cancer) cells, MDA-MB-468 (breast cancer) cells, PC3
(prostate cancer) cells, SaOS-2 (bone cancer) cells, SH-SY5Y
(neuroblastoma, cloned from a myeloma) cells, T-47D (breast cancer)
cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma)
cells, National Cancer Institute's 60 cancer cell line panel
(NCI60), vero (African green monkey Chlorocebus kidney epithelial
cell line) cells, MC3T3 (embryonic calvarium) cells, GH3 (pituitary
tumor) cells, PC12 (pheochromocytoma) cells, dog MDCK kidney
epithelial cells, Xenopus A6 kidney epithelial cells, zebrafish AB9
cells, and Sf9 insect epithelial cells.
[0352] Additional examples of adherent cells are shown in Table 1
and catalogued, for example, in "A Catalog of in Vitro Cell Lines,
Transplantable Animal and Human Tumors and Yeast," The Division of
Cancer Treatment and Diagnosis (DCTD), National Cancer Institute
(2013), and in Abaan et al., "The exomes of the NCI-60 panel: a
genomic resource for cancer biology and systems pharmacology,"
Cancer Research 73(14):4372-82, 2013, the entire contents of each
of which are incorporated by reference herein.
TABLE-US-00001 TABLE 1 Examples of adherent cells Cell Organ of
Line Species Origin Disease BT549 Human Breast Ductal Carcinoma HS
578T Human Breast Carcinoma MCF7 Human Breast Adenocarcinoma
MDA-MB-231 Human Breast Adenocarcinoma MDA-MB-468 Human Breast
Adenocarcinoma T-47D Human Breast Ductal Carcinoma SF268 Human CNS
Anaplastic Astrocytoma SF295 Human CNS Glioblastoma-Multiforme
SF539 Human CNS Glioblastoma SNB-19 Human CNS Glioblastoma SNB-75
Human CNS Astrocytoma U251 Human CNS Glioblastoma Colo205 Human
Colon Dukes' type D, Colorectal adenocarcinoma HCC 2998 Human Colon
Carcinoma HCT-116 Human Colon Carcinoma HCT-15 Human Colon Dukes'
type C, Colorectal adenocarcinoma HT29 Human Colon Colorectal
adenocarcinoma KM12 Human Colon Adenocarcinoma, Grade III SW620
Human Colon Adenocarcinoma 786-O Human Kidney renal cell
adenocarcinoma A498 Human Kidney Adenocarcinoma ACHN Human Kidney
renal cell adenocarcinoma CAKI Human Kidney clear cell carcinoma
RXF 393 Human Kidney Poorly Differentiated Hypernephroma SN12C
Human Kidney Carcinoma TK-10 Human Kidney Spindle Cell carcinoma
UO-31 Human Kidney Carcinoma A549 Human Lung Adenocarcinoma EKVX
Human Lung Adenocarcinoma HOP-62 Human Lung Adenocarcinoma HOP-92
Human Lung Large Cell, Undifferentiated NCI-H226 Human Lung
squamous cell carcinoma; mesothelioma NCI-H23 Human Lung
adenocarcinoma; non-small cell lung cancer NCI-H460 Human Lung
carcinoma; large cell lung cancer NCI-H522 Human Lung
adenocarcinoma; non-small cell lung cancer LOX IMVI Human Melanoma
Malignant Amelanotic melanoma M14 Human Melanoma malignant melanoma
MALME-3M Human Melanoma malignant melanoma MDA-MB-435 Human
Melanoma Adenocarcinoma SK-MEL-2 Human Melanoma malignant melanoma
SK-MEL-28 Human Melanoma malignant melanoma SK-MEL-5 Human Melanoma
malignant melanoma UACC-257 Human Melanoma malignant melanoma
UACC-62 Human Melanoma malignant melanoma IGROV1 Human Ovary
Cystoadenocarcinoma OVCAR-3 Human Ovary Adenocarcinoma OVCAR-4
Human Ovary Adenocarcinoma OVCAR-5 Human Ovary Adenocarcinoma
OVCAR-8 Human Ovary Adenocarcinoma SK-OV-3 Human Ovary
Adenocarcinoma NCI-ADR-RES Human Ovary Adenocarcinoma DU145 Human
Prostate Carcinoma PC-3 Human Prostate grade IV, adenocarcinoma
[0353] In some embodiments, the adherent cells are cells that
correspond to one or more of the following cell lines: BT549, HS
578T, MCF7, MDA-MB-231, MDA-MB-468, T-47D, SF268, SF295, SF539,
SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29,
KM12, SW620, 786-O, A498, ACHN, CAM, RXF 393, SN12C, TK-10, UO-31,
A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522,
LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28,
SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5,
OVCAR-8, SK-OV-3, NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa,
KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D,
THP-1, U87, vero, MC3T3, GH3, PC12, dog MDCK kidney epithelial,
Xenopus A6 kidney epithelial, zebrafish AB9, and Sf9 insect
epithelial cell lines.
[0354] (13) Tissue Permeabilization
[0355] In some embodiments, a biological sample can be
permeabilized to facilitate transfer of analytes out of the sample,
and/or to facilitate transfer of species (such as capture probes)
into the sample. If a sample is not permeabilized sufficiently, the
amount of analyte captured from the sample may be too low to enable
adequate analysis. Conversely, if the tissue sample is too
permeable, the relative spatial relationship of the analytes within
the tissue sample can be lost. Hence, a balance between
permeabilizing the tissue sample enough to obtain good signal
intensity while still maintaining the spatial resolution of the
analyte distribution in the sample is desirable.
[0356] In general, a biological sample can be permeabilized by
exposing the sample to one or more permeabilizing agents. Suitable
agents for this purpose include, but are not limited to, organic
solvents (e.g., acetone, ethanol, and methanol), cross-linking
agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton
X-100.TM., Tween-20.TM., or sodium dodecyl sulfate (SDS)), and
enzymes (e.g., trypsin, proteases (e.g., proteinase K). In some
embodiments, the detergent is an anionic detergent (e.g., SDS or
N-lauroylsarcosine sodium salt solution). In some embodiments, the
biological sample can be permeabilized using any of the methods
described herein (e.g., using any of the detergents described
herein, e.g., SDS and/or N-lauroylsarcosine sodium salt solution)
before or after enzymatic treatment (e.g., treatment with any of
the enzymes described herein, e.g., trypin, proteases (e.g., pepsin
and/or proteinase K)).
[0357] In some embodiments, a biological sample can be
permeabilized by exposing the sample to greater than about 1.0 w/v
(e.g., greater than about 2.0 w/v %, greater than about 3.0 w/v %,
greater than about 4.0 w/v %, greater than about 5.0 w/v %, greater
than about 6.0 w/v %, greater than about 7.0 w/v %, greater than
about 8.0 w/v %, greater than about 9.0 w/v %, greater than about
10.0 w/v %, greater than about 11.0 w/v %, greater than about 12.0
w/v %, or greater than about 13.0 w/v %) sodium dodecyl sulfate
(SDS) and/or N-lauroylsarcosine or N-lauroylsarcosine sodium salt.
In some embodiments, a biological sample can be permeabilized by
exposing the sample (e.g., for about 5 minutes to about 1 hour,
about 5 minutes to about 40 minutes, about 5 minutes to about 30
minutes, about 5 minutes to about 20 minutes, or about 5 minutes to
about 10 minutes) to about 1.0 w/v % to about 14.0 w/v % (e.g.,
about 2.0 w/v % to about 14.0 w/v %, about 2.0 w/v % to about 12.0
w/v %, about 2.0 w/v % to about 10.0 w/v %, about 4.0 w/v % to
about 14.0 w/v %, about 4.0 w/v % to about 12.0 w/v %, about 4.0
w/v % to about 10.0 w/v %, about 6.0 w/v % to about 14.0 w/v %,
about 6.0 w/v % to about 12.0 w/v %, about 6.0 w/v % to about 10.0
w/v %, about 8.0 w/v % to about 14.0 w/v %, about 8.0 w/v % to
about 12.0 w/v %, about 8.0 w/v % to about 10.0 w/v %, about 10.0%
w/v % to about 14.0 w/v %, about 10.0 w/v % to about 12.0 w/v %, or
about 12.0 w/v % to about 14.0 w/v %) SDS and/or N-lauroylsarcosine
salt solution and/or proteinase K (e.g., at a temperature of about
4% to about 35.degree. C., about 4.degree. C. to about 25.degree.
C., about 4.degree. C. to about 20.degree. C., about 4.degree. C.
to about 10.degree. C., about 10.degree. C. to about 25.degree. C.,
about 10.degree. C. to about 20.degree. C., about 10.degree. C. to
about 15.degree. C., about 35.degree. C. to about 50.degree. C.,
about 35.degree. C. to about 45.degree. C., about 35.degree. C. to
about 40.degree. C., about 40.degree. C. to about 50.degree. C.,
about 40.degree. C. to about 45.degree. C., or about 45.degree. C.
to about 50.degree. C.).
[0358] In some embodiments, the biological sample can be incubated
with a permeabilizing agent to facilitate permeabilization of the
sample. Additional methods for sample permeabilization are
described, for example, in Jamur et al., Method Mol. Biol.
588:63-66, 2010, the entire contents of which are incorporated
herein by reference.
[0359] Lysis Reagents
[0360] In some embodiments, the biological sample can be
permeabilized by adding one or more lysis reagents to the sample.
Examples of suitable lysis agents include, but are not limited to,
bioactive reagents such as lysis enzymes that are used for lysis of
different cell types, e.g., gram positive or negative bacteria,
plants, yeast, mammalian, such as lysozymes, achromopeptidase,
lysostaphin, labiase, kitalase, lyticase, and a variety of other
commercially available lysis enzymes.
[0361] Other lysis agents can additionally or alternatively be
added to the biological sample to facilitate permeabilization. For
example, surfactant-based lysis solutions can be used to lyse
sample cells. Lysis solutions can include ionic surfactants such
as, for example, sarcosyl and sodium dodecyl sulfate (SDS). More
generally, chemical lysis agents can include, without limitation,
organic solvents, chelating agents, detergents, surfactants, and
chaotropic agents.
[0362] In some embodiments, the biological sample can be
permeabilized by non-chemical permeabilization methods.
Non-chemical permeabilization methods are known in the art. For
example, non-chemical permeabilization methods that can be used
include, but are not limited to, physical lysis techniques such as
electroporation, mechanical permeabilization methods (e.g., bead
beating using a homogenizer and grinding balls to mechanically
disrupt sample tissue structures), acoustic permeabilization (e.g.,
sonication), and thermal lysis techniques such as heating to induce
thermal permeabilization of the sample.
[0363] Proteases
[0364] In some embodiments, a medium, solution, or permeabilization
solution may contain one or more proteases. In some embodiments, a
biological sample treated with a protease capable of degrading
histone proteins can result in the generation of fragmented genomic
DNA. The fragmented genomic DNA can be captured using the same
capture domain (e.g., capture domain having a poly(T) sequence)
used to capture mRNA. In some embodiments, a biological sample is
treated with a protease capable of degrading histone proteins and
an RNA protectant prior to spatial profiling in order to facilitate
the capture of both genomic DNA and mRNA.
[0365] In some embodiments, a biological sample is permeabilized by
exposing the sample to a protease capable of degrading histone
proteins. As used herein, the term "histone protein" typically
refers to a linker histone protein (e.g., H1) and/or a core histone
protein (e.g., H2A, H2B, H3, and H4). In some embodiments, a
protease degrades linker histone proteins, core histone proteins,
or linker histone proteins and core histone proteins. Any suitable
protease capable of degrading histone proteins in a biological
sample can be used. Non-limiting examples of proteases capable of
degrading histone proteins include proteases inhibited by leupeptin
and TLCK (Tosyl-L-lysyl-chloromethane hydrochloride), a protease
encoded by the EUO gene from Chlamydia trachomatis serovar A,
granzyme A, a serine protease (e.g., trypsin or trypsin-like
protease, neutral serine protease, elastase, cathepsin G), an
aspartyl protease (e.g., cathepsin D), a peptidase family C1 enzyme
(e.g., cathepsin L), pepsin, proteinase K, a protease that is
inhibited by the diazomethane inhibitor Z-Phe-Phe-CHN(2) or the
epoxide inhibitor E-64, a lysosomal protease, or an azurophilic
enzyme (e.g., cathepsin G, elastase, proteinase 3, neutral serine
protease). In some embodiments, a serine protease is a trypsin
enzyme, trypsin-like enzyme or a functional variant or derivative
thereof (e.g., P00761; C0HK48; Q8IYP2; Q8BW11; Q6IE06; P35035;
P00760; P06871; Q90627; P16049; P07477; P00762; P35031; P19799;
P35036; Q29463; P06872; Q90628; P07478; P07146; P00763; P35032;
P70059; P29786; P35037; Q90629; P35030; P08426; P35033; P35038;
P12788; P29787; P35039; P35040; Q8NHM4; P35041; P35043; P35044;
P54624; P04814; P35045; P32821; P54625; P35004; P35046; P32822;
P35047; C0HKA5; C0HKA2; P54627; P35005; C0HKA6; C0HKA3; P52905;
P83348; P00765; P35042; P81071; P35049; P51588; P35050; P35034;
P35051; P24664; P35048; P00764; P00775; P54628; P42278; P54629;
P42279; Q91041; P54630; P42280; C0HKA4) or a combination thereof.
In some embodiments, a trypsin enzyme is P00761, P00760, Q29463, or
a combination thereof. In some embodiments, a protease capable of
degrading one or more histone proteins comprises an amino acid
sequence with at least 80% sequence identity to P00761, P00760, or
Q29463.
[0366] In some embodiments, a protease capable of degrading one or
more histone proteins comprises an amino acid sequence with at
least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity to P00761, P00760, or Q29463. A protease may be considered
a functional variant if it has at least 50% e.g., at least 60%,
70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity
relative to the activity of the protease in condition optimum for
the enzyme. In some embodiments, the enzymatic treatment with
pepsin enzyme, or pepsin like enzyme, can include:
P03954/PEPA1_MACFU; P28712/PEPA1_RABIT; P27677/PEPA2_MACFU;
P27821/PEPA2_RABIT; PODJD8/PEPA3_HUMAN; P27822/PEPA3_RABIT;
PODJD7/PEPA4_HUMAN; P27678/PEPA4_MACFU; P28713/PEPA4_RABIT;
PODJD9/PEPA5_HUMAN; Q9D106/PEPA5_MOUSE; P27823/PEPAF_RABIT;
P00792/PEPA_BOVIN; Q9N2D4/PEPA_CALJA; Q9GMY6/PEPA_CANLF;
P00793/PEPA_CHICK; P11489/PEPA_MACMU; P00791/PEPA_PIG;
Q9GMY7/PEPA_RHIFE; Q9GMY8/PEPA_SORUN; P81497/PEPA_SUNMU;
P13636/PEPA_URSTH and functional variants and derivatives thereof,
or a combination thereof. In some embodiments, the pepsin enzyme
can include: P00791/PEPA_PIG; P00792/PEPA_BOVIN, functional
variants, derivatives, or combinations thereof.
[0367] Additionally, the protease may be contained in a reaction
mixture (solution), which also includes other components (e.g.,
buffer, salt, chelator (e.g., EDTA), and/or detergent (e.g., SDS,
N-Lauroylsarcosine sodium salt solution)). The reaction mixture may
be buffered, having a pH of about 6.5-8.5, e.g., about 7.0-8.0.
Additionally, the reaction mixture may be used at any suitable
temperature, such as about 10-50.degree. C., e.g., about
10-44.degree. C., 11-43.degree. C., 12-42.degree. C., 13-41.degree.
C., 14-40.degree. C., 15-39.degree. C., 16-38.degree. C.,
17-37.degree. C., e.g., about 10.degree. C., 12.degree. C.,
15.degree. C., 18.degree. C., 20.degree. C., 22.degree. C.,
25.degree. C., 28.degree. C., 30.degree. C., 33.degree. C.,
35.degree. C. or 37.degree. C., preferably about 35-45.degree. C.,
e.g., about 37.degree. C.
[0368] Other Reagents
[0369] In some embodiments, a permeabilization solution can contain
additional reagents or a biological sample may be treated with
additional reagents in order to optimize biological sample
permeabilization. In some embodiments, an additional reagent is an
RNA protectant. As used herein, the term "RNA protectant" typically
refers to a reagent that protects RNA from RNA nucleases (e.g.,
RNases). Any appropriate RNA protectant that protects RNA from
degradation can be used. A non-limiting example of a RNA protectant
includes organic solvents (e.g., at least 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95% v/v organic solvent), which include, without
limitation, ethanol, methanol, propan-2-ol, acetone,
trichloroacetic acid, propanol, polyethylene glycol, acetic acid,
or a combination thereof. In some embodiments, a RNA protectant
includes ethanol, methanol and/or propan-2-ol, or a combination
thereof. In some embodiments, a RNA protectant includes RNAlater
ICE (ThermoFisher Scientific). In some embodiments, the RNA
protectant comprises at least about 60% ethanol. In some
embodiments, the RNA protectant comprises about 60-95% ethanol,
about 0-35% methanol and about 0-35% propan-2-ol, wherein the total
amount of organic solvent in the medium is not more than about 95%.
In some embodiments, the RNA protectant comprises about 60-95%
ethanol, about 5-20% methanol and about 5-20% propan-2-ol, wherein
the total amount of organic solvent in the medium is not more than
about 95%.
[0370] In some embodiments, the RNA protectant includes a salt. The
salt may include ammonium sulfate, ammonium bisulfate, ammonium
chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium
iron (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate,
copper (II) sulfate, lithium chloride, lithium acetate, lithium
sulfate, magnesium sulfate, magnesium chloride, manganese sulfate,
manganese chloride, potassium chloride, potassium sulfate, sodium
chloride, sodium acetate, sodium sulfate, zinc chloride, zinc
acetate and zinc sulfate. In some embodiments, the salt is a
sulfate salt, for example, ammonium sulfate, ammonium bisulfate,
cesium sulfate, cadmium sulfate, cesium iron (II) sulfate, chromium
(III) sulfate, cobalt (II) sulfate, copper (II) sulfate, lithium
sulfate, magnesium sulfate, manganese sulfate, potassium sulfate,
sodium sulfate, or zinc sulfate. In some embodiments, the salt is
ammonium sulfate. The salt may be present at a concentration of
about 20 g/100 ml of medium or less, such as about 15 g/100 ml, 10
g/100 ml, 9 g/100 ml, 8 g/100 ml, 7 g/100 ml, 6 g/100 ml, 5 g/100
ml or less, e.g., about 4 g, 3 g, 2 g or 1 g/100 ml.
[0371] Additionally, the RNA protectant may be contained in a
medium that further includes a chelator (e.g., EDTA), a buffer
(e.g., sodium citrate, sodium acetate, potassium citrate, or
potassium acetate, preferably sodium acetate), and/or buffered to a
pH between about 4-8 (e.g., about 5).
[0372] In some embodiments, the biological sample is treated with
one or more RNA protectants before, contemporaneously with, or
after permeabilization. For example, a biological sample is treated
with one or more RNA protectants prior to treatment with one or
more permeabilization reagents (e.g., one or more proteases). In
another example, a biological sample is treated with a solution
including one or more RNA protectants and one or more
permeabilization reagents (e.g., one or more proteases). In yet
another example, a biological sample is treated with one or more
RNA protectants after the biological sample has been treated with
one or more permeabilization reagents (e.g., one or more
proteases). In some embodiments, a biological sample is treated
with one or more RNA protectants prior to fixation.
[0373] In some embodiments, identifying the location of the
captured analyte in the biological sample includes a nucleic acid
extension reaction. In some embodiments where a capture probe
captures a fragmented genomic DNA molecule, a nucleic acid
extension reaction includes DNA polymerase. For example, a nucleic
acid extension reaction includes using a DNA polymerase to extend
the capture probe that is hybridized to the captured analyte (e.g.,
fragmented genomic DNA) using the captured analyte (e.g.,
fragmented genomic DNA) as a template. The product of the extension
reaction includes a spatially-barcoded analyte (e.g.,
spatially-barcoded fragmented genomic DNA). The spatially-barcoded
analyte (e.g., spatially-barcoded fragmented genomic DNA) can be
used to identify the spatial location of the analyte in the
biological sample. Any DNA polymerase that is capable of extending
the capture probe using the captured analyte as a template can be
used for the methods described herein. Non-limiting examples of DNA
polymerases include T7 DNA polymerase; Bsu DNA polymerase; and E.
coli DNA Polymerase pol I.
[0374] Diffusion-Resistant Media
[0375] In some embodiments, a diffusion-resistant medium, typically
used to limit diffusion of analytes, can include at least one
permeabilization reagent. For example, the diffusion-resistant
medium (e.g., a hydrogel) can include wells (e.g., micro-, nano-,
or picowells or pores) containing a permeabilization buffer or
reagents. In some embodiments, the diffusion-resistant medium
(e.g., a hydrogel) is soaked in permeabilization buffer prior to
contacting the hydrogel with a sample. In some embodiments, the
hydrogel or other diffusion-resistant medium can contain dried
reagents or monomers to deliver permeabilization reagents when the
diffusion-resistant medium is applied to a biological sample. In
some embodiments, the diffusion-resistant medium, (e.g., hydrogel)
is covalently attached to a solid substrate (e.g., an acrylated
glass slide).
[0376] In some embodiments, the hydrogel can be modified to both
deliver permeabilization reagents and contain capture. For example,
a hydrogel film can be modified to include spatially-barcoded
capture probes. The spatially-barcoded hydrogel film is then soaked
in permeabilization buffer before contacting the spatially-barcoded
hydrogel film to the sample. In another example, a hydrogel can be
modified to include spatially-barcoded capture probes and designed
to serve as a porous membrane (e.g., a permeable hydrogel) when
exposed to permeabilization buffer or any other biological sample
preparation reagent. The permeabilization reagent diffuses through
the spatially-barcoded permeable hydrogel and permeabilizes the
biological sample on the other side of the hydrogel. The analytes
then diffuse into the spatially-barcoded hydrogel after exposure to
permeabilization reagents. In such cases, the spatially-barcoded
hydrogel (e.g., porous membrane) is facilitating the diffusion of
the biological analytes in the biological sample into the hydrogel.
In some embodiments, biological analytes diffuse into the hydrogel
before exposure to permeabilization reagents (e.g., when secreted
analytes are present outside of the biological sample or in
instances where a biological sample is lysed or permeabilized by
other means prior to addition of permeabilization reagents). In
some embodiments, the permeabilization reagent is flowed over the
hydrogel at a variable flow rate (e.g., any flow rate that
facilitates diffusion of the permeabilization reagent across the
spatially-barcoded hydrogel). In some embodiments, the
permeabilization reagents are flowed through a microfluidic chamber
or channel over the spatially-barcoded hydrogel. In some
embodiments, after using flow to introduce permeabilization
reagents to the biological sample, biological sample preparation
reagents can be flowed over the hydrogel to further facilitate
diffusion of the biological analytes into the spatially-barcoded
hydrogel. The spatially-barcoded hydrogel film thus delivers
permeabilization reagents to a sample surface in contact with the
spatially-barcoded hydrogel, enhancing analyte migration and
capture. In some embodiments, the spatially-barcoded hydrogel is
applied to a sample and placed in a permeabilization bulk solution.
In some embodiments, the hydrogel film soaked in permeabilization
reagents is sandwiched between a sample and a spatially-barcoded
array. In some embodiments, target analytes are able to diffuse
through the permeabilizing reagent soaked hydrogel and hybridize or
bind the capture probes on the other side of the hydrogel. In some
embodiments, the thickness of the hydrogel is proportional to the
resolution loss. In some embodiments, wells (e.g., micro-, nano-,
or picowells) can contain spatially-barcoded capture probes and
permeabilization reagents and/or buffer. In some embodiments,
spatially-barcoded capture probes and permeabilization reagents are
held between spacers. In some embodiments, the sample is punch,
cut, or transferred into the well, wherein a target analyte
diffuses through the permeabilization reagent/buffer and to the
spatially-barcoded capture probes. In some embodiments, resolution
loss may be proportional to gap thickness (e.g., the amount of
permeabilization buffer between the sample and the capture probes).
In some embodiments, the diffusion-resistant medium (e.g.,
hydrogel) is between approximately 50-500 micrometers thick
including 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50
micrometers thick, or any thickness within 50 and 500
micrometers.
[0377] In some embodiments, a biological sample is exposed to a
porous membrane (e.g., a permeable hydrogel) to aid in
permeabilization and limit diffusive analyte losses, while allowing
permeabilization reagents to reach a sample. Membrane chemistry and
pore volume can be manipulated to minimize analyte loss. In some
embodiments, the porous membrane may be made of glass, silicon,
paper, hydrogel, polymer monoliths, or other material. In some
embodiments, the material may be naturally porous. In some
embodiments, the material may have pores or wells etched into solid
material. In some embodiments, the permeabilization reagents are
flowed through a microfluidic chamber or channel over the porous
membrane. In some embodiments, the flow controls the sample's
access to the permeabilization reagents. In some embodiments, the
porous membrane is a permeable hydrogel. For example, a hydrogel is
permeable when permeabilization reagents and/or biological sample
preparation reagents can pass through the hydrogel using diffusion.
Any suitable permeabilization reagents and/or biological sample
preparation reagents described herein can be used under conditions
sufficient to release analytes (e.g., nucleic acid, protein,
metabolites, lipids, etc.) from the biological sample. In some
embodiments, a hydrogel is exposed to the biological sample on one
side and permeabilization reagent on the other side. The
permeabilization reagent diffuses through the permeable hydrogel
and permeabilizes the biological sample on the other side of the
hydrogel. In some embodiments, permeabilization reagents are flowed
over the hydrogel at a variable flow rate (e.g., any flow rate that
facilitates diffusion of the permeabilization reagent across the
hydrogel). In some embodiments, the permeabilization reagents are
flowed through a microfluidic chamber or channel over the hydrogel.
Flowing permeabilization reagents across the hydrogel enables
control of the concentration of reagents. In some embodiments,
hydrogel chemistry and pore volume can be tuned to enhance
permeabilization and limit diffusive analyte losses.
[0378] In some embodiments, a porous membrane is sandwiched between
a spatially-barcoded array and the sample, wherein permeabilization
solution is applied over the porous membrane. The permeabilization
reagents diffuse through the pores of the membrane and into the
biological sample. In some embodiments, the biological sample can
be placed on a substrate (e.g., a glass slide). Biological analytes
then diffuse through the porous membrane and into to the space
containing the capture probes. In some embodiments, the porous
membrane is modified to include capture probes. For example, the
capture probes can be attached to a surface of the porous membrane
using any of the methods described herein. In another example, the
capture probes can be embedded in the porous membrane at any depth
that allows interaction with a biological analyte. In some
embodiments, the porous membrane is placed onto a biological sample
in a configuration that allows interaction between the capture
probes on the porous membrane and the biological analytes from the
biological sample. For example, the capture probes are located on
the side of the porous membrane that is proximal to the biological
sample. In such cases, permeabilization reagents on the other side
of the porous membrane diffuse through the porous membrane into the
location containing the biological sample and the capture probes in
order to facilitate permeabilization of the biological sample
(e.g., also facilitating capture of the biological analytes by the
capture probes). In some embodiments, the porous membrane is
located between the sample and the capture probes. In some
embodiments, the permeabilization reagents are flowed through a
microfluidic chamber or channel over the porous membrane.
[0379] Selective Permeabilization/Selective Lysis
[0380] In some embodiments, biological samples can be processed to
selectively release an analyte from a subcellular region of a cell
according to established methods. In some embodiments, a method
provided herein can include detecting at least one biological
analyte present in a subcellular region of a cell in a biological
sample. As used herein, a "subcellular region" can refer to any
subcellular region. For example, a subcellular region can refer to
cytosol, a mitochondria, a nucleus, a nucleolus, an endoplasmic
reticulum, a lysosome, a vesicle, a Golgi apparatus, a plastid, a
vacuole, a ribosome, cytoskeleton, or combinations thereof. In some
embodiments, the subcellular region comprises at least one of
cytosol, a nucleus, a mitochondria, and a microsome. In some
embodiments, the subcellular region is cytosol. In some
embodiments, the subcellular region is a nucleus. In some
embodiments, the subcellular region is a mitochondria. In some
embodiments, the subcellular region is a microsome.
[0381] For example, a biological analyte can be selectively
released from a subcellular region of a cell by selective
permeabilization or selective lysing. In some embodiments,
"selective permeabilization" can refer to a permeabilization method
that can permeabilize a membrane of a subcellular region while
leaving a different subcellular region substantially intact (e.g.,
biological analytes are not released from subcellular region due to
the applied permeabilization method). Non-limiting examples of
selective permeabilization methods include using electrophoresis
and/or applying a permeabilization reagent. In some embodiments,
"selective lysing" can refer to a lysis method that can lyse a
membrane of a subcellular region while leaving a different
subcellular region substantially intact (e.g., biological analytes
are not released from subcellular region due to the applied lysis
method). Several methods for selective permeabilization or lysis
are known to one of skill in the art including the methods
described in Lu et al. Lab Chip. 2005 January; 5(1):23-9; Niklas et
al. Anal Biochem. 2011 Sep. 15; 416(2):218-27; Cox and Emili. Nat
Protoc. 2006; 1(4):1872-8; Chiang et al. J Biochem. Biophys.
Methods. 2000 Nov. 20; 46(1-2):53-68; and Yamauchi and Herr et al.
Microsyst. Nanoeng. 2017; 3. pii: 16079; each of which is
incorporated herein by reference in its entirety.
[0382] In some embodiments, "selective permeabilization" or
"selective lysis" refer to the selective permeabilization or
selective lysis of a specific cell type. For example, "selective
permeabilization" or "selective lysis" can refer to lysing one cell
type while leaving a different cell type substantially intact
(e.g., biological analytes are not released from the cell due to
the applied permeabilization or lysis method). A cell that is a
"different cell type" than another cell can refer to a cell from a
different taxonomic kingdom, a prokaryotic cell versus a eukaryotic
cell, a cell from a different tissue type, etc. Many methods are
known to one of skill in the art for selectively permeabilizing or
lysing different cell types. Non-limiting examples include applying
a permeabilization reagent, electroporation, and/or sonication.
See, e.g., International Application No. WO 2012/168003; Han et al.
Microsyst Nanoeng. 2019 Jun. 17; 5:30; Gould et al. Oncotarget.
2018 Mar. 20; 9(21): 15606-15615; Oren and Shai. Biochemistry. 1997
Feb. 18; 36(7):1826-35; Algayer et al. Molecules. 2019 May 31;
24(11). pii: E2079; Hipp et al. Leukemia. 2017 October;
31(10):2278; International Application No. WO 2012/168003; and U.S.
Pat. No. 7,785,869; all of which are incorporated by reference
herein in their entireties.
[0383] In some embodiments, applying a selective permeabilization
or lysis reagent comprises contacting the biological sample with a
hydrogel comprising the permeabilization or lysis reagent.
[0384] In some embodiments, the biological sample is contacted with
two or more arrays (e.g., flexible arrays, as described herein).
For example, after a subcellular region is permeabilized and a
biological analyte from the subcellular region is captured on a
first array, the first array can be removed, and a biological
analyte from a different subcellular region can be captured on a
second array.
[0385] (14) Selective Enrichment of RNA Species
[0386] In some embodiments, where RNA is the analyte, one or more
RNA analyte species of interest can be selectively enriched (e.g.,
Adiconis, et. al., Comparative analysis of RNA sequencing methods
for degraded and low-input samples, Nature, vol. 10, July 2013,
623-632, herein incorporated by reference in its entirety). For
example, one or more species of RNA can be selected by addition of
one or more oligonucleotides to the sample. In some embodiments,
the additional oligonucleotide is a sequence used for priming a
reaction by a polymerase. For example, one or more primer sequences
with sequence complementarity to one or more RNAs of interest can
be used to amplify the one or more RNAs of interest, thereby
selectively enriching these RNAs. In some embodiments, an
oligonucleotide with sequence complementarity to the complementary
strand of captured RNA (e.g., cDNA) can bind to the cDNA. For
example, biotinylated oligonucleotides with sequence complementary
to one or more cDNAs of interest binds to the cDNA and can be
selected using biotinylation-streptavidin affinity using any of a
variety of methods known to the field (e.g., streptavidin
beads).
[0387] Alternatively, one or more species of RNA (e.g., ribosomal
and/or mitochondrial RNA) can be down-selected (e.g., removed,
depleted) using any of a variety of methods. Non-limiting examples
of a hybridization and capture method of ribosomal RNA depletion
include RiboMinus.TM., RiboCop.TM., and Ribo-Zero.TM.. Another
non-limiting RNA depletion method involves hybridization of
complementary DNA oligonucleotides to unwanted RNA followed by
degradation of the RNA/DNA hybrids using RNase H. Non-limiting
examples of a hybridization and degradation method include
NEBNext.RTM. rRNA depletion, NuGEN AnyDeplete, TruSeq.TM.. Another
non-limiting ribosomal RNA depletion method includes ZapR.TM.
digestion, for example SMARTer. In the SMARTer method, random
nucleic acid adapters are hybridized to RNA for first-strand
synthesis and tailing by reverse transcriptase, followed by
template switching and extension by reverse transcriptase.
Additionally, first round PCR amplification adds full-length
Illumina sequencing adapters (e.g., Illumina indexes). Ribosomal
RNA is cleaved by ZapR v2 and R probes v2. A second round of PCR is
performed, amplifying non-rRNA molecules (e.g., cDNA). Parts or
steps of these ribosomal depletion protocols/kits can be further
combined with the methods described herein to optimize protocols
for a specific biological sample.
[0388] In depletion protocols, probes can be administered to a
sample that selectively hybridize to ribosomal RNA (rRNA), thereby
reducing the pool and concentration of rRNA in the sample. Probes
can be administered to a biological sample that selectively
hybridize to mitochondria RNA (mtRNA), thereby reducing the pool
and concentration of mtRNA in the sample. In some embodiments,
probes complementary to mitochondrial RNA can be added during cDNA
synthesis, or probes complementary to both ribosomal and
mitochondrial RNA can be added during cDNA synthesis. Subsequent
application of capture probes to the sample can result in improved
capture of other types of RNA due to a reduction in non-specific
RNA (e.g., down-selected RNA) present in the sample. Additionally
and alternatively, duplex-specific nuclease (DSN) treatment can
remove rRNA (see, e.g., Archer, et al, Selective and flexible
depletion of problematic sequences from RNA-seq libraries at the
cDNA stage, BMC Genomics, 15 401, (2014), the entire contents of
which are incorporated herein by reference). Furthermore,
hydroxyapatite chromatography can remove abundant species (e.g.,
rRNA) (see, e.g., Vandernoot, V. A., cDNA normalization by
hydroxyapatite chromatography to enrich transcriptome diversity in
RNA-seq applications, Biotechniques, 53(6) 373-80, (2012), the
entire contents of which are incorporated herein by reference).
[0389] (15) Other Reagents
[0390] Additional reagents can be added to a biological sample to
perform various functions prior to analysis of the biological
sample. In some embodiments, nuclease inhibitors such as DNase and
RNase inactivating agents or protease inhibitors, and/or chelating
agents such as EDTA, can be added to the biological sample. In
other embodiments nucleases, such as DNase or RNAse, or proteases,
such as pepsin or proteinase K, are added to the sample. In some
embodiments, additional reagents may be dissolved in a solution or
applied as a medium to the sample. In some embodiments, additional
reagents (e.g., pepsin) may be dissolved in HCl prior to applying
to the sample.
[0391] In some embodiments, the biological sample can be treated
with one or more enzymes. For example, one or more endonucleases to
fragment DNA, DNA polymerase enzymes, and dNTPs used to amplify
nucleic acids can be added. Other enzymes that can also be added to
the biological sample include, but are not limited to, polymerase,
transposase, ligase, and DNAse, and RNAse.
[0392] In some embodiments, reverse transcriptase enzymes can be
added to the sample, including enzymes with terminal transferase
activity, primers, and template switch oligonucleotides (TSOs).
Template switching can be used to increase the length of a cDNA,
e.g., by appending a predefined nucleic acid sequence to the cDNA.
In some embodiments, the appended nucleic acid sequence comprises
one or more ribonucleotides.
[0393] In some embodiments, additional reagents can be added to
improve the recovery of one or more target molecules (e.g., cDNA
molecules, mRNA transcripts). For example, addition of carrier RNA
to a RNA sample workflow process can increase the yield of
extracted RNA/DNA hybrids from the biological sample. In some
embodiments, carrier molecules are useful when the concentration of
input or target molecules is low as compared to remaining
molecules. Generally, single target molecules cannot form a
precipitate, and addition of the carrier molecules can help in
forming a precipitate. Some target molecule recovery protocols use
carrier RNA to prevent small amounts of target nucleic acids
present in the sample from being irretrievably bound. In some
embodiments, carrier RNA can be added immediately prior to a second
strand synthesis step. In some embodiments, carrier RNA can be
added immediately prior to a second strand cDNA synthesis on
oligonucleotides released from an array. In some embodiments,
carrier RNA can be added immediately prior to a post in vitro
transcription clean-up step. In some embodiments, carrier RNA can
be added prior to amplified RNA purification and quantification. In
some embodiments, carrier RNA can be added before RNA
quantification. In some embodiments, carrier RNA can be added
immediately prior to both a second strand cDNA synthesis and a post
in vitro transcription clean-up step.
[0394] (16) Pre-Processing for Capture Probe Interaction
[0395] In some embodiments, analytes in a biological sample can be
pre-processed prior to interaction with a capture probe. For
example, prior to interaction with capture probes, polymerization
reactions catalyzed by a polymerase (e.g., DNA polymerase or
reverse transcriptase) are performed in the biological sample. In
some embodiments, a primer for the polymerization reaction includes
a functional group that enhances hybridization with the capture
probe. The capture probes can include appropriate capture domains
to capture biological analytes of interest (e.g., poly(dT) sequence
to capture poly(A) mRNA).
[0396] In some embodiments, biological analytes are pre-processed
for library generation via next generation sequencing. For example,
analytes can be pre-processed by addition of a modification (e.g.,
ligation of sequences that allow interaction with capture probes).
In some embodiments, analytes (e.g., DNA or RNA) are fragmented
using fragmentation techniques (e.g., using transposases and/or
fragmentation buffers).
[0397] Fragmentation can be followed by a modification of the
analyte. For example, a modification can be the addition through
ligation of an adapter sequence that allows hybridization with the
capture probe. In some embodiments, where the analyte of interest
is RNA, poly(A) tailing is performed. Addition of a poly(A) tail to
RNA that does not contain a poly(A) tail can facilitate
hybridization with a capture probe that includes a capture domain
with a functional amount of poly(dT) sequence.
[0398] In some embodiments, prior to interaction with capture
probes, ligation reactions catalyzed by a ligase are performed in
the biological sample. In some embodiments, ligation can be
performed by chemical ligation. In some embodiments, the ligation
can be performed using click chemistry as further described below.
In some embodiments, the capture domain includes a DNA sequence
that has complementarity to a RNA molecule, where the RNA molecule
has complementarity to a second DNA sequence, and where the RNA-DNA
sequence complementarity is used to ligate the second DNA sequence
to the DNA sequence in the capture domain. In these embodiments,
direct detection of RNA molecules is possible.
[0399] In some embodiments, prior to interaction with capture
probes, target-specific reactions are performed in the biological
sample. Examples of target specific reactions include, but are not
limited to, ligation of target specific adaptors, probes and/or
other oligonucleotides, target specific amplification using primers
specific to one or more analytes, and target-specific detection
using in situ hybridization, DNA microscopy, and/or antibody
detection. In some embodiments, a capture probe includes capture
domains targeted to target-specific products (e.g., amplification
or ligation).
II. General Spatial Array-Based Analytical Methodology
[0400] Provided herein are methods, apparatus, systems, and
compositions for spatial array-based analysis of biological
samples.
[0401] (a) Spatial Analysis Methods
[0402] Array-based spatial analysis methods involve the transfer of
one or more analytes from a biological sample to an array of
features on a substrate, where each feature is associated with a
unique spatial location on the array. Subsequent analysis of the
transferred analytes includes determining the identity of the
analytes and the spatial location of each analyte within the
biological sample. The spatial location of each analyte within the
biological sample is determined based on the feature to which each
analyte is bound on the array, and the feature's relative spatial
location within the array.
[0403] There are at least two general methods to associate a
spatial barcode with one or more neighboring cells, such that the
spatial barcode identifies the one or more cells, and/or contents
of the one or more cells, as associated with a particular spatial
location. One general method is to promote analytes out of a cell
and towards the spatially-barcoded array. FIG. 1 depicts an
exemplary embodiment of this general method. In FIG. 1, the
spatially-barcoded array populated with capture probes (as
described further herein) is contacted with a biological sample
101, and biological sample is permeabilized, allowing the analyte
to migrate away from the sample and toward the array. The analyte
interacts with a capture probe on the spatially-barcoded array 102.
Once the analyte hybridizes/is bound to the capture probe, the
sample is optionally removed from the array and the capture probes
are analyzed in order to obtain spatially-resolved analyte
information 103.
[0404] Another general method is to cleave the spatially-barcoded
capture probes from an array, and promote the spatially-barcoded
capture probes towards and/or into or onto the biological sample.
FIG. 2 depicts an exemplary embodiment of this general method, the
spatially-barcoded array populated with capture probes (as
described further herein) can be contacted with a sample 201. The
spatially-barcoded capture probes are cleaved and then interact
with cells within the provided biological sample 202. The
interaction can be a covalent or non-covalent cell-surface
interaction. The interaction can be an intracellular interaction
facilitated by a delivery system or a cell penetration peptide.
Once the spatially-barcoded capture probe is associated with a
particular cell, the sample can be optionally removed for analysis.
The sample can be optionally dissociated before analysis. Once the
tagged cell is associated with the spatially-barcoded capture
probe, the capture probes can be analyzed to obtain
spatially-resolved information about the tagged cell 203.
[0405] FIG. 3 shows an exemplary workflow that includes preparing a
biological sample on a spatially-barcoded array 301. Sample
preparation may include placing the sample on a slide, fixing the
sample, and/or staining the biological sample for imaging. The
stained sample can be then imaged on the array 302 using both
brightfield (to image the sample hematoxylin and eosin stain) and
fluorescence (to image features) modalities. Optionally, the sample
can be destained prior to permeabilization. In some embodiments,
analytes are then released from the sample and capture probes
forming the spatially-barcoded array hybridize or bind the released
analytes 303. The sample is then removed from the array 304 and the
capture probes cleaved from the array 305. The biological sample
and array are then optionally imaged a second time in both
modalities 305B while the analytes are reverse transcribed into
cDNA, and an amplicon library is prepared 306 and sequenced 307.
The two sets of images are then spatially-overlaid in order to
correlate spatially-identified biological sample information 308.
When the sample and array are not imaged a second time, 305B, a
spot coordinate file is supplied instead. The spot coordinate file
replaces the second imaging step 305B. Further, amplicon library
preparation 306 can be performed with a unique PCR adapter and
sequenced 307.
[0406] FIG. 4 shows another exemplary workflow that utilizes a
spatially-barcoded array on a substrate, where spatially-barcoded
capture probes are clustered at areas called features. The
spatially-barcoded capture probes can include a cleavage domain,
one or more functional domains, a spatial barcode, a unique
molecular identifier, and a capture domain. The spatially-barcoded
capture probes can also include a 5' end modification for
reversible attachment to the substrate. The spatially-barcoded
array is contacted with a biological sample 401, and the sample is
permeabilized through application of permeabilization reagents 402.
Permeabilization reagents may be administered by placing the
array/sample assembly within a bulk solution. Alternatively,
permeabilization reagents may be administered to the sample via a
diffusion-resistant medium and/or a physical barrier such as a lid,
wherein the sample is sandwiched between the diffusion-resistant
medium and/or barrier and the array-containing substrate. The
analytes are migrated toward the spatially-barcoded capture array
using any number of techniques disclosed herein. For example,
analyte migration can occur using a diffusion-resistant medium lid
and passive migration. As another example, analyte migration can be
active migration, using an electrophoretic transfer system, for
example. Once the analytes are in close proximity to the
spatially-barcoded capture probes, the capture probes can hybridize
or otherwise bind a target analyte 403. The biological sample can
be optionally removed from the array 404.
[0407] The capture probes can be optionally cleaved from the array
405, and the captured analytes can be spatially-barcoded by
performing a reverse transcriptase first strand cDNA reaction. A
first strand cDNA reaction can be optionally performed using
template switching oligonucleotides. For example, a template
switching oligonucleotide can hybridize to a poly(C) tail added to
a 3'end of the cDNA by a reverse transcriptase enzyme in a template
independent manner. The original mRNA template and template
switching oligonucleotide can then be denatured from the cDNA and
the spatially-barcoded capture probe can then hybridize with the
cDNA and a complement of the cDNA can be generated. The first
strand cDNA can then be purified and collected for downstream
amplification steps. The first strand cDNA can be amplified using
PCR 406, where the forward and reverse primers flank the spatial
barcode and analyte regions of interest, generating a library
associated with a particular spatial barcode. In some embodiments,
the cDNA comprises a sequencing by synthesis (SBS) primer sequence.
The library amplicons are sequenced and analyzed to decode spatial
information 407.
[0408] Using the methods, compositions, systems, kits, and devices
described herein, RNA transcripts present in biological samples
(e.g., tissue samples) can be used for spatial transcriptome
analysis. In particular, in some cases, the barcoded
oligonucleotides may be configured to prime, replicate, and
consequently yield barcoded extension products from an RNA
template, or derivatives thereof. For example, in some cases, the
barcoded oligonucleotides may include mRNA specific priming
sequences, e.g., poly-T primer segments that allow priming and
replication of mRNA in a reverse transcription reaction or other
targeted priming sequences. Alternatively or additionally, random
RNA priming may be carried out using random N-mer primer segments
of the barcoded oligonucleotides. Reverse transcriptases (RTs) can
use an RNA template and a primer complementary to the 3' end of the
RNA template to direct the synthesis of the first strand
complementary DNA (cDNA). Many RTs can be used in this reverse
transcription reactions, including, for example, avian
myeloblastosis virus (AMV) reverse transcriptase, moloney murine
leukemia virus (M-MuLV or MMLV), and other variants thereof. Some
recombinant M-MuLV reverse transcriptase, such as, for example,
PROTOSCRIPT.RTM. II reverse transcriptase, can have reduced RNase H
activity and increased thermostability when compared to its wild
type counterpart, and provide higher specificity, higher yield of
cDNA and more full-length cDNA products with up to 12 kilobase (kb)
in length. In some embodiments, the reverse transcriptase enzyme is
a mutant reverse transcriptase enzyme such as, but not limited to,
mutant MMLV reverse transcriptase. In another embodiment, the
reverse transcriptase is a mutant MMLV reverse transcriptase such
as, but not limited to, one or more variants described in US Patent
Publication No. 20180312822, which is incorporated herein by
reference in its entirety.
[0409] FIG. 5 depicts an exemplary workflow where the biological
sample is removed from the spatially-barcoded array and the
spatially-barcoded capture probes are removed from the array for
barcoded analyte amplification and library preparation. Another
embodiment includes performing first strand synthesis using
template switching oligonucleotides on the spatially-barcoded array
without cleaving the capture probes. In this embodiment, sample
preparation 501 and permeabilization 502 are performed as described
elsewhere herein. Once the capture probes capture the analyte(s),
first strand cDNA created by template switching and reverse
transcriptase 503 is then denatured and the second strand is then
extended 504. The second strand cDNA is then denatured from the
first strand cDNA, neutralized, and transferred to a tube 505. cDNA
quantification and amplification can be performed using standard
techniques discussed herein. The cDNA can then be subjected to
library preparation 506 and indexing 507, including fragmentation,
end-repair, and a-tailing, and indexing PCR steps.
[0410] In a non-limiting example of the workflows described above,
a biological sample (e.g., tissue section), can be fixed with
methanol, stained with hematoxylin and eosin, and imaged.
Optionally, the sample can be destained prior to permeabilization.
The images can be used to map spatial gene expression patterns back
to the biological sample. A permeabilization enzyme can be used to
permeabilize the biological sample directly on the slide. Analytes
(e.g., polyadenylated mRNA) released from the overlying cells of
the biological sample can be captured by capture probes within a
capture area on a substrate. Reverse transcription (RT) reagents
can be added to permeabilized biological samples. Incubation with
the RT reagents can produce spatially-barcoded full-length cDNA
from the captured analytes (e.g., polyadenylated mRNA). Second
strand reagents (e.g., second strand primers, enzymes) can be added
to the biological sample on the slide to initiate second strand
synthesis. The resulting cDNA can be denatured from the capture
probe template and transferred (e.g., to a clean tube) for
amplification, and/or library construction. The spatially-barcoded,
full-length cDNA can be amplified via PCR prior to library
construction. The cDNA can then be enzymatically fragmented and
size-selected in order to optimize the cDNA amplicon size. P5, P7,
i7, and i5 can be used as sample indexes, and TruSeq Read 2 can be
added via End Repair, A-tailing, Adaptor Ligation, and PCR. The
cDNA fragments can then be sequenced using paired-end sequencing
using TruSeq Read 1 and TruSeq Read 2 as sequencing primer
sites.
[0411] In some embodiments, performing correlative analysis of data
produced by this workflow, and other workflows described herein,
can yield over 95% correlation of genes expressed across two
capture areas (e.g., 95% or greater, 96% or greater, 97% or
greater, 98% or greater, or 99% or greater). When performing the
described workflows using single cell RNA sequencing of nuclei, in
some embodiments, correlative analysis of the data can yield over
90% (e.g., over 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%) correlation of genes expressed across two capture areas.
[0412] In some embodiments, after cDNA is generated (e.g., by
reverse transcription) the cDNA can be amplified directly on the
substrate surface. Generating multiple copies of the cDNA (e.g.,
cDNA synthesized from captured analytes) via amplification directly
on the substrate surface can improve final sequencing library
complexity. Thus, in some embodiments, cDNA can be amplified
directly on the substrate surface by isothermal nucleic acid
amplification. In some embodiments, isothermal nucleic acid
amplification can amplify RNA or DNA.
[0413] In some embodiments, isothermal amplification can be faster
than a standard PCR reaction. In some embodiments, isothermal
amplification can be linear amplification (e.g., asymmetrical with
a single primer), or exponential amplification (e.g., with two
primers). In some embodiments, isothermal nucleic acid
amplification can be performed by a template-switching
oligonucleotide primer. In some embodiments, the template switching
oligonucleotide adds a common sequence onto the 5' end of the RNA
being reverse transcribed. For example, after a capture probe
interacts with an analyte (e.g., mRNA) and reverse transcription is
performed such that additional nucleotides are added to the end of
the cDNA creating a 3' overhang as described herein. In some
embodiments, a template switching oligonucleotide hybridizes to
untemplated poly(C) nucleotides added by a reverse transcriptase to
continue replication to the 5' end of the template switching
oligonucleotide, thereby generating full-length cDNA ready for
further amplification. In some embodiments, the template switching
oligonucleotide adds a common 5' sequence to full-length cDNA that
is used for cDNA amplification (e.g., a reverse complement of the
template switching oligonucleotide).
[0414] In some embodiments, once a full-length cDNA molecule is
generated, the template switching oligonucleotide can serve as a
primer in a cDNA amplification reaction (e.g., with a DNA
polymerase). In some embodiments, double stranded cDNA (e.g., first
strand cDNA and second strand reverse complement cDNA) can be
amplified via isothermal amplification with either a helicase or
recombinase, followed by a strand displacing DNA polymerase. The
strand displacing DNA polymerase can generate a displaced second
strand resulting in an amplified product.
[0415] In any of isothermal amplification methods described herein,
barcode exchange (e.g., spatial barcode) can occur after the first
amplification cycle where there are unused capture probes on the
substrate surface. In some embodiments, the free 3'OH end of the
unused capture probes can be blocked by any suitable 3'OH blocking
method. In some embodiments, the 3'OH can be blocked by hairpin
ligation.
[0416] Isothermal nucleic acid amplification can be used in
addition to, or as an alternative to standard PCR reactions (e.g.,
a PCR reaction that requires heating to about 95.degree. C. to
denature double stranded DNA). Isothermal nucleic acid
amplification generally does not require the use of a thermocycler,
however in some embodiments, isothermal amplification can be
performed in a thermocycler. In some embodiments, isothermal
amplification can be performed from about 35.degree. C. to about
75.degree. C. In some embodiments, isothermal amplification can be
performed from about 40.degree. C., about 45.degree. C., about
50.degree. C., about 55.degree. C., about 60.degree. C., about
65.degree. C., or about 70.degree. C. or anywhere in between
depending on the polymerase and auxiliary enzymes used.
[0417] Isothermal nucleic acid amplification techniques are known
in the art, and can be used alone or in combination with any of the
spatial methods described herein. For example, non-limiting
examples of suitable isothermal nucleic acid amplification
techniques include transcription mediated amplification, nucleic
acid sequence-based amplification, signal mediated amplification of
RNA technology, strand displacement amplification, rolling circle
amplification, loop-mediated isothermal amplification of DNA
(LAMP), isothermal multiple displacement amplification, recombinase
polymerase amplification, helicase-dependent amplification, single
primer isothermal amplification, and circular helicase-dependent
amplification (See, e.g., Gill and Ghaemi, Nucleic acid isothermal
amplification technologies: a review, Nucleosides, Nucleotides,
& Nucleic Acids, 27(3), 224-43, doi: 10.1080/15257770701845204
(2008), which is incorporated herein by reference in its
entirety).
[0418] In some embodiments, the isothermal nucleic acid
amplification is helicase-dependent nucleic acid amplification.
Helicase-dependent isothermal nucleic acid amplification is
described in Vincent, et. al., Helicase-dependent isothermal DNA
amplification, EMBO Rep., 795-800 (2004) and U.S. Pat. No.
7,282,328, which are both incorporated herein by reference in their
entireties. Further, helicase-dependent nucleic acid amplification
on a substrate (e.g., on-chip) is described in Andresen, et. al.,
Helicase-dependent amplification: use in OnChip amplification and
potential for point-of-care diagnostics, Expert Rev Mol Diagn., 9,
645-650, doi: 10.1586/erm.09.46 (2009), which is incorporated
herein by reference in its entirety. In some embodiments, the
isothermal nucleic acid amplification is recombinase polymerase
nucleic acid amplification. Recombinase polymerase nucleic acid
amplification is described in Piepenburg, et al., DNA Detection
Using Recombinant Proteins, PLoS Biol., 4, 7 e204 (2006) and Li,
et. al., Review: a comprehensive summary of a decade development of
the recombinase polymerase amplification, Analyst, 144, 31-67, doi:
10.1039/C8AN01621F (2019), both of which are incorporated herein by
reference in their entireties.
[0419] Generally, isothermal amplification techniques use standard
PCR reagents (e.g., buffer, dNTPs etc.) known in the art. Some
isothermal amplification techniques can require additional
reagents. For example, helicase dependent nucleic acid
amplification uses a single-strand binding protein and an accessory
protein. In another example, recombinase polymerase nucleic acid
amplification uses recombinase (e.g., T4 UvsX), recombinase loading
factor (e.g., TF UvsY), single-strand binding protein (e.g., T4
gp32), crowding agent (e.g., PEG-35K), and ATP.
[0420] After isothermal nucleic acid amplification of the
full-length cDNA described by any of the methods herein, the
isothermally amplified cDNAs (e.g., single-stranded or
double-stranded) can be recovered from the substrate, and
optionally followed by amplification with typical cDNA PCR in
microcentrifuge tubes. The sample can then be used with any of the
spatial methods described herein.
[0421] (i) Immunohistochemistry and Immunofluorescence
[0422] In some embodiments, immunofluorescence or
immunohistochemistry protocols (direct and indirect staining
techniques) can be performed as a part of, or in addition to, the
exemplary spatial workflows presented herein. For example, tissue
sections can be fixed according to methods described herein. The
biological sample can be transferred to an array (e.g., capture
probe array), wherein analytes (e.g., proteins) are probed using
immunofluorescence protocols. For example, the sample can be
rehydrated, blocked, and permeabilized (3.times.SSC, 2% BSA, 0.1%
Triton X, 1 U/.mu.l RNAse inhibitor for 10 min at 4.degree. C.)
before being stained with fluorescent primary antibodies (1:100 in
3.times.SSC, 2% BSA, 0.1% Triton X, 1 U/.mu.l RNAse inhibitor for
30 min at 4.degree. C.). The biological sample can be washed,
coverslipped (in glycerol+1 U/.mu.l RNAse inhibitor), imaged (e.g.,
using a confocal microscope or other apparatus capable of
fluorescent detection), washed, and processed according to analyte
capture or spatial workflows described herein.
[0423] As used herein, an "antigen retrieval buffer" can improve
antibody capture in IF/IHC protocols. An exemplary protocol for
antigen retrieval can be preheating the antigen retrieval buffer
(e.g., to 95.degree. C.), immersing the biological sample in the
heated antigen retrieval buffer for a predetermined time, and then
removing the biological sample from the antigen retrieval buffer
and washing the biological sample.
[0424] In some embodiments, optimizing permeabilization can be
useful for identifying intracellular analytes. Permeabilization
optimization can include selection of permeabilization agents,
concentration of permeabilization agents, and permeabilization
duration. Tissue permeabilization is discussed elsewhere
herein.
[0425] In some embodiments, blocking an array and/or a biological
sample in preparation of labeling the biological sample decreases
unspecific binding of the antibodies to the array and/or biological
sample (decreases background). Some embodiments provide for
blocking buffers/blocking solutions that can be applied before
and/or during application of the label, wherein the blocking buffer
can include a blocking agent, and optionally a surfactant and/or a
salt solution. In some embodiments, a blocking agent can be bovine
serum albumin (BSA), serum, gelatin (e.g., fish gelatin), milk
(e.g., non-fat dry milk), casein, polyethylene glycol (PEG),
polyvinyl alcohol (PVA), or polyvinylpyrrolidone (PVP), biotin
blocking reagent, a peroxidase blocking reagent, levamisole,
Carnoy's solution, glycine, lysine, sodium borohydride, pontamine
sky blue, Sudan Black, trypan blue, FITC blocking agent, and/or
acetic acid. The blocking buffer/blocking solution can be applied
to the array and/or biological sample prior to and/or during
labeling (e.g., application of fluorophore-conjugated antibodies)
to the biological sample.
[0426] In some embodiments, additional steps or optimizations can
be included in performing IF/IHC protocols in conjunction with
spatial arrays. Additional steps or optimizations can be included
in performing spatially-tagged analyte capture agent workflows
discussed herein.
[0427] In some embodiments, provided herein are methods for
spatially detecting an analyte (e.g., detecting the location of an
analyte, e.g., a biological analyte) from a biological sample
(e.g., an analyte present in a biological sample, such as a tissue
section) that include: (a) providing a biological sample on a
substrate; (b) staining the biological sample on the substrate,
imaging the stained biological sample, and selecting the biological
sample or subsection of the biological sample (e.g., region of
interest) to subject to analysis; (c) providing an array comprising
one or more pluralities of capture probes on a substrate; (d)
contacting the biological sample with the array, thereby allowing a
capture probe of the one or more pluralities of capture probes to
capture the analyte of interest; and (e) analyzing the captured
analyte, thereby spatially detecting the analyte of interest. Any
variety of staining and imaging techniques as described herein or
known in the art can be used in accordance with methods described
herein. In some embodiments, the staining includes optical labels
as described herein, including, but not limited to, fluorescent,
radioactive, chemiluminescent, calorimetric, or colorimetric
detectable labels. In some embodiments, the staining includes a
fluorescent antibody directed to a target analyte (e.g., cell
surface or intracellular proteins) in the biological sample. In
some embodiments, the staining includes an immunohistochemistry
stain directed to a target analyte (e.g., cell surface or
intracellular proteins) in the biological sample. In some
embodiments, the staining includes a chemical stain such as
hematoxylin and eosin (H&E) or periodic acid-schiff (PAS). In
some embodiments, significant time (e.g., days, months, or years)
can elapse between staining and/or imaging the biological sample
and performing analysis. In some embodiments, reagents for
performing analysis are added to the biological sample before,
contemporaneously with, or after the array is contacted to the
biological sample. In some embodiments, step (d) includes placing
the array onto the biological sample. In some embodiments, the
array is a flexible array where the plurality of spatially-barcoded
features (e.g., a substrate with capture probes, a bead with
capture probes) are attached to a flexible substrate. In some
embodiments, measures are taken to slow down a reaction (e.g.,
cooling the temperature of the biological sample or using enzymes
that preferentially perform their primary function at lower or
higher temperature as compared to their optimal functional
temperature) before the array is contacted with the biological
sample. In some embodiments, step (e) is performed without bringing
the biological sample out of contact with the array. In some
embodiments, step (e) is performed after the biological sample is
no longer in contact with the array. In some embodiments, the
biological sample is tagged with an analyte capture agent before,
contemporaneously with, or after staining and/or imaging of the
biological sample. In such cases, significant time (e.g., days,
months, or years) can elapse between staining and/or imaging and
performing analysis. In some embodiments, the array is adapted to
facilitate biological analyte migration from the stained and/or
imaged biological sample onto the array (e.g., using any of the
materials or methods described herein). In some embodiments, a
biological sample is permeabilized before being contacted with an
array. In some embodiments, the rate of permeabilization is slowed
prior to contacting a biological sample with an array (e.g., to
limit diffusion of analytes away from their original locations in
the biological sample). In some embodiments, modulating the rate of
permeabilization (e.g., modulating the activity of a
permeabilization reagent) can occur by modulating a condition that
the biological sample is exposed to (e.g., modulating temperature,
pH, and/or light). In some embodiments, modulating the rate of
permeabilization includes use of external stimuli (e.g., small
molecules, enzymes, and/or activating reagents) to modulate the
rate of permeabilization. For example, a permeabilization reagent
can be provided to a biological sample prior to contact with an
array, which permeabilization reagent is inactive until a condition
(e.g., temperature, pH, and/or light) is changed or an external
stimulus (e.g., a small molecule, an enzyme, and/or an activating
reagent) is provided.
[0428] In some embodiments, provided herein are methods for
spatially detecting an analyte (e.g., detecting the location of an
analyte, e.g., a biological analyte) from a biological sample
(e.g., present in a biological sample such as a tissue section)
that include: (a) providing a biological sample on a substrate; (b)
staining the biological sample on the substrate, imaging the
stained biological sample, and selecting the biological sample or
subsection of the biological sample (e.g., a region of interest) to
subject to spatial transcriptomic analysis; (c) providing an array
comprising one or more pluralities of capture probes on a
substrate; (d) contacting the biological sample with the array,
thereby allowing a capture probe of the one or more pluralities of
capture probes to capture the biological analyte of interest; and
(e) analyzing the captured biological analyte, thereby spatially
detecting the biological analyte of interest.
[0429] (b) Capture Probes
[0430] A "capture probe" refers to any molecule capable of
capturing (directly or indirectly) and/or labelling an analyte
(e.g., an analyte of interest) in a biological sample. In some
embodiments, the capture probe is a nucleic acid or a polypeptide.
In some embodiments, the capture probe is a conjugate (e.g., an
oligonucleotide-antibody conjugate). In some embodiments, the
capture probe includes a barcode (e.g., a spatial barcode and/or a
unique molecular identifier (UMI)) and a capture domain.
[0431] FIG. 6 is a schematic diagram showing an example of a
capture probe, as described herein. As shown, the capture probe 602
is optionally coupled to a feature 601 by a cleavage domain 603,
such as a disulfide linker. The capture probe can include
functional sequences that are useful for subsequent processing,
such as functional sequence 604, which can include a sequencer
specific flow cell attachment sequence, e.g., a P5 sequence, as
well as functional sequence 606, which can include sequencing
primer sequences, e.g., a R1 primer binding site. In some
embodiments, sequence 604 is a P7 sequence and sequence 606 is a R2
primer binding site. A spatial barcode 605 can be included within
the capture probe for use in barcoding the target analyte. The
functional sequences can generally be selected for compatibility
with any of a variety of different sequencing systems, e.g., 454
Sequencing, Ion Torrent Proton or PGM, Illumina X10, PacBio,
Nanopore, etc., and the requirements thereof. In some embodiments,
functional sequences can be selected for compatibility with
non-commercialized sequencing systems. Examples of such sequencing
systems and techniques, for which suitable functional sequences can
be used, include (but are not limited to) Roche 454 sequencing, Ion
Torrent Proton or PGM sequencing, Illumina X10 sequencing, PacBio
SMRT sequencing, and Oxford Nanopore sequencing. Further, in some
embodiments, functional sequences can be selected for compatibility
with other sequencing systems, including non-commercialized
sequencing systems.
[0432] In some embodiments, the spatial barcode 605, functional
sequences 604 (e.g., flow cell attachment sequence) and 606 (e.g.,
sequencing primer sequences) can be common to all of the probes
attached to a given feature. The spatial barcode can also include a
capture domain 607 to facilitate capture of a target analyte.
[0433] (i) Capture Domain
[0434] As discussed above, each capture probe includes at least one
capture domain. The "capture domain" can be an oligonucleotide, a
polypeptide, a small molecule, or any combination thereof, that
binds specifically to a desired analyte. In some embodiments, a
capture domain can be used to capture or detect a desired
analyte.
[0435] In some embodiments, the capture domain is a functional
nucleic acid sequence configured to interact with one or more
analytes, such as one or more different types of nucleic acids
(e.g., RNA molecules and DNA molecules). In some embodiments, the
functional nucleic acid sequence can include an N-mer sequence
(e.g., a random N-mer sequence), which N-mer sequences are
configured to interact with a plurality of DNA molecules. In some
embodiments, the functional sequence can include a poly(T)
sequence, which poly(T) sequences are configured to interact with
messenger RNA (mRNA) molecules via the poly(A) tail of an mRNA
transcript. In some embodiments, the functional nucleic acid
sequence is the binding target of a protein (e.g., a transcription
factor, a DNA binding protein, or a RNA binding protein), where the
analyte of interest is a protein.
[0436] Capture probes can include ribonucleotides and/or
deoxyribonucleotides as well as synthetic nucleotide residues that
are capable of participating in Watson-Crick type or analogous base
pair interactions. In some embodiments, the capture domain is
capable of priming a reverse transcription reaction to generate
cDNA that is complementary to the captured RNA molecules. In some
embodiments, the capture domain of the capture probe can prime a
DNA extension (polymerase) reaction to generate DNA that is
complementary to the captured DNA molecules. In some embodiments,
the capture domain can template a ligation reaction between the
captured DNA molecules and a surface probe that is directly or
indirectly immobilized on the substrate. In some embodiments, the
capture domain can be ligated to one strand of the captured DNA
molecules. For example, SplintR ligase along with RNA or DNA
sequences (e.g., degenerate RNA) can be used to ligate a
single-stranded DNA or RNA to the capture domain. In some
embodiments, ligases with RNA-templated ligase activity, e.g.,
SplintR ligase, T4 RNA ligase 2 or KOD ligase, can be used to
ligate a single-stranded DNA or RNA to the capture domain. In some
embodiments, a capture domain includes a splint oligonucleotide. In
some embodiments, a capture domain captures a splint
oligonucleotide.
[0437] In some embodiments, the capture domain is located at the 3'
end of the capture probe and includes a free 3' end that can be
extended, e.g., by template dependent polymerization, to form an
extended capture probe as described herein. In some embodiments,
the capture domain includes a nucleotide sequence that is capable
of hybridizing to nucleic acid, e.g., RNA or other analyte, present
in the cells of the biological sample contacted with the array. In
some embodiments, the capture domain can be selected or designed to
bind selectively or specifically to a target nucleic acid. For
example, the capture domain can be selected or designed to capture
mRNA by way of hybridization to the mRNA poly(A) tail. Thus, in
some embodiments, the capture domain includes a poly(T) DNA
oligonucleotide, e.g., a series of consecutive deoxythymidine
residues linked by phosphodiester bonds, which is capable of
hybridizing to the poly(A) tail of mRNA. In some embodiments, the
capture domain can include nucleotides that are functionally or
structurally analogous to a poly(T) tail. For example, a poly(U)
oligonucleotide or an oligonucleotide included of deoxythymidine
analogues. In some embodiments, the capture domain includes at
least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In
some embodiments, the capture domain includes at least 25, 30, or
35 nucleotides.
[0438] In some embodiments, a capture probe includes a capture
domain having a sequence that is capable of binding to mRNA and/or
genomic DNA. For example, the capture probe can include a capture
domain that includes a nucleic acid sequence (e.g., a poly(T)
sequence) capable of binding to a poly(A) tail of an mRNA and/or to
a poly(A) homopolymeric sequence present in genomic DNA. In some
embodiments, a homopolymeric sequence is added to an mRNA molecule
or a genomic DNA molecule using a terminal transferase enzyme in
order to produce an analyte that has a poly(A) or poly(T) sequence.
For example, a poly(A) sequence can be added to an analyte (e.g., a
fragment of genomic DNA) thereby making the analyte capable of
capture by a poly(T) capture domain.
[0439] In some embodiments, random sequences, e.g., random hexamers
or similar sequences, can be used to form all or a part of the
capture domain. For example, random sequences can be used in
conjunction with poly(T) (or poly(T) analogue) sequences. Thus,
where a capture domain includes a poly(T) (or a "poly(T)-like")
oligonucleotide, it can also include a random oligonucleotide
sequence (e.g., "poly(T)-random sequence" probe). This can, for
example, be located 5' or 3' of the poly(T) sequence, e.g., at the
3' end of the capture domain. The poly(T)-random sequence probe can
facilitate the capture of the mRNA poly(A) tail. In some
embodiments, the capture domain can be an entirely random sequence.
In some embodiments, degenerate capture domains can be used.
[0440] In some embodiments, a pool of two or more capture probes
form a mixture, where the capture domain of one or more capture
probes includes a poly(T) sequence and the capture domain of one or
more capture probes includes random sequences. In some embodiments,
a pool of two or more capture probes form a mixture where the
capture domain of one or more capture probes includes poly(T)-like
sequence and the capture domain of one or more capture probes
includes random sequences. In some embodiments, a pool of two or
more capture probes form a mixture where the capture domain of one
or more capture probes includes a poly(T)-random sequences and the
capture domain of one or more capture probes includes random
sequences. In some embodiments, probes with degenerate capture
domains can be added to any of the preceding combinations listed
herein. In some embodiments, probes with degenerate capture domains
can be substituted for one of the probes in each of the pairs
described herein.
[0441] The capture domain can be based on a particular gene
sequence or particular motif sequence or common/conserved sequence,
that it is designed to capture (i.e., a sequence-specific capture
domain). Thus, in some embodiments, the capture domain is capable
of binding selectively to a desired sub-type or subset of nucleic
acid, for example a particular type of RNA, such as mRNA, rRNA,
tRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, RNase
P, RNase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA,
piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other
types of RNA. In a non-limiting example, the capture domain can be
capable of binding selectively to a desired subset of ribonucleic
acids, for example, microbiome RNA, such as 16S rRNA.
[0442] In some embodiments, a capture domain includes an "anchor"
or "anchoring sequence", which is a sequence of nucleotides that is
designed to ensure that the capture domain hybridizes to the
intended analyte. In some embodiments, an anchor sequence includes
a sequence of nucleotides, including a 1-mer, 2-mer, 3-mer or
longer sequence. In some embodiments, the short sequence is random.
For example, a capture domain including a poly(T) sequence can be
designed to capture an mRNA. In such embodiments, an anchoring
sequence can include a random 3-mer (e.g., GGG) that helps ensure
that the poly(T) capture domain hybridizes to an mRNA. In some
embodiments, an anchoring sequence can be VN, N, or NN.
Alternatively, the sequence can be designed using a specific
sequence of nucleotides. In some embodiments, the anchor sequence
is at the 3' end of the capture domain. In some embodiments, the
anchor sequence is at the 5' end of the capture domain.
[0443] In some embodiments, capture domains of capture probes are
blocked prior to contacting the biological sample with the array,
and blocking probes are used when the nucleic acid in the
biological sample is modified prior to its capture on the array. In
some embodiments, the blocking probe is used to block or modify the
free 3' end of the capture domain. In some embodiments, blocking
probes can be hybridized to the capture probes to mask the free 3'
end of the capture domain, e.g., hairpin probes, partially double
stranded probes, or complementary sequences. In some embodiments,
the free 3' end of the capture domain can be blocked by chemical
modification, e.g., addition of an azidomethyl group as a
chemically reversible capping moiety such that the capture probes
do not include a free 3' end. Blocking or modifying the capture
probes, particularly at the free 3' end of the capture domain,
prior to contacting the biological sample with the array, prevents
modification of the capture probes, e.g., prevents the addition of
a poly(A) tail to the free 3' end of the capture probes.
[0444] Non-limiting examples of 3' modifications include dideoxy
C-3' (3'-ddC), 3' inverted dT, 3' C3 spacer, 3' Amino, and 3'
phosphorylation. In some embodiments, the nucleic acid in the
biological sample can be modified such that it can be captured by
the capture domain. For example, an adaptor sequence (including a
binding domain capable of binding to the capture domain of the
capture probe) can be added to the end of the nucleic acid, e.g.,
fragmented genomic DNA. In some embodiments, this is achieved by
ligation of the adaptor sequence or extension of the nucleic acid.
In some embodiments, an enzyme is used to incorporate additional
nucleotides at the end of the nucleic acid sequence, e.g., a
poly(A) tail. In some embodiments, the capture probes can be
reversibly masked or modified such that the capture domain of the
capture probe does not include a free 3' end. In some embodiments,
the 3' end is removed, modified, or made inaccessible so that the
capture domain is not susceptible to the process used to modify the
nucleic acid of the biological sample, e.g., ligation or
extension.
[0445] In some embodiments, the capture domain of the capture probe
is modified to allow the removal of any modifications of the
capture probe that occur during modification of the nucleic acid
molecules of the biological sample. In some embodiments, the
capture probes can include an additional sequence downstream of the
capture domain, e.g., 3' to the capture domain, namely a blocking
domain.
[0446] In some embodiments, the capture domain of the capture probe
can be a non-nucleic acid domain. Examples of suitable capture
domains that are not exclusively nucleic-acid based include, but
are not limited to, proteins, peptides, aptamers, antigens,
antibodies, and molecular analogs that mimic the functionality of
any of the capture domains described herein.
[0447] (ii) Cleavage Domain
[0448] Each capture probe can optionally include at least one
cleavage domain. The cleavage domain represents the portion of the
probe that is used to reversibly attach the probe to an array
feature, as will be described further herein. Further, one or more
segments or regions of the capture probe can optionally be released
from the array feature by cleavage of the cleavage domain. As an
example, spatial barcodes and/or universal molecular identifiers
(UMIs) can be released by cleavage of the cleavage domain.
[0449] FIG. 7 is a schematic illustrating a cleavable capture
probe, wherein the cleaved capture probe can enter into a
non-permeabilized cell and bind to analytes within the sample. The
capture probe 701 contains a cleavage domain 702, a cell
penetrating peptide 703, a reporter molecule 704, and a disulfide
bond (--S--S--). 705 represents all other parts of a capture probe,
for example a spatial barcode and a capture domain.
[0450] In some embodiments, the cleavage domain linking the capture
probe to a feature is a disulfide bond. A reducing agent can be
added to break the disulfide bonds, resulting in release of the
capture probe from the feature. As another example, heating can
also result in degradation of the cleavage domain and release of
the attached capture probe from the array feature. In some
embodiments, laser radiation is used to heat and degrade cleavage
domains of capture probes at specific locations. In some
embodiments, the cleavage domain is a photo-sensitive chemical bond
(e.g., a chemical bond that dissociates when exposed to light such
as ultraviolet light).
[0451] Oligonucleotides with photo-sensitive chemical bonds (e.g.,
photo-cleavable linkers) have various advantages. They can be
cleaved efficiently and rapidly (e.g., in nanoseconds and
milliseconds). In some cases, photo-masks can be used such that
only specific regions of the array are exposed to cleavable stimuli
(e.g., exposure to UV light, exposure to light, exposure to heat
induced by laser). When a photo-cleavable linker is used, the
cleavable reaction is triggered by light, and can be highly
selective to the linker and consequently biorthogonal. Typically,
wavelength absorption for the photocleavable linker is located in
the near-UV range of the spectrum. In some embodiments,
.lamda..sub.max of the photocleavable linker is from about 300 nm
to about 400 nm, or from about 310 nm to about 365 nm. In some
embodiments, .lamda.max of the photocleavable linker is about 300
nm, about 312 nm, about 325 nm, about 330 nm, about 340 nm, about
345 nm, about 355 nm, about 365 nm, or about 400 nm.
[0452] Non-limiting examples of a photo-sensitive chemical bond
that can be used in a cleavage domain include those described in
Leriche et al. Bioorg Med Chem. 2012 Jan. 15; 20(2):571-82 and U.S.
Publication No. 2017/0275669, both of which are incorporated by
reference herein in their entireties. For example, linkers that
comprise photo-sensitive chemical bonds include
3-amino-3-(2-nitrophenyl)propionic acid (ANP), phenacyl ester
derivatives, 8-quinolinyl benzenesulfonate, dicoumarin,
6-bromo-7-alkixycoumarin-4-ylmethoxycarbonyl, a bimane-based
linker, and a bis-arylhydrazone based linker. In some embodiments,
the photo-sensitive bond is part of a cleavable linker such as an
ortho-nitrobenzyl (ONB) linker below:
##STR00001##
wherein:
[0453] X is selected from 0 and NH;
[0454] R.sup.1 is selected from H and C.sub.1-3 alkyl;
[0455] R.sup.2 is selected from H and C.sub.1-3 alkoxy;
[0456] n is 1, 2, or 3; and
[0457] a and b each represent either the point of attachment of the
linker to the substrate, or the point of attachment of the linker
to the capture probe.
[0458] In some embodiments, at least one spacer is included between
the substrate and the ortho-nitrobenzyl (ONB) linker, and at least
one spacer is included between the ortho-nitrobenzyl (ONB) linker
and the capture probe. In some aspects of these embodiments, the
spacer comprises at least one group selected from C1-6 alkylene,
C2-6 alkenylene, C2-6 alkynylene, C.dbd.O, O, S, NH,
--(C.dbd.O)O--, --(C.dbd.O)NH--, --S--S--, ethylene glycol,
polyethyleneglycol, propylene glycol, and polypropyleneglycol, or
any combination thereof. In some embodiments, X is O. In some
embodiments, X is NH. In some embodiments, R.sup.1 is H. In some
embodiments, R.sup.1 is C.sub.1-3 alkyl. In some embodiments,
R.sup.1 is methyl. In some embodiments, R.sup.2 is H. In some
embodiments, R.sup.2 is C1-3 alkoxy. In some embodiments, R.sup.2
is methoxy. In some embodiments, R.sup.1 is H and R.sup.2 is H. In
some embodiments, R.sup.1 is H and R.sup.2 is methoxy. In some
embodiments, R.sup.1 is methyl and R.sup.2 is H. In some
embodiments, R.sup.1 is methyl and R.sup.2 is methoxy.
[0459] In some embodiments, the photocleavable linker has
formula:
##STR00002##
[0460] In some embodiments, the photocleavable linker has
formula:
##STR00003##
[0461] In some embodiments, the photocleavable linker has
formula:
##STR00004##
[0462] In some embodiments, the photocleavable linker has
formula:
##STR00005##
[0463] In some embodiments, the photocleavable linker has
formula:
##STR00006##
[0464] Without being bound to any particular theory, it is believed
that excitation of the ortho-nitrobenzyl (ONB) linker leads to
Norrish-type hydrogen abstraction in the .gamma.-position, followed
by formation of azinic acid, which is highly reactive and
rearranges into nitroso compound, resulting in the complete
cleavage of the linker, as shown on the following scheme:
##STR00007##
[0465] In some embodiments, the photocleavable linker is
3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker:
##STR00008##
[0466] wherein X, R.sup.2, n, a, and b are as described herein for
the ortho-nitrobenzyl (ONB) linker.
[0467] In some embodiments, the photocleavable linker has
formula:
##STR00009##
[0468] In some embodiments, the photocleavable linker is phenacyl
ester linker:
##STR00010##
wherein a and b are as described herein for the ortho-nitrobenzyl
(ONB) linker.
[0469] Other examples of photo-sensitive chemical bonds that can be
used in a cleavage domain include halogenated nucleosides such as
bromodeoxyuridine (BrdU). Brdu is an analog of thymidine that can
be readily incorporated into oligonucleotides (e.g., in the
cleavage domain of a capture probe), and is sensitive to UVB light
(280-320 nm range). Upon exposure to UVB light, a photo-cleavage
reaction occurs (e.g., at a nucleoside immediately 5' to the site
of Brdu incorporation (Doddridge et al. Chem. Comm., 1998,
18:1997-1998 and Cook et al. Chemistry and Biology. 1999,
6:451-459)) that results in release of the capture probe from the
feature.
[0470] Other examples of cleavage domains include labile chemical
bonds such as, but not limited to, ester linkages (e.g., cleavable
with an acid, a base, or hydroxylamine), a vicinal diol linkage
(e.g., cleavable via sodium periodate), a Diels-Alder linkage
(e.g., cleavable via heat), a sulfone linkage (e.g., cleavable via
a base), a silyl ether linkage (e.g., cleavable via an acid), a
glycosidic linkage (e.g., cleavable via an amylase), a peptide
linkage (e.g., cleavable via a protease), an abasic or
apurinic/apyrimidinic (AP) site (e.g., cleavable with an alkali or
an AP endonuclease), or a phosphodiester linkage (e.g., cleavable
via a nuclease (e.g., DNAase)).
[0471] In some embodiments, the cleavage domain includes a sequence
that is recognized by one or more enzymes capable of cleaving a
nucleic acid molecule, e.g., capable of breaking the phosphodiester
linkage between two or more nucleotides. A bond can be cleavable
via other nucleic acid molecule targeting enzymes, such as
restriction enzymes (e.g., restriction endonucleases). For example,
the cleavage domain can include a restriction endonuclease
(restriction enzyme) recognition sequence. Restriction enzymes cut
double-stranded or single stranded DNA at specific recognition
nucleotide sequences known as restriction sites. In some
embodiments, a rare-cutting restriction enzyme, e.g., enzymes with
a long recognition site (at least 8 base pairs in length), is used
to reduce the possibility of cleaving elsewhere in the capture
probe.
[0472] In some embodiments, the cleavage domain includes a poly(U)
sequence which can be cleaved by a mixture of Uracil DNA
glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII,
commercially known as the USER.TM. enzyme. Releasable capture
probes can be available for reaction once released. Thus, for
example, an activatable capture probe can be activated by releasing
the capture probes from a feature.
[0473] In some embodiments, where the capture probe is attached
indirectly to a substrate, e.g., via a surface probe, the cleavage
domain includes one or more mismatch nucleotides, so that the
complementary parts of the surface probe and the capture probe are
not 100% complementary (for example, the number of mismatched base
pairs can be one, two, or three base pairs). Such a mismatch is
recognized, e.g., by the MutY and T7 endonuclease I enzymes, which
results in cleavage of the nucleic acid molecule at the position of
the mismatch. As described herein a "surface probe" can be any
moiety present on the surface of the substrate capable of attaching
to an agent (e.g., a capture probe). In some embodiments, the
surface probe is an oligonucleotide. In some embodiments, the
surface probe is part of the capture probe.
[0474] In some embodiments, where the capture probe is attached
(e.g., immobilized) to a feature indirectly, e.g., via a surface
probe, the cleavage domain includes a nickase recognition site or
sequence. Nickases are endonucleases which cleave only a single
strand of a DNA duplex. Thus, the cleavage domain can include a
nickase recognition site close to the 5' end of the surface probe
(and/or the 5' end of the capture probe) such that cleavage of the
surface probe or capture probe destabilizes the duplex between the
surface probe and capture probe thereby releasing the capture
probe) from the feature.
[0475] Nickase enzymes can also be used in some embodiments where
the capture probe is attached (e.g., immobilized) to the feature
directly. For example, the substrate can be contacted with a
nucleic acid molecule that hybridizes to the cleavage domain of the
capture probe to provide or reconstitute a nickase recognition
site, e.g., a cleavage helper probe. Thus, contact with a nickase
enzyme will result in cleavage of the cleavage domain thereby
releasing the capture probe from the feature. Such cleavage helper
probes can also be used to provide or reconstitute cleavage
recognition sites for other cleavage enzymes, e.g., restriction
enzymes.
[0476] Some nickases introduce single-stranded nicks only at
particular sites on a DNA molecule, by binding to and recognizing a
particular nucleotide recognition sequence. A number of
naturally-occurring nickases have been discovered, of which at
present the sequence recognition properties have been determined
for at least four. Nickases are described in U.S. Pat. No.
6,867,028, which is incorporated herein by reference in its
entirety. In general, any suitable nickase can be used to bind to a
complementary nickase recognition site of a cleavage domain.
Following use, the nickase enzyme can be removed from the assay or
inactivated following release of the capture probes to prevent
unwanted cleavage of the capture probes.
[0477] Examples of suitable capture domains that are not
exclusively nucleic-acid based include, but are not limited to,
proteins, peptides, aptamers, antigens, antibodies, and molecular
analogs that mimic the functionality of any of the capture domains
described herein.
[0478] In some embodiments, a cleavage domain is absent from the
capture probe. Examples of substrates with attached capture probes
lacking a cleavage domain are described for example in Macosko et
al., (2015) Cell 161, 1202-1214, the entire contents of which are
incorporated herein by reference.
[0479] In some embodiments, the region of the capture probe
corresponding to the cleavage domain can be used for some other
function. For example, an additional region for nucleic acid
extension or amplification can be included where the cleavage
domain would normally be positioned. In such embodiments, the
region can supplement the functional domain or even exist as an
additional functional domain. In some embodiments, the cleavage
domain is present but its use is optional.
[0480] (iii) Functional Domain
[0481] Each capture probe can optionally include at least one
functional domain. Each functional domain typically includes a
functional nucleotide sequence for a downstream analytical step in
the overall analysis procedure.
[0482] In some embodiments, the capture probe can include a
functional domain for attachment to a sequencing flow cell, such
as, for example, a P5 sequence for Illumina.RTM. sequencing. In
some embodiments, the capture probe or derivative thereof can
include another functional domain, such as, for example, a P7
sequence for attachment to a sequencing flow cell for Illumina.RTM.
sequencing. The functional domains can be selected for
compatibility with a variety of different sequencing systems, e.g.,
454 Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and
the requirements thereof.
[0483] In some embodiments, the functional domain includes a
primer. The primer can include an R1 primer sequence for
Illumina.RTM. sequencing, and in some embodiments, an R2 primer
sequence for Illumina.RTM. sequencing. Examples of such capture
probes and uses thereof are described in U.S. Patent Publication
Nos. 2014/0378345 and 2015/0376609, the entire contents of each of
which are incorporated herein by reference.
[0484] (iv) Spatial Barcode
[0485] As discussed above, the capture probe can include one or
more spatial barcodes (e.g., two or more, three or more, four or
more, five or more) spatial barcodes. A "spatial barcode" is a
contiguous nucleic acid segment or two or more non-contiguous
nucleic acid segments that function as a label or identifier that
conveys or is capable of conveying spatial information. In some
embodiments, a capture probe includes a spatial barcode that
possesses a spatial aspect, where the barcode is associated with a
particular location within an array or a particular location on a
substrate.
[0486] A spatial barcode can be part of an analyte, or independent
from an analyte (e.g., part of the capture probe). A spatial
barcode can be a tag attached to an analyte (e.g., a nucleic acid
molecule) or a combination of a tag in addition to an endogenous
characteristic of the analyte (e.g., size of the analyte or end
sequence(s)). A spatial barcode can be unique. In some embodiments
where the spatial barcode is unique, the spatial barcode functions
both as a spatial barcode and as a unique molecular identifier
(UMI), associated with one particular capture probe.
[0487] Spatial barcodes can have a variety of different formats.
For example, spatial barcodes can include polynucleotide spatial
barcodes; random nucleic acid and/or amino acid sequences; and
synthetic nucleic acid and/or amino acid sequences. In some
embodiments, a spatial barcode is attached to an analyte in a
reversible or irreversible manner. In some embodiments, a spatial
barcode is added to, for example, a fragment of a DNA or RNA sample
before, during, and/or after sequencing of the sample. In some
embodiments, a spatial barcode allows for identification and/or
quantification of individual sequencing-reads. In some embodiments,
a spatial barcode is a used as a fluorescent barcode for which
fluorescently labeled oligonucleotide probes hybridize to the
spatial barcode.
[0488] In some embodiments, the spatial barcode is a nucleic acid
sequence that does not substantially hybridize to analyte nucleic
acid molecules in a biological sample. In some embodiments, the
spatial barcode has less than 80% sequence identity (e.g., less
than 70%, 60%, 50%, or less than 40% sequence identity) to the
nucleic acid sequences across a substantial part (e.g., 80% or
more) of the nucleic acid molecules in the biological sample.
[0489] The spatial barcode sequences can include from about 6 to
about 20 or more nucleotides within the sequence of the capture
probes. In some embodiments, the length of a spatial barcode
sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 nucleotides or longer. In some embodiments, the length
of a spatial barcode sequence can be at least about 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In
some embodiments, the length of a spatial barcode sequence is at
most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
nucleotides or shorter.
[0490] These nucleotides can be completely contiguous, e.g., in a
single stretch of adjacent nucleotides, or they can be separated
into two or more separate subsequences that are separated by 1 or
more nucleotides. Separated spatial barcode subsequences can be
from about 4 to about 16 nucleotides in length. In some
embodiments, the spatial barcode subsequence can be about 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some
embodiments, the spatial barcode subsequence can be at least about
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer.
In some embodiments, the spatial barcode subsequence can be at most
about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or
shorter.
[0491] For multiple capture probes that are attached to a common
array feature, the one or more spatial barcode sequences of the
multiple capture probes can include sequences that are the same for
all capture probes coupled to the feature, and/or sequences that
are different across all capture probes coupled to the feature.
[0492] FIG. 8 is a schematic diagram of an exemplary multiplexed
spatially-barcoded feature. In FIG. 8, the feature 801 can be
coupled to spatially-barcoded capture probes, wherein the
spatially-barcoded probes of a particular feature can possess the
same spatial barcode, but have different capture domains designed
to associate the spatial barcode of the feature with more than one
target analyte. For example, a feature may be coupled to four
different types of spatially-barcoded capture probes, each type of
spatially-barcoded capture probe possessing the spatial barcode
802. One type of capture probe associated with the feature includes
the spatial barcode 802 in combination with a poly(T) capture
domain 803, designed to capture mRNA target analytes. A second type
of capture probe associated with the feature includes the spatial
barcode 802 in combination with a random N-mer capture domain 804
for gDNA analysis. A third type of capture probe associated with
the feature includes the spatial barcode 802 in combination with a
capture domain complementary to the capture domain on an analyte
capture agent capture agent barcode domain 805. A fourth type of
capture probe associated with the feature includes the spatial
barcode 802 in combination with a capture probe that can
specifically bind a nucleic acid molecule 806 that can function in
a CRISPR assay (e.g., CRISPR/Cas9). While only four different
capture probe-barcoded constructs are shown in FIG. 8,
capture-probe barcoded constructs can be tailored for analyses of
any given analyte associated with a nucleic acid and capable of
binding with such a construct. For example, the schemes shown in
FIG. 8 can also be used for concurrent analysis of other analytes
disclosed herein, including, but not limited to: (a) mRNA, a
lineage tracing construct, cell surface or intracellular proteins
and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g.,
ATAC-seq, DNase-seq, and/or MNase-seq) cell surface or
intracellular proteins and metabolites, and a perturbation agent
(e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or
antisense oligonucleotide as described herein); (c) mRNA, cell
surface or intracellular proteins and/or metabolites, a barcoded
labelling agent (e.g., the MHC multimers described herein), and a
V(D)J sequence of an immune cell receptor (e.g., T-cell receptor).
In some embodiments, a perturbation agent can be a small molecule,
an antibody, a drug, an aptamer, a miRNA, a physical environmental
(e.g., temperature change), or any other known perturbation
agents.
[0493] Capture probes attached to a single array feature can
include identical (or common) spatial barcode sequences, different
spatial barcode sequences, or a combination of both. Capture probes
attached to a feature can include multiple sets of capture probes.
Capture probes of a given set can include identical spatial barcode
sequences. The identical spatial barcode sequences can be different
from spatial barcode sequences of capture probes of another
set.
[0494] The plurality of capture probes can include spatial barcode
sequences (e.g., nucleic acid barcode sequences) that are
associated with specific locations on a spatial array. For example,
a first plurality of capture probes can be associated with a first
region, based on a spatial barcode sequence common to the capture
probes within the first region, and a second plurality of capture
probes can be associated with a second region, based on a spatial
barcode sequence common to the capture probes within the second
region. The second region may or may not be associated with the
first region. Additional pluralities of capture probes can be
associated with spatial barcode sequences common to the capture
probes within other regions. In some embodiments, the spatial
barcode sequences can be the same across a plurality of capture
probe molecules.
[0495] In some embodiments, multiple different spatial barcodes are
incorporated into a single arrayed capture probe. For example, a
mixed but known set of spatial barcode sequences can provide a
stronger address or attribution of the spatial barcodes to a given
spot or location, by providing duplicate or independent
confirmation of the identity of the location. In some embodiments,
the multiple spatial barcodes represent increasing specificity of
the location of the particular array point.
[0496] (v) Unique Molecular Identifier
[0497] The capture probe can include one or more (e.g., two or
more, three or more, four or more, five or more) Unique Molecular
Identifiers (UMIs). A unique molecular identifier is a contiguous
nucleic acid segment or two or more non-contiguous nucleic acid
segments that function as a label or identifier for a particular
analyte, or for a capture probe that binds a particular analyte
(e.g., via the capture domain).
[0498] A UMI can be unique. A UMI can include one or more specific
polynucleotides sequences, one or more random nucleic acid and/or
amino acid sequences, and/or one or more synthetic nucleic acid
and/or amino acid sequences.
[0499] In some embodiments, the UMI is a nucleic acid sequence that
does not substantially hybridize to analyte nucleic acid molecules
in a biological sample. In some embodiments, the UMI has less than
80% sequence identity (e.g., less than 70%, 60%, 50%, or less than
40% sequence identity) to the nucleic acid sequences across a
substantial part (e.g., 80% or more) of the nucleic acid molecules
in the biological sample.
[0500] The UMI can include from about 6 to about 20 or more
nucleotides within the sequence of the capture probes. In some
embodiments, the length of a UMI sequence can be about 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.
In some embodiments, the length of a UMI sequence can be at least
about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
nucleotides or longer. In some embodiments, the length of a UMI
sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 nucleotides or shorter.
[0501] These nucleotides can be completely contiguous, i.e., in a
single stretch of adjacent nucleotides, or they can be separated
into two or more separate subsequences that are separated by 1 or
more nucleotides. Separated UMI subsequences can be from about 4 to
about 16 nucleotides in length. In some embodiments, the UMI
subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16 nucleotides or longer. In some embodiments, the UMI subsequence
can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
nucleotides or longer. In some embodiments, the UMI subsequence can
be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
nucleotides or shorter.
[0502] In some embodiments, a UMI is attached to an analyte in a
reversible or irreversible manner. In some embodiments, a UMI is
added to, for example, a fragment of a DNA or RNA sample before,
during, and/or after sequencing of the analyte. In some
embodiments, a UMI allows for identification and/or quantification
of individual sequencing-reads. In some embodiments, a UMI is a
used as a fluorescent barcode for which fluorescently labeled
oligonucleotide probes hybridize to the UMI.
[0503] (vi) Other Aspects of Capture Probes
[0504] For capture probes that are attached to an array feature, an
individual array feature can include one or more capture probes. In
some embodiments, an individual array feature includes hundreds or
thousands of capture probes. In some embodiments, the capture
probes are associated with a particular individual feature, where
the individual feature contains a capture probe including a spatial
barcode unique to a defined region or location on the array.
[0505] In some embodiments, a particular feature can contain
capture probes including more than one spatial barcode (e.g., one
capture probe at a particular feature can include a spatial barcode
that is different than the spatial barcode included in another
capture probe at the same particular feature, while both capture
probes include a second, common spatial barcode), where each
spatial barcode corresponds to a particular defined region or
location on the array. For example, multiple spatial barcode
sequences associated with one particular feature on an array can
provide a stronger address or attribution to a given location by
providing duplicate or independent confirmation of the location. In
some embodiments, the multiple spatial barcodes represent
increasing specificity of the location of the particular array
point. In a non-limiting example, a particular array point can be
coded with two different spatial barcodes, where each spatial
barcode identifies a particular defined region within the array,
and an array point possessing both spatial barcodes identifies the
sub-region where two defined regions overlap, e.g., such as the
overlapping portion of a Venn diagram.
[0506] In another non-limiting example, a particular array point
can be coded with three different spatial barcodes, where the first
spatial barcode identifies a first region within the array, the
second spatial barcode identifies a second region, where the second
region is a subregion entirely within the first region, and the
third spatial barcode identifies a third region, where the third
region is a subregion entirely within the first and second
subregions.
[0507] In some embodiments, capture probes attached to array
features are released from the array features for sequencing.
Alternatively, in some embodiments, capture probes remain attached
to the array features, and the probes are sequenced while remaining
attached to the array features (e.g., via in situ sequencing).
Further aspects of the sequencing of capture probes are described
in subsequent sections of this disclosure.
[0508] In some embodiments, an array feature can include different
types of capture probes attached to the feature. For example, the
array feature can include a first type of capture probe with a
capture domain designed to bind to one type of analyte, and a
second type of capture probe with a capture domain designed to bind
to a second type of analyte. In general, array features can include
one or more (e.g., two or more, three or more, four or more, five
or more, six or more, eight or more, ten or more, 12 or more, 15 or
more, 20 or more, 30 or more, 50 or more) different types of
capture probes attached to a single array feature.
[0509] In some embodiments, the capture probe is nucleic acid. In
some embodiments, the capture probe is attached to the array
feature via its 5' end. In some embodiments, the capture probe
includes from the 5' to 3' end: one or more barcodes (e.g., a
spatial barcode and/or a UMI) and one or more capture domains. In
some embodiments, the capture probe includes from the 5' to 3' end:
one barcode (e.g., a spatial barcode or a UMI) and one capture
domain. In some embodiments, the capture probe includes from the 5'
to 3' end: a cleavage domain, a functional domain, one or more
barcodes (e.g., a spatial barcode and/or a UMI), and a capture
domain. In some embodiments, the capture probe includes from the 5'
to 3' end: a cleavage domain, a functional domain, one or more
barcodes (e.g., a spatial barcode and/or a UMI), a second
functional domain, and a capture domain. In some embodiments, the
capture probe includes from the 5' to 3' end: a cleavage domain, a
functional domain, a spatial barcode, a UMI, and a capture domain.
In some embodiments, the capture probe does not include a spatial
barcode. In some embodiments, the capture probe does not include a
UMI. In some embodiments, the capture probe includes a sequence for
initiating a sequencing reaction.
[0510] In some embodiments, the capture probe is immobilized on a
feature via its 3' end. In some embodiments, the capture probe
includes from the 3' to 5' end: one or more barcodes (e.g., a
spatial barcode and/or a UMI) and one or more capture domains. In
some embodiments, the capture probe includes from the 3' to 5' end:
one barcode (e.g., a spatial barcode or a UMI) and one capture
domain. In some embodiments, the capture probe includes from the 3'
to 5' end: a cleavage domain, a functional domain, one or more
barcodes (e.g., a spatial barcode and/or a UMI), and a capture
domain. In some embodiments, the capture probe includes from the 3'
to 5' end: a cleavage domain, a functional domain, a spatial
barcode, a UMI, and a capture domain.
[0511] In some embodiments, a capture probe includes an in situ
synthesized oligonucleotide. The in situ synthesized
oligonucleotide can be attached to a substrate, or to a feature on
a substrate. In some embodiments, the in situ synthesized
oligonucleotide includes one or more constant sequences, one or
more of which serves as a priming sequence (e.g., a primer for
amplifying target nucleic acids). The in situ synthesized
oligonucleotide can, for example, include a constant sequence at
the 3'end that is attached to a substrate, or attached to a feature
on a substrate. Additionally or alternatively, the in situ
synthesized oligonucleotide can include a constant sequence at the
free 5' end. In some embodiments, the one or more constant
sequences can be a cleavable sequence. In some embodiments, the in
situ synthesized oligonucleotide includes a barcode sequence, e.g.,
a variable barcode sequence. The barcode can be any of the barcodes
described herein. The length of the barcode can be approximately 8
to 16 nucleotides (e.g., 8, 9, 10, 11, 12, 13, 14, 15, or 16
nucleotides). The length of the in situ synthesized oligonucleotide
can be less than 100 nucleotides (e.g., less than 90, 80, 75, 70,
60, 50, 45, 40, 35, 30, 25 or 20 nucleotides). In some instances,
the length of the in situ synthesized oligonucleotide is about 20
to about 40 nucleotides. Exemplary in situ synthesized
oligonucleotides are produced by Affymetrix. In some embodiments,
the in situ synthesized oligonucleotide is attached to a feature of
an array.
[0512] Additional oligonucleotides can be ligated to an in situ
synthesized oligonucleotide to generate a capture probe. For
example, a primer complementary to a portion of the in situ
synthesized oligonucleotide (e.g., a constant sequence in the
oligonucleotide) can be used to hybridize an additional
oligonucleotide and extend (using the in situ synthesized
oligonucleotide as a template e.g., a primer extension reaction) to
form a double stranded oligonucleotide and to further create a 3'
overhang. In some embodiments, the 3' overhang can be created by
template-independent ligases (e.g., terminal deoxynucleotidyl
transferase (TdT) or poly(A) polymerase). An additional
oligonucleotide comprising one or more capture domains can be
ligated to the 3' overhang using a suitable enzyme (e.g., a ligase)
and a splint oligonucleotide, to generate a capture probe. Thus, in
some embodiments, a capture probe is a product of two or more
oligonucleotide sequences, (e.g., the in situ synthesized
oligonucleotide and the additional oligonucleotide) that are
ligated together. In some embodiments, one of the oligonucleotide
sequences is an in situ synthesized oligonucleotide.
[0513] In some embodiments, the capture probe can be prepared using
a splint oligonucleotide (e.g., any of the splint oligonucleotides
described herein). Two or more oligonucleotides can be ligated
together using a splint oligonucleotide and any variety of ligases
known in the art or described herein (e.g., SplintR ligase).
[0514] One of the oligonucleotides can include, for example, a
constant sequence (e.g., a sequence complementary to a portion of a
splint oligonucleotide), a degenerate sequence, and/or a capture
domain (e.g., as described herein). One of the oligonucleotides can
also include a sequence compatible for ligating or hybridizing to
an analyte of interest in the biological sample. An analyte of
interest (e.g., an mRNA) can also be used as a splint
oligonucleotide to ligate further oligonucleotides onto the capture
probe. In some embodiments, the capture probe is generated by
having an enzyme add polynucleotides at the end of an
oligonucleotide sequence. The capture probe can include a
degenerate sequence, which can function as a unique molecular
identifier.
[0515] A degenerate sequence, which is a sequence in which some
positions of a nucleotide sequence contain a number of possible
bases. A degenerate sequence can be a degenerate nucleotide
sequence including about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides.
In some embodiments, a nucleotide sequence contains 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, or more degenerate positions within the
nucleotide sequence. In some embodiments, the degenerate sequence
is used as a UMI.
[0516] In some embodiments, a capture probe includes a restriction
endonuclease recognition sequence or a sequence of nucleotides
cleavable by specific enzyme activities. For example, uracil
sequences can be enzymatically cleaved from a nucleotide sequence
using uracil DNA glycosylase (UDG) or Uracil Specific Excision
Reagent (USER). As another example, other modified bases (e.g.,
modified by methylation) can be recognized and cleaved by specific
endonucleases. The capture probes can be subjected to an enzymatic
cleavage, which removes the blocking domain and any of the
additional nucleotides that are added to the 3' end of the capture
probe during the modification process. Removal of the blocking
domain reveals and/or restores the free 3' end of the capture
domain of the capture probe. In some embodiments, additional
nucleotides can be removed to reveal and/or restore the 3' end of
the capture domain of the capture probe.
[0517] In some embodiments, a blocking domain can be incorporated
into the capture probe when it is synthesized, or after its
synthesis. The terminal nucleotide of the capture domain is a
reversible terminator nucleotide (e.g., 3'-O-blocked reversible
terminator and 3'-unblocked reversible terminator), and can be
included in the capture probe during or after probe synthesis.
[0518] (vii) Extended Capture Probes
[0519] An "extended capture probe" is a capture probe with an
enlarged nucleic acid sequence. For example, where the capture
probe includes nucleic acid, an "extended 3' end" indicates that
further nucleotides were added to the most 3' nucleotide of the
capture probe to extend the length of the capture probe, for
example, by standard polymerization reactions utilized to extend
nucleic acid molecules including templated polymerization catalyzed
by a polymerase (e.g., a DNA polymerase or reverse
transcriptase).
[0520] In some embodiments, extending the capture probe includes
generating cDNA from the captured (hybridized) RNA. This process
involves synthesis of a complementary strand of the hybridized
nucleic acid, e.g., generating cDNA based on the captured RNA
template (the RNA hybridized to the capture domain of the capture
probe). Thus, in an initial step of extending the capture probe,
e.g., the cDNA generation, the captured (hybridized) nucleic acid,
e.g., RNA, acts as a template for the extension, e.g., reverse
transcription, step.
[0521] In some embodiments, the capture probe is extended using
reverse transcription. For example, reverse transcription includes
synthesizing cDNA (complementary or copy DNA) from RNA, e.g.,
(messenger RNA), using a reverse transcriptase. In some
embodiments, reverse transcription is performed while the tissue is
still in place, generating an analyte library, where the analyte
library includes the spatial barcodes from the adjacent capture
probes. In some embodiments, the capture probe is extended using
one or more DNA polymerases.
[0522] In some embodiments, the capture domain of the capture probe
includes a primer for producing the complementary strand of the
nucleic acid hybridized to the capture probe, e.g., a primer for
DNA polymerase and/or reverse transcription. The nucleic acid,
e.g., DNA and/or cDNA, molecules generated by the extension
reaction incorporate the sequence of the capture probe. The
extension of the capture probe, e.g., a DNA polymerase and/or
reverse transcription reaction, can be performed using a variety of
suitable enzymes and protocols.
[0523] In some embodiments, a full-length DNA, e.g., cDNA, molecule
is generated. In some embodiments, a "full-length" DNA molecule
refers to the whole of the captured nucleic acid molecule. However,
if the nucleic acid, e.g., RNA, was partially degraded in the
tissue sample, then the captured nucleic acid molecules will not be
the same length as the initial RNA in the tissue sample. In some
embodiments, the 3' end of the extended probes, e.g., first strand
cDNA molecules, is modified. For example, a linker or adaptor can
be ligated to the 3' end of the extended probes. This can be
achieved using single stranded ligation enzymes such as T4 RNA
ligase or Circligase.TM. (available from Epicentre Biotechnologies,
Madison, Wis.). In some embodiments, template switching
oligonucleotides are used to extend cDNA in order to generate a
full-length cDNA (or as close to a full-length cDNA as possible).
In some embodiments, a second strand synthesis helper probe (a
partially double stranded DNA molecule capable of hybridizing to
the 3' end of the extended capture probe), can be ligated to the 3'
end of the extended probe, e.g., first strand cDNA, molecule using
a double stranded ligation enzyme such as T4 DNA ligase. Other
enzymes appropriate for the ligation step are known in the art and
include, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp.
(strain 9.degree.N) DNA ligase (9.degree.N.TM. DNA ligase, New
England Biolabs), Ampligase.TM. (available from Epicentre
Biotechnologies, Madison, Wis.), and SplintR (available from New
England Biolabs, Ipswich, Mass.). In some embodiments, a
polynucleotide tail, e.g., a poly(A) tail, is incorporated at the
3' end of the extended probe molecules. In some embodiments, the
polynucleotide tail is incorporated using a terminal transferase
active enzyme.
[0524] In some embodiments, double-stranded extended capture probes
are treated to remove any unextended capture probes prior to
amplification and/or analysis, e.g., sequence analysis. This can be
achieved by a variety of methods, e.g., using an enzyme to degrade
the unextended probes, such as an exonuclease enzyme, or
purification columns.
[0525] In some embodiments, extended capture probes are amplified
to yield quantities that are sufficient for analysis, e.g., via DNA
sequencing. In some embodiments, the first strand of the extended
capture probes (e.g., DNA and/or cDNA molecules) acts as a template
for the amplification reaction (e.g., a polymerase chain
reaction).
[0526] In some embodiments, the amplification reaction incorporates
an affinity group onto the extended capture probe (e.g., RNA-cDNA
hybrid) using a primer including the affinity group. In some
embodiments, the primer includes an affinity group and the extended
capture probes includes the affinity group. The affinity group can
correspond to any of the affinity groups described previously.
[0527] In some embodiments, the extended capture probes including
the affinity group can be coupled to an array feature specific for
the affinity group. In some embodiments, the substrate can include
an antibody or antibody fragment. In some embodiments, the array
feature includes avidin or streptavidin and the affinity group
includes biotin. In some embodiments, the array feature includes
maltose and the affinity group includes maltose-binding protein. In
some embodiments, the array feature includes maltose-binding
protein and the affinity group includes maltose. In some
embodiments, amplifying the extended capture probes can function to
release the extended probes from the array feature, insofar as
copies of the extended probes are not attached to the array
feature.
[0528] In some embodiments, the extended capture probe or
complement or amplicon thereof is released from an array feature.
The step of releasing the extended capture probe or complement or
amplicon thereof from an array feature can be achieved in a number
of ways. In some embodiments, an extended capture probe or a
complement thereof is released from the feature by nucleic acid
cleavage and/or by denaturation (e.g., by heating to denature a
double-stranded molecule).
[0529] In some embodiments, the extended capture probe or
complement or amplicon thereof is released from the array feature
by physical means. For example, methods for inducing physical
release include denaturing double stranded nucleic acid molecules.
Another method for releasing the extended capture probes is to use
a solution that interferes with the hydrogen bonds of the double
stranded molecules. In some embodiments, the extended capture probe
is released by applying heated water such as water or buffer of at
least 85.degree. C., e.g., at least 90, 91, 92, 93, 94, 95, 96, 97,
98, or 99.degree. C. In some embodiments, a solution including
salts, surfactants, etc. that can further destabilize the
interaction between the nucleic acid molecules is added to release
the extended capture probe from the array feature. In some
embodiments, a formamide solution can be used to destabilize the
interaction between nucleic acid molecules to release the extended
capture probe from the array feature.
[0530] (viii) Analyte Capture Agents
[0531] This disclosure also provides methods and materials for
using analyte capture agents for spatial profiling of biological
analytes (e.g., mRNA, genomic DNA, accessible chromatin, and cell
surface or intracellular proteins and/or metabolites). As used
herein, an "analyte capture agent" (also referred to previously at
times as a "cell labelling" agent") refers to an agent that
interacts with an analyte (e.g., an analyte in a sample) and with a
capture probe (e.g., a capture probe attached to a substrate) to
identify the analyte. In some embodiments, the analyte capture
agent includes an analyte binding moiety and a capture agent
barcode domain.
[0532] FIG. 9 is a schematic diagram of an exemplary analyte
capture agent 902 comprised of an analyte binding moiety 904 and a
capture agent barcode domain 908. An analyte binding moiety 904 is
a molecule capable of binding to an analyte 906 and interacting
with a spatially-barcoded capture probe. The analyte binding moiety
can bind to the analyte 906 with high affinity and/or with high
specificity. The analyte capture agent can include a capture agent
barcode domain 908, a nucleotide sequence (e.g., an
oligonucleotide), which can hybridize to at least a portion or an
entirety of a capture domain of a capture probe. The analyte
binding moiety 904 can include a polypeptide and/or an aptamer
(e.g., an oligonucleotide or peptide molecule that binds to a
specific target analyte). The analyte binding moiety 904 can
include an antibody or antibody fragment (e.g., an antigen-binding
fragment).
[0533] As used herein, the term "analyte binding moiety" refers to
a molecule or moiety capable of binding to a macromolecular
constituent (e.g., an analyte, e.g., a biological analyte). In some
embodiments of any of the spatial profiling methods described
herein, the analyte binding moiety of the analyte capture agent
that binds to a biological analyte can include, but is not limited
to, an antibody, or an epitope binding fragment thereof, a cell
surface receptor binding molecule, a receptor ligand, a small
molecule, a bi-specific antibody, a bi-specific T-cell engager, a
T-cell receptor engager, a B-cell receptor engager, a pro-body, an
aptamer, a monobody, an affimer, a darpin, and a protein scaffold,
or any combination thereof. The analyte binding moiety can bind to
the macromolecular constituent (e.g., analyte) with high affinity
and/or with high specificity. The analyte binding moiety can
include a nucleotide sequence (e.g., an oligonucleotide), which can
correspond to at least a portion or an entirety of the analyte
binding moiety. The analyte binding moiety can include a
polypeptide and/or an aptamer (e.g., a polypeptide and/or an
aptamer that binds to a specific target molecule, e.g., an
analyte). The analyte binding moiety can include an antibody or
antibody fragment (e.g., an antigen-binding fragment) that binds to
a specific analyte (e.g., a polypeptide).
[0534] In some embodiments, an analyte binding moiety of an analyte
capture agent includes one or more antibodies or antigen binding
fragments thereof. The antibodies or antigen binding fragments
including the analyte binding moiety can specifically bind to a
target analyte. In some embodiments, the analyte is a protein
(e.g., a protein on a surface of the biological sample (e.g., a
cell) or an intracellular protein). In some embodiments, a
plurality of analyte capture agents comprising a plurality of
analyte binding moieties bind a plurality of analytes present in a
biological sample. In some embodiments, the plurality of analytes
includes a single species of analyte (e.g., a single species of
polypeptide). In some embodiments in which the plurality of
analytes includes a single species of analyte, the analyte binding
moieties of the plurality of analyte capture agents are the same.
In some embodiments in which the plurality of analytes includes a
single species of analyte, the analyte binding moieties of the
plurality of analyte capture agents are the different (e.g.,
members of the plurality of analyte capture agents can have two or
more species of analyte binding moieties, wherein each of the two
or more species of analyte binding moieties binds a single species
of analyte, e.g., at different binding sites). In some embodiments,
the plurality of analytes includes multiple different species of
analyte (e.g., multiple different species of polypeptides).
[0535] An analyte capture agent can include an analyte binding
moiety. The analyte binding moiety can be an antibody. Exemplary,
non-limiting antibodies that can be used as analyte binding
moieties in an analyte capture agent or that can be used in the
IHC/IF applications disclosed herein include any of the following
including variations thereof: A-ACT, A-AT, ACTH,
Actin-Muscle-specific, Actin-Smooth Muscle (SMA), AE1, AE1/AE3,
AE3, AFP, AKT Phosphate, ALK-1, Amyloid A, Androgen Receptor,
Annexin A1, B72.3, BCA-225, BCL-1 (Cyclin D1), BCL-1/CD20, BCL-2,
BCL-2/BCL-6, BCL-6, Ber-EP4, Beta-amyloid, Beta-catenin, BG8 (Lewis
Y), BOB-1, CA 19.9, CA 125, CAIX, Calcitonin, Caldesmon, Calponin,
Calretinin, CAM 5.2, CAM 5.2/AE1, CD1a, CD2, CD3 (M), CD3 (P),
CD3/CD20, CD4, CD5, CD7, CD8, CD10, CD14, CD15, CD20, CD21, CD22,
CD 23, CD25, CD30, CD31, CD33, CD34, CD35, CD43, CD45 (LCA),
CD45RA, CD56, CD57, CD61, CD68, CD71, CD74, CD79a, CD99, CD117
(c-KIT), CD123, CD138, CD163, CDX-2, CDX-2/CK-7, CEA (M), CEA (P),
Chromogranin A, Chymotrypsin, CK-5, CK-5/6, CK-7, CK-7/TTF-1,
CK-14, CK-17, CK-18, CK-19, CK-20, CK-HMW, CK-LMW, CMV-IH, COLL-IV,
COX-2, D2-40, DBA44, Desmin, DOG1, EBER-ISH, EBV (LMP1),
E-Cadherin, EGFR, EMA, ER, ERCC1, Factor VIII (vWF), Factor XIIIa,
Fascin, FLI-1, FHS, Galectin-3, Gastrin, GCDFP-15, GFAP, Glucagon,
Glycophorin A, Glypican-3, Granzyme B, Growth Hormone (GH), GST,
HAM 56, HMBE-1, HBP, HCAg, HCG, Hemoglobin A, HEP B CORE (HBcAg),
HEP B SURF, (HBsAg), HepPar1, HER2, Herpes I, Herpes II, HHV-8,
HLA-DR, HMB 45, HPL, HPV-IHC, HPV (6/11)-ISH, HPV (16/18)-ISH, HPV
(31/33)-ISH, HPV WSS-ISH, HPV High-ISH, HPV Low-ISH, HPV High &
Low-ISH, IgA, IgD, IgG, IgG4, IgM, Inhibin, Insulin, JC Virus-ISH,
Kappa-ISH, KER PAN, Ki-67, Lambda-IHC, Lambda-ISH, LH, Lipase,
Lysozyme (MURA), Mammaglobin, MART-1, MBP, M-Cell Tryptase, MEL-5,
Melan-A, Melan-A/Ki-67, Mesothelin, MiTF, MLH-1, MOC-31, MPO,
MSH-2, MSH-6, MUC1, MUC2, MUC4, MUCSAC, MUM-1, MYO D1, Myogenin,
Myoglobin, Myoin Heavy Chain, Napsin A, NB84a, NEW-N, NF, NK1-C3,
NPM, NSE, OCT-2, OCT-3/4, OSCAR, p16, p21, p27/Kip1, p53, p57, p63,
p120, P504S, Pan Melanoma, PANC.POLY, Parvovirus B19, PAX-2, PAX-5,
PAX-5/CD43, PAX=5/CD5, PAX-8, PC, PD1, Perforin, PGP 9.5, PLAP,
PMS-2, PR, Prolactin, PSA, PSAP, PSMA, PTEN, PTH, PTS, RB, RCC, S6,
S100, Serotonin, Somatostatin, Surfactant (SP-A), Synaptophysin,
Synuclein, TAU, TCL-1, TCR beta, TdT, Thrombomodulin,
Thyroglobulin, TIA-1, TOXO, TRAP, TriView.TM. breast, TriView.TM.
prostate, Trypsin, TS, TSH, TTF-1, Tyrosinase, Ubiqutin, Uroplakin,
VEGF, Villin, Vimentin (VIM), VIP, VZV, WTI (M) N-Terminus, WTI (P)
C-Terminus, ZAP-70.
[0536] Further, exemplary, non-limiting antibodies that can be used
as analyte binding moieties in an analyte capture agent or that can
be used in the IHC/IF applications disclosed herein include any of
the following antibodies (and variations thereof) to: cell surface
proteins, intracellular proteins, kinases (e.g., AGC kinase family
(e.g., AKT1, AKT2, PDK1, Protein Kinase C, ROCK1, ROCK2, SGK3),
CAMK kinase family (e.g., AMPK1, AMPK2, CAMK, Chk1, Chk2, Zip), CK1
kinase family, TK kinase family (e.g., Ab12, AXL, CD167, CD246/ALK,
c-Met, CSK, c-Src, EGFR, ErbB2 (HER2/neu), ErbB3, ErbB4, FAK, Fyn,
LCK, Lyn, PKT7, Syk, Zap70), STE kinase family (e.g., ASK1, MAPK,
MEK1, MEK2, MEK3 MEK4, MEK5, PAK1, PAK2, PAK4, PAK6), CMGC kinase
family (e.g., Cdk2, Cdk4, Cdk5, Cdk6, Cdk7, Cdk9, Erkl, GSK3,
Jnk/MAPK8, Jnk2/MAPK9, JNK3/MAPK10, p38/MAPK), and TKL kinase
family (e.g., ALK1, ILK1, IRAK1, IRAK2, IRAK3, IRAK4, LIMK1, LIMK2,
M3K11, RAF1, RIP1, RIP3, VEGFR1, VEGFR2, VEGFR3), Aurora A kinase,
Aurora B kinase, IKK, Nemo-like kinase, PINK, PLK3, ULK2, WEE1,
transcription factors (e.g., FOXP3, ATF3, BACH1, EGR, ELF3, FOXA1,
FOXA2, FOX01, GATA), growth factor receptors, tumor suppressors
(e.g., anti-p53, anti-BLM, anti-Cdk2, anti-Chk2, anti-BRCA-1,
anti-NBS1, anti-BRCA-2, anti-WRN, anti-PTEN, anti-WT1,
anti-p38).
[0537] In some embodiments, analyte capture agents are capable of
binding to analytes present inside a cell. In some embodiments,
analyte capture agents are capable of binding to cell surface
analytes that can include, without limitation, a receptor, an
antigen, a surface protein, a transmembrane protein, a cluster of
differentiation protein, a protein channel, a protein pump, a
carrier protein, a phospholipid, a glycoprotein, a glycolipid, a
cell-cell interaction protein complex, an antigen-presenting
complex, a major histocompatibility complex, an engineered T-cell
receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen
receptor, an extracellular matrix protein, a posttranslational
modification (e.g., phosphorylation, glycosylation, ubiquitination,
nitrosylation, methylation, acetylation or lipidation) state of a
cell surface protein, a gap junction, and an adherens junction. In
some embodiments, the analyte capture agents are capable of binding
to cell surface analytes that are post-translationally modified. In
such embodiments, analyte capture agents can be specific for cell
surface analytes based on a given state of posttranslational
modification (e.g., phosphorylation, glycosylation, ubiquitination,
nitrosylation, methylation, acetylation or lipidation), such that a
cell surface analyte profile can include posttranslational
modification information of one or more analytes.
[0538] In some embodiments, the analyte capture agent includes a
capture agent barcode domain that is conjugated or otherwise
attached to the analyte binding moiety. In some embodiments, the
capture agent barcode domain is covalently-linked to the analyte
binding moiety. In some embodiments, a capture agent barcode domain
is a nucleic acid sequence. In some embodiments, a capture agent
barcode domain includes an analyte binding moiety barcode and an
analyte capture sequence.
[0539] As used herein, the term "analyte binding moiety barcode"
refers to a barcode that is associated with or otherwise identifies
the analyte binding moiety. In some embodiments, by identifying an
analyte binding moiety and its associated analyte binding moiety
barcode, the analyte to which the analyte binding moiety binds can
also be identified. An analyte binding moiety barcode can be a
nucleic acid sequence of a given length and/or sequence that is
associated with the analyte binding moiety. An analyte binding
moiety barcode can generally include any of the variety of aspects
of barcodes described herein. For example, an analyte capture agent
that is specific to one type of analyte can have coupled thereto a
first capture agent barcode domain (e.g., that includes a first
analyte binding moiety barcode), while an analyte capture agent
that is specific to a different analyte can have a different
capture agent barcode domain (e.g., that includes a second barcode
analyte binding moiety barcode) coupled thereto. In some aspects,
such a capture agent barcode domain can include an analyte binding
moiety barcode that permits identification of the analyte binding
moiety to which the capture agent barcode domain is coupled. The
selection of the capture agent barcode domain can allow significant
diversity in terms of sequence, while also being readily attachable
to most analyte binding moieties (e.g., antibodies or aptamers) as
well as being readily detected, (e.g., using sequencing or array
technologies).
[0540] In some embodiments, the capture agent barcode domain of an
analyte capture agent includes an analyte capture sequence. As used
herein, the term "analyte capture sequence" refers to a region or
moiety configured to hybridize to, bind to, couple to, or otherwise
interact with a capture domain of a capture probe. In some
embodiments, an analyte capture sequence includes a nucleic acid
sequence that is complementary to or substantially complementary to
the capture domain of a capture probe such that the analyte capture
sequence hybridizes to the capture domain of the capture probe. In
some embodiments, an analyte capture sequence comprises a poly(A)
nucleic acid sequence that hybridizes to a capture domain that
comprises a poly(T) nucleic acid sequence. In some embodiments, an
analyte capture sequence comprises a poly(T) nucleic acid sequence
that hybridizes to a capture domain that comprises a poly(A)
nucleic acid sequence. In some embodiments, an analyte capture
sequence comprises a non-homopolymeric nucleic acid sequence that
hybridizes to a capture domain that comprises a non-homopolymeric
nucleic acid sequence that is complementary (or substantially
complementary) to the non-homopolymeric nucleic acid sequence of
the analyte capture region.
[0541] In some embodiments of any of the spatial analysis methods
described herein that employ an analyte capture agent, the capture
agent barcode domain can be directly coupled to the analyte binding
moiety, or they can be attached to a bead, molecular lattice, e.g.,
a linear, globular, cross-slinked, or other polymer, or other
framework that is attached or otherwise associated with the analyte
binding moiety, which allows attachment of multiple capture agent
barcode domains to a single analyte binding moiety. Attachment
(coupling) of the capture agent barcode domains to the analyte
binding moieties can be achieved through any of a variety of direct
or indirect, covalent or non-covalent associations or attachments.
For example, in the case of a capture agent barcode domain coupled
to an analyte binding moiety that includes an antibody or
antigen-binding fragment, such capture agent barcode domains can be
covalently attached to a portion of the antibody or antigen-binding
fragment using chemical conjugation techniques (e.g.,
Lightning-Link.RTM. antibody labelling kits available from Innova
Biosciences). In some embodiments, a capture agent barcode domain
can be coupled to an antibody or antigen-binding fragment using
non-covalent attachment mechanisms (e.g., using biotinylated
antibodies and oligonucleotides or beads that include one or more
biotinylated linker(s), coupled to oligonucleotides with an avidin
or streptavidin linker.) Antibody and oligonucleotide biotinylation
techniques can be used, and are described for example in Fang et
al., Nucleic Acids Res. (2003), 31(2): 708-715, the entire contents
of which are incorporated by reference herein. Likewise, protein
and peptide biotinylation techniques have been developed and can be
used, and are described for example in U.S. Pat. No. 6,265,552, the
entire contents of which are incorporated by reference herein.
Furthermore, click reaction chemistry such as a
methyltetrazine-PEG5-NHS ester reaction, a TCO-PEG4-NHS ester
reaction, or the like, can be used to couple capture agent barcode
domains to analyte binding moieties. The reactive moiety on the
analyte binding moiety can also include amine for targeting
aldehydes, amine for targeting maleimide (e.g., free thiols), azide
for targeting click chemistry compounds (e.g., alkynes), biotin for
targeting streptavidin, phosphates for targeting EDC, which in turn
targets active ester (e.g., NH2). The reactive moiety on the
analyte binding moiety can be a chemical compound or group that
binds to the reactive moiety on the analyte binding moiety.
Exemplary strategies to conjugate the analyte binding moiety to the
capture agent barcode domain include the use of commercial kits
(e.g., Solulink, Thunder link), conjugation of mild reduction of
hinge region and maleimide labelling, stain-promoted click
chemistry reaction to labeled amides (e.g., copper-free), and
conjugation of periodate oxidation of sugar chain and amine
conjugation. In the cases where the analyte binding moiety is an
antibody, the antibody can be modified prior to or
contemporaneously with conjugation of the oligonucleotide. For
example, the antibody can be glycosylated with a chemical
substrate-permissive mutant of .beta.-1,4-galactosyltransferase,
GalT (Y289L) and azide-bearing uridine
diphosphate-N-acetylgalactosamine analog uridine
diphosphate--GalNAz. The modified antibody can be conjugated to an
oligonucleotide with a dibenzocyclooctyne-PEG4-NHS group. In some
embodiments, certain steps (e.g., COOH activation (e.g., EDC) and
homobifunctional cross linkers) can be avoided to prevent the
analyte binding moieties from conjugating to themselves. In some
embodiments of any of the spatial profiling methods described
herein, the analyte capture agent (e.g., analyte binding moiety
coupled to an oligonucleotide) can be delivered into the cell,
e.g., by transfection (e.g., using transfectamine, cationic
polymers, calcium phosphate or electroporation), by transduction
(e.g., using a bacteriophage or recombinant viral vector), by
mechanical delivery (e.g., magnetic beads), by lipid (e.g.,
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporter
proteins. An analyte capture agent can be delivered into a cell
using exosomes. For example, a first cell can be generated that
releases exosomes comprising an analyte capture agent. An analyte
capture agent can be attached to an exosome membrane. An analyte
capture agent can be contained within the cytosol of an exosome.
Released exosomes can be harvested and provided to a second cell,
thereby delivering the analyte capture agent into the second cell.
An analyte capture agent can be releasable from an exosome membrane
before, during, or after delivery into a cell. In some embodiments,
the cell is permeabilized to allow the analyte capture agent to
couple with intracellular constituents (such as, without
limitation, intracellular proteins, metabolites, and nuclear
membrane proteins). Following intracellular delivery, analyte
capture agents can be used to analyze intracellular constituents as
described herein.
[0542] In some embodiments of any of the spatial profiling methods
described herein, the capture agent barcode domain coupled to an
analyte capture agent can include modifications that render it
non-extendable by a polymerase. In some embodiments, when binding
to a capture domain of a capture probe or nucleic acid in a sample
for a primer extension reaction, the capture agent barcode domain
can serve as a template, not a primer. When the capture agent
barcode domain also includes a barcode (e.g., an analyte binding
moiety barcode), such a design can increase the efficiency of
molecular barcoding by increasing the affinity between the capture
agent barcode domain and unbarcoded sample nucleic acids, and
eliminate the potential formation of adaptor artifacts. In some
embodiments, the capture agent barcode domain can include a random
N-mer sequence that is capped with modifications that render it
non-extendable by a polymerase. In some cases, the composition of
the random N-mer sequence can be designed to maximize the binding
efficiency to free, unbarcoded ssDNA molecules. The design can
include a random sequence composition with a higher GC content, a
partial random sequence with fixed G or C at specific positions,
the use of guanosines, the use of locked nucleic acids, or any
combination thereof.
[0543] A modification for blocking primer extension by a polymerase
can be a carbon spacer group of different lengths or a
dideoxynucleotide. In some embodiments, the modification can be an
abasic site that has an apurine or apyrimidine structure, a base
analog, or an analogue of a phosphate backbone, such as a backbone
of N-(2-aminoethyl)-glycine linked by amide bonds, tetrahydrofuran,
or 1',2'-Dideoxyribose. The modification can also be a uracil base,
2'OMe modified RNA, C3-18 spacers (e.g., structures with 3-18
consecutive carbon atoms, such as C3 spacer), ethylene glycol
multimer spacers (e.g., spacer 18 (hexa-ethyleneglycol spacer),
biotin, di-deoxynucleotide triphosphate, ethylene glycol, amine, or
phosphate.
[0544] In some embodiments of any of the spatial profiling methods
described herein, the capture agent barcode domain coupled to the
analyte binding moiety includes a cleavable domain. For example,
after the analyte capture agent binds to an analyte (e.g., a cell
surface analyte), the capture agent barcode domain can be cleaved
and collected for downstream analysis according to the methods as
described herein. In some embodiments, the cleavable domain of the
capture agent barcode domain includes a U-excising element that
allows the species to release from the bead. In some embodiments,
the U-excising element can include a single-stranded DNA (ssDNA)
sequence that contains at least one uracil. The species can be
attached to a bead via the ssDNA sequence. The species can be
released by a combination of uracil-DNA glycosylase (e.g., to
remove the uracil) and an endonuclease (e.g., to induce an ssDNA
break). If the endonuclease generates a 5' phosphate group from the
cleavage, then additional enzyme treatment can be included in
downstream processing to eliminate the phosphate group, e.g., prior
to ligation of additional sequencing handle elements, e.g.,
Illumina full P5 sequence, partial P5 sequence, full R1 sequence,
and/or partial R1 sequence.
[0545] In some embodiments, multiple different species of analytes
(e.g., polypeptides) from the biological sample can be subsequently
associated with the one or more physical properties of the
biological sample. For example, the multiple different species of
analytes can be associated with locations of the analytes in the
biological sample. Such information (e.g., proteomic information
when the analyte binding moiety(ies) recognizes a polypeptide(s))
can be used in association with other spatial information (e.g.,
genetic information from the biological sample, such as DNA
sequence information, transcriptome information (i.e., sequences of
transcripts), or both). For example, a cell surface protein of a
cell can be associated with one or more physical properties of the
cell (e.g., a shape, size, activity, or a type of the cell). The
one or more physical properties can be characterized by imaging the
cell. The cell can be bound by an analyte capture agent comprising
an analyte binding moiety that binds to the cell surface protein
and an analyte binding moiety barcode that identifies that analyte
binding moiety, and the cell can be subjected to spatial analysis
(e.g., any of the variety of spatial analysis methods described
herein). For example, the analyte capture agent bound to the cell
surface protein can be bound to a capture probe (e.g., a capture
probe on an array), which capture probe includes a capture domain
that interacts with an analyte capture sequence present on the
capture agent barcode domain of the analyte capture agent. All or
part of the capture agent barcode domain (including the analyte
binding moiety barcode) can be copied with a polymerase using a 3'
end of the capture domain as a priming site, generating an extended
capture probe that includes the all or part of complementary
sequence that corresponds to the capture probe (including a spatial
barcode present on the capture probe) and a copy of the analyte
binding moiety barcode. In some embodiments, an analyte capture
agent with an extended capture agent barcode domain that includes a
sequence complementary to a spatial barcode of a capture probe is
called a "spatially-tagged analyte capture agent."
[0546] In some embodiments, the spatial array with spatially-tagged
analyte capture agents can be contacted with a sample, where the
analyte capture agent(s) associated with the spatial array capture
the target analyte(s). The analyte capture agent(s) containing the
extended capture probe(s), which includes a sequence complementary
to the spatial barcode(s) of the capture probe(s) and the analyte
binding moiety barcode(s), can then be denatured from the capture
probe(s) of the spatial array. This allows the spatial array to be
reused. The sample can be dissociated into non-aggregated cells
(e.g., single cells) and analyzed by the single cell/droplet
methods described herein. The spatially-tagged analyte capture
agent can be sequenced to obtain the nucleic acid sequence of the
spatial barcode of the capture probe and the analyte binding moiety
barcode of the analyte capture agent. The nucleic acid sequence of
the extended capture probe can thus be associated with an analyte
(e.g., cell surface protein), and in turn, with the one or more
physical properties of the cell (e.g., a shape or cell type). In
some embodiments, the nucleic acid sequence of the extended capture
probe can be associated with an intracellular analyte of a nearby
cell, where the intracellular analyte was released using any of the
cell permeabilization or analyte migration techniques described
herein.
[0547] In some embodiments of any of the spatial profiling methods
described herein, the capture agent barcode domains released from
the analyte capture agents can then be subjected to sequence
analysis to identify which analyte capture agents were bound to
analytes. Based upon the capture agent barcode domains that are
associated with a feature (e.g., a feature at a particular
location) on a spatial array and the presence of the analyte
binding moiety barcode sequence, an analyte profile can be created
for a biological sample. Profiles of individual cells or
populations of cells can be compared to profiles from other cells,
e.g., `normal` cells, to identify variations in analytes, which can
provide diagnostically relevant information. In some embodiments,
these profiles can be useful in the diagnosis of a variety of
disorders that are characterized by variations in cell surface
receptors, such as cancer and other disorders.
[0548] FIG. 10 is a schematic diagram depicting an exemplary
interaction between a feature-immobilized capture probe 1024 and an
analyte capture agent 1026. The feature-immobilized capture probe
1024 can include a spatial barcode 1008 as well as one or more
functional sequences 1006 and 1010, as described elsewhere herein.
The capture probe can also include a capture domain 1012 that is
capable of binding to an analyte capture agent 1026. The analyte
capture agent 1026 can include a functional sequence 1018, capture
agent barcode domain 1016, and an analyte capture sequence 1014
that is capable of binding to the capture domain 1012 of the
capture probe 1024. The analyte capture agent can also include a
linker 1020 that allows the capture agent barcode domain 1016 to
couple to the analyte binding moiety 1022.
[0549] In some embodiments of any of the spatial profiling methods
described herein, the methods are used to identify immune cell
profiles. Immune cells express various adaptive immunological
receptors relating to immune function, such as T cell receptors
(TCRs) and B cell receptors (BCRs). T cell receptors and B cell
receptors play a part in the immune response by specifically
recognizing and binding to antigens and aiding in their
destruction.
[0550] The T cell receptor, or TCR, is a molecule found on the
surface of T cells that is generally responsible for recognizing
fragments of antigen as peptides bound to major histocompatibility
complex (MHC) molecules. The TCR is generally a heterodimer of two
chains, each of which is a member of the immunoglobulin
superfamily, possessing an N-terminal variable (V) domain, and a C
terminal constant domain. In humans, in 95% of T cells, the TCR
consists of an alpha (.alpha.) and beta (.beta.) chain, whereas in
5% of T cells, the TCR consists of gamma and delta
(.gamma./.delta.) chains. This ratio can change during ontogeny and
in diseased states as well as in different species. When the TCR
engages with antigenic peptide and MHC (peptide/MHC or pMHC), the T
lymphocyte is activated through signal transduction.
[0551] Each of the two chains of a TCR contains multiple copies of
gene segments--a variable `V` gene segment, a diversity `D` gene
segment, and a joining `J` gene segment. The TCR alpha chain (TCRa)
is generated by recombination of V and J segments, while the beta
chain (TCRb) is generated by recombination of V, D, and J segments.
Similarly, generation of the TCR gamma chain involves recombination
of V and J gene segments, while generation of the TCR delta chain
occurs by recombination of V, D, and J gene segments. The
intersection of these specific regions (V and J for the alpha or
gamma chain, or V, D and J for the beta or delta chain) corresponds
to the CDR3 region that is important for antigen-MHC recognition.
Complementarity determining regions (e.g., CDR1, CDR2, and CDR3),
or hypervariable regions, are sequences in the variable domains of
antigen receptors (e.g., T cell receptor and immunoglobulin) that
can complement an antigen. Most of the diversity of CDRs is found
in CDR3, with the diversity being generated by somatic
recombination events during the development of T lymphocytes. A
unique nucleotide sequence that arises during the gene arrangement
process can be referred to as a clonotype.
[0552] The B cell receptor, or BCR, is a molecule found on the
surface of B cells. The antigen binding portion of a BCR is
composed of a membrane-bound antibody that, like most antibodies
(e.g., immunoglobulins), has a unique and randomly determined
antigen-binding site. The antigen binding portion of a BCR includes
membrane-bound immunoglobulin molecule of one isotype (e.g., IgD,
IgM, IgA, IgG, or IgE). When a B cell is activated by its first
encounter with a cognate antigen, the cell proliferates and
differentiates to generate a population of antibody-secreting
plasma B cells and memory B cells. The various immunoglobulin
isotypes differ in their biological features, structure, target
specificity, and distribution. A variety of molecular mechanisms
exist to generate initial diversity, including genetic
recombination at multiple sites.
[0553] The BCR is composed of two genes IgH and IgK (or IgL) coding
for antibody heavy and light chains. Immunoglobulins are formed by
recombination among gene segments, sequence diversification at the
junctions of these segments, and point mutations throughout the
gene. Each heavy chain gene contains multiple copies of three
different gene segments--a variable `V` gene segment, a diversity
`D` gene segment, and a joining 1' gene segment. Each light chain
gene contains multiple copies of two different gene segments for
the variable region of the protein--a variable `V` gene segment and
a joining `J` gene segment.
[0554] The recombination can generate a molecule with one of each
of the V, D, and J segments. Furthermore, several bases can be
deleted and others added (called N and P nucleotides) at each of
the two junctions, thereby generating further diversity. After B
cell activation, a process of affinity maturation through somatic
hypermutation occurs. In this process, progeny cells of the
activated B cells accumulate distinct somatic mutations throughout
the gene with higher mutation concentration in the CDR regions
leading to the generation of antibodies with higher affinity to the
antigens.
[0555] In addition to somatic hypermutation, activated B cells
undergo the process of isotype switching. Antibodies with the same
variable segments can have different forms (isotypes) depending on
the constant segment. Whereas all naive B cells express IgM (or
IgD), activated B cells mostly express IgG but also IgM, IgA, and
IgE. This expression switching from IgM (and/or IgD) to IgG, IgA,
or IgE occurs through a recombination event causing one cell to
specialize in producing a specific isotype. A unique nucleotide
sequence that arises during the gene arrangement process can
similarly be referred to as a clonotype.
[0556] Certain methods described herein are utilized to analyze the
various sequences of TCRs and BCRs from immune cells, for example,
various clonotypes. In some embodiments, the methods are used to
analyze the sequence of a TCR alpha chain, a TCR beta chain, a TCR
delta chain, a TCR gamma chain, or any fragment thereof (e.g.,
variable regions including V(D)J or VJ regions, constant regions,
transmembrane regions, fragments thereof, combinations thereof, and
combinations of fragments thereof). In some embodiments, the
methods described herein can be used to analyze the sequence of a B
cell receptor heavy chain, B cell receptor light chain, or any
fragment thereof (e.g., variable regions including V(D)J or VJ
regions, constant regions, transmembrane regions, fragments
thereof, combinations thereof, and combinations of fragments
thereof).
[0557] Where immune cells are to be analyzed, primer sequences
useful in any of the various operations for attaching barcode
sequences and/or amplification reactions can include gene specific
sequences which target genes or regions of genes of immune cell
proteins, for example immune receptors. Such gene sequences
include, but are not limited to, sequences of various T cell
receptor alpha variable genes (TRAV genes), T cell receptor alpha
joining genes (TRAJ genes), T cell receptor alpha constant genes
(TRAC genes), T cell receptor beta variable genes (TRBV genes), T
cell receptor beta diversity genes (TRBD genes), T cell receptor
beta joining genes (TRBJ genes), T cell receptor beta constant
genes (TRBC genes), T cell receptor gamma variable genes (TRGV
genes), T cell receptor gamma joining genes (TRGJ genes), T cell
receptor gamma constant genes (TRGC genes), T cell receptor delta
variable genes (TRDV genes), T cell receptor delta diversity genes
(TRDD genes), T cell receptor delta joining genes (TRDJ genes), and
T cell receptor delta constant genes (TRDC genes).
[0558] In some embodiments, the analyte binding moiety is based on
the Major Histocompatibility Complex (MHC) class I or class II. In
some embodiments, the analyte binding moiety is an MHC multimer
including, without limitation, MHC dextramers, MHC tetramers, and
MHC pentamers (see, for example, U.S. Patent Application
Publication Nos. US 2018/0180601 and US 2017/0343545, the entire
contents of each of which are incorporated herein by reference.
MHCs (e.g., a soluble MHC monomer molecule), including full or
partial MHC-peptides, can be used as analyte binding moieties of
analyte capture agents that are coupled to capture agent barcode
domains that include an analyte binding moiety barcode that
identifies its associated MHC (and, thus, for example, the MHC's
TCR binding partner). In some embodiments, MHCs are used to analyze
one or more cell-surface features of a T-cell, such as a TCR. In
some cases, multiple MHCs are associated together in a larger
complex (MHC multi-mer) to improve binding affinity of MHCs to TCRs
via multiple ligand binding synergies.
[0559] FIGS. 11A, 11B, and 11C are schematics illustrating how
streptavidin cell tags can be utilized in an array-based system to
produce a spatially-barcoded cell or cellular contents. For
example, as shown in FIG. 11, peptide-bound major
histocompatibility complex (pMHCs) can be individually associated
with biotin and bound to a streptavidin moiety such that the
streptavidin moiety comprises multiple pMHC moieties. Each of these
moieties can bind to a TCR such that the streptavidin binds to a
target T-cell via multiple MCH/TCR binding interactions. Multiple
interactions synergize and can substantially improve binding
affinity. Such improved affinity can improve labelling of T-cells
and also reduce the likelihood that labels will dissociate from
T-cell surfaces. As shown in FIG. 11B, a capture agent barcode
domain 1101 can be modified with streptavidin 1102 and contacted
with multiple molecules of biotinylated MHC 1103 (such as a pMHC)
such that the biotinylated MHC 1103 molecules are coupled with the
streptavidin conjugated capture agent barcode domain 1101. The
result is a barcoded MHC multimer complex 1105. As shown in FIG.
11B, the capture agent barcode domain sequence 1101 can identify
the MHC as its associated label and also includes optional
functional sequences such as sequences for hybridization with other
oligonucleotides. As shown in FIG. 11C, one example oligonucleotide
is capture probe 1106 that comprises a complementary sequence
(e.g., rGrGrG corresponding to C C C), a barcode sequence and other
functional sequences, such as, for example, a UMI, an adapter
sequence (e.g., comprising a sequencing primer sequence (e.g., R1
or a partial R1 ("pR1")), a flow cell attachment sequence (e.g., P5
or P7 or partial sequences thereof)), etc. In some cases, capture
probe 1106 may at first be associated with a feature (e.g., a gel
bead) and released from the feature. In other embodiments, capture
probe 1106 can hybridize with a capture agent barcode domain 1101
of the MHC-oligonucleotide complex 1105. The hybridized
oligonucleotides (Spacer C C C and Spacer rGrGrG) can then be
extended in primer extension reactions such that constructs
comprising sequences that correspond to each of the two spatial
barcode sequences (the spatial barcode associated with the capture
probe, and the barcode associated with the MHC-oligonucleotide
complex) are generated. In some cases, one or both of these
corresponding sequences may be a complement of the original
sequence in capture probe 1106 or capture agent barcode domain
1101. In other embodiments, the capture probe and the capture agent
barcode domain are ligated together. The resulting constructs can
be optionally further processed (e.g., to add any additional
sequences and/or for clean-up) and subjected to sequencing. As
described elsewhere herein, a sequence derived from the capture
probe 1106 spatial barcode sequence may be used to identify a
feature and the sequence derived from spatial barcode sequence on
the capture agent barcode domain 1101 may be used to identify the
particular peptide MHC complex 1104 bound on the surface of the
cell (e.g., when using MHC-peptide libraries for screening immune
cells or immune cell populations).
[0560] (c) Substrates
[0561] For the spatial array-based analytical methods described
herein, a substrate functions as a support for direct or indirect
attachment of capture probes to features of the array. In addition,
in some embodiments, a substrate (e.g., the same substrate or a
different substrate) can be used to provide support to a biological
sample, particularly, for example, a thin tissue section.
Accordingly, a "substrate" is a support that is insoluble in
aqueous liquid and which allows for positioning of biological
samples, analytes, features, and/or capture probes on the
substrate.
[0562] Further, a "substrate" as used herein, and when not preceded
by the modifier "chemical", refers to a member with at least one
surface that generally functions to provide physical support for
biological samples, analytes, and/or any of the other chemical
and/or physical moieties, agents, and structures described herein.
Substrates can be formed from a variety of solid materials,
gel-based materials, colloidal materials, semi-solid materials
(e.g., materials that are at least partially cross-linked),
materials that are fully or partially cured, and materials that
undergo a phase change or transition to provide physical support.
Examples of substrates that can be used in the methods and systems
described herein include, but are not limited to, slides (e.g.,
slides formed from various glasses, slides formed from various
polymers), hydrogels, layers and/or films, membranes (e.g., porous
membranes), flow cells, cuvettes, wafers, plates, or combinations
thereof. In some embodiments, substrates can optionally include
functional elements such as recesses, protruding structures,
microfluidic elements (e.g., channels, reservoirs, electrodes,
valves, seals), and various markings, as will be discussed in
further detail below.
[0563] (i) Substrate Attributes
[0564] A substrate can generally have any suitable form or format.
For example, a substrate can be flat, curved, e.g., convexly or
concavely curved towards the area where the interaction between a
biological sample, e.g., tissue sample, and a substrate takes
place. In some embodiments, a substrate is flat, e.g., planar,
chip, or slide. A substrate can contain one or more patterned
surfaces within the substrate (e.g., channels, wells, projections,
ridges, divots, etc.).
[0565] A substrate can be of any desired shape. For example, a
substrate can be typically a thin, flat shape (e.g., a square or a
rectangle). In some embodiments, a substrate structure has rounded
corners (e.g., for increased safety or robustness). In some
embodiments, a substrate structure has one or more cut-off corners
(e.g., for use with a slide clamp or cross-table). In some
embodiments, where a substrate structure is flat, the substrate
structure can be any appropriate type of support having a flat
surface (e.g., a chip or a slide such as a microscope slide).
[0566] Substrates can optionally include various structures such
as, but not limited to, projections, ridges, and channels. A
substrate can be micropatterned to limit lateral diffusion (e.g.,
to prevent overlap of spatial barcodes). A substrate modified with
such structures can be modified to allow association of analytes,
features (e.g., beads), or probes at individual sites. For example,
the sites where a substrate is modified with various structures can
be contiguous or non-contiguous with other sites.
[0567] In some embodiments, the surface of a substrate can be
modified so that discrete sites are formed that can only have or
accommodate a single feature. In some embodiments, the surface of a
substrate can be modified so that features adhere to random
sites.
[0568] In some embodiments, the surface of a substrate is modified
to contain one or more wells, using techniques such as (but not
limited to) stamping, microetching, or molding techniques. In some
embodiments in which a substrate includes one or more wells, the
substrate can be a concavity slide or cavity slide. For example,
wells can be formed by one or more shallow depressions on the
surface of the substrate. In some embodiments, where a substrate
includes one or more wells, the wells can be formed by attaching a
cassette (e.g., a cassette containing one or more chambers) to a
surface of the substrate structure.
[0569] In some embodiments, the structures of a substrate (e.g.,
wells or features) can each bear a different capture probe.
Different capture probes attached to each structure can be
identified according to the locations of the structures in or on
the surface of the substrate. Exemplary substrates include arrays
in which separate structures are located on the substrate
including, for example, those having wells that accommodate
features.
[0570] In some embodiments where the substrate is modified to
contain one or more structures, including but not limited to,
wells, projections, ridges, features, or markings, the structures
can include physically altered sites. For example, a substrate
modified with various structures can include physical properties,
including, but not limited to, physical configurations, magnetic or
compressive forces, chemically functionalized sites, chemically
altered sites, and/or electrostatically altered sites. In some
embodiments where the substrate is modified to contain various
structures, including but not limited to wells, projections,
ridges, features, or markings, the structures are applied in a
pattern. Alternatively, the structures can be randomly
distributed.
[0571] The substrate (e.g., or a bead or a feature on an array) can
include tens to hundreds of thousands or millions of individual
oligonucleotide molecules (e.g., at least about 10,000, 50,000,
100,000, 500,000, 1,000,000, 10,000,000, 100,000,000,
1,000,000,000, or 10,000,000,000 oligonucleotide molecules).
[0572] In some embodiments, a substrate includes one or more
markings on a surface of a substrate, e.g., to provide guidance for
correlating spatial information with the characterization of the
analyte of interest. For example, a substrate can be marked with a
grid of lines (e.g., to allow the size of objects seen under
magnification to be easily estimated and/or to provide reference
areas for counting objects). In some embodiments, fiducial markers
can be included on a substrate. Such markings can be made using
techniques including, but not limited to, printing, sand-blasting,
and depositing on the surface.
[0573] In some embodiments, imaging can be performed using one or
more fiducial markers, i.e., objects placed in the field of view of
an imaging system which appear in the image produced. Fiducial
markers are typically used as a point of reference or measurement
scale. Fiducial markers can include, but are not limited to,
detectable labels such as fluorescent, radioactive,
chemiluminescent, and colorimetric labels. The use of fiducial
markers to stabilize and orient biological samples is described,
for example, in Carter et al., Applied Optics 46:421-427, 2007),
the entire contents of which are incorporated herein by reference.
In some embodiments, a fiducial marker can be a physical particle
(e.g., a nanoparticle, a microsphere, a nanosphere, a bead, or any
of the other exemplary physical particles described herein or known
in the art).
[0574] In some embodiments, a fiducial marker can be present on a
substrate to provide orientation of the biological sample. In some
embodiments, a microsphere can be coupled to a substrate to aid in
orientation of the biological sample. In some examples, a
microsphere coupled to a substrate can produce an optical signal
(e.g., fluorescence). In another example, a microsphere can be
attached to a portion (e.g., corner) of an array in a specific
pattern or design (e.g., hexagonal design) to aid in orientation of
a biological sample on an array of features on the substrate. In
some embodiments, a quantum dot can be coupled to the substrate to
aid in the orientation of the biological sample. In some examples,
a quantum dot coupled to a substrate can produce an optical
signal.
[0575] In some embodiments, a fiducial marker can be an immobilized
molecule with which a detectable signal molecule can interact to
generate a signal. For example, a marker nucleic acid can be linked
or coupled to a chemical moiety capable of fluorescing when
subjected to light of a specific wavelength (or range of
wavelengths). Such a marker nucleic acid molecule can be contacted
with an array before, contemporaneously with, or after the tissue
sample is stained to visualize or image the tissue section.
Although not required, it can be advantageous to use a marker that
can be detected using the same conditions (e.g., imaging
conditions) used to detect a labelled cDNA.
[0576] In some embodiments, fiducial markers are included to
facilitate the orientation of a tissue sample or an image thereof
in relation to an immobilized capture probes on a substrate. Any
number of methods for marking an array can be used such that a
marker is detectable only when a tissue section is imaged. For
instance, a molecule, e.g., a fluorescent molecule that generates a
signal, can be immobilized directly or indirectly on the surface of
a substrate. Markers can be provided on a substrate in a pattern
(e.g., an edge, one or more rows, one or more lines, etc.).
[0577] In some embodiments, a fiducial marker can be randomly
placed in the field of view. For example, an oligonucleotide
containing a fluorophore can be randomly printed, stamped,
synthesized, or attached to a substrate (e.g., a glass slide) at a
random position on the substrate. A tissue section can be contacted
with the substrate such that the oligonucleotide containing the
fluorophore contacts, or is in proximity to, a cell from the tissue
section or a component of the cell (e.g., an mRNA or DNA molecule).
An image of the substrate and the tissue section can be obtained,
and the position of the fluorophore within the tissue section image
can be determined (e.g., by reviewing an optical image of the
tissue section overlaid with the fluorophore detection). In some
embodiments, fiducial markers can be precisely placed in the field
of view (e.g., at known locations on a substrate). In this
instance, a fiducial marker can be stamped, attached, or
synthesized on the substrate and contacted with a biological
sample. Typically, an image of the sample and the fiducial marker
is taken, and the position of the fiducial marker on the substrate
can be confirmed by viewing the image.
[0578] In some embodiments, a fiducial marker can be an immobilized
molecule (e.g., a physical particle) attached to the substrate. For
example, a fiducial marker can be a nanoparticle, e.g., a nanorod,
a nanowire, a nanocube, a nanopyramid, or a spherical nanoparticle.
In some examples, the nanoparticle can be made of a heavy metal
(e.g., gold). In some embodiments, the nanoparticle can be made
from diamond. In some embodiments, the fiducial marker can be
visible by eye.
[0579] As noted herein, any of the fiducial markers described
herein (e.g., microspheres, beads, or any of the other physical
particles described herein) can be located at a portion (e.g.,
corner) of an array in a specific pattern or design (e.g.,
hexagonal design) to aid in orientation of a biological sample on
an array of features on the substrate. In some embodiments, the
fiducial markers located at a portion (e.g., corner) of an array
(e.g., an array on a substrate) can be patterned or designed in at
least 1, at least 2, at least 3, or at least 4 unique patterns. In
some examples, the fiducial markers located at the corners of the
array (e.g., an array on a substrate) can have four unique patterns
of fiducial markers.
[0580] In some examples, fiducial markers can surround the array.
In some embodiments the fiducial markers allow for detection of,
e.g., mirroring. In some embodiments, the fiducial markers may
completely surround the array. In some embodiments, the fiducial
markers may not completely surround the array. In some embodiments,
the fiducial markers identify the corners of the array. In some
embodiments, one or more fiducial markers identify the center of
the array. In some embodiments, the fiducial markers comprise
patterned spots, wherein the diameter of one or more patterned spot
fiducial markers is approximately 100 micrometers. The diameter of
the fiducial markers can be any useful diameter including, but not
limited to, 50 micrometers to 500 micrometers in diameter. The
fiducial markers may be arranged in such a way that the center of
one fiducial marker is between 100 micrometers and 200 micrometers
from the center of one or more other fiducial markers surrounding
the array. In some embodiments, the array with the surrounding
fiducial markers is approximately 8 mm by 8 mm. In some
embodiments, the array without the surrounding fiducial markers is
smaller than 8 mm by 50 mm.
[0581] In some embodiments, an array can be enclosed within a
frame. Put another way, the perimeter of an array can have fiducial
markers such that the array is enclosed, or substantially enclosed.
In some embodiments, the perimeter of an array can be fiducial
markers (e.g., any fiducial marker described herein). In some
embodiments, the perimeter of an array can be uniform. For example,
the fiducial markings can connect, or substantially connect,
consecutive corners of an array in such a fashion that the
non-corner portion of the array perimeter is the same on all sides
(e.g., four sides) of the array. In some embodiments, the fiducial
markers attached to the non-corner portions of the perimeter can be
pattered or designed to aid in the orientation of the biological
sample on the array. In some embodiments, the particles attached to
the non-corner portions of the perimeter can be patterned or
designed in at least 1, at least 2, at least 3, or at least 4
patterns. In some embodiments, the patterns can have at least 2, at
least 3, or at least 4 unique patterns of fiducial markings on the
non-corner portion of the array perimeter.
[0582] In some embodiments, an array can include at least two
fiducial markers (e.g., at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
12, at least 15, at least 20, at least 30, at least 40, at least
50, at least 60, at least 70, at least 80, at least 90, at least
100 fiducial markers or more (e.g., several hundred, several
thousand, or tens of thousands of fiducial markers)) in distinct
positions on the surface of a substrate. Fiducial markers can be
provided on a substrate in a pattern (e.g., an edge, one or more
rows, one or more lines, etc.).
[0583] A wide variety of different substrates can be used for the
foregoing purposes. In general, a substrate can be any suitable
support material. Exemplary substrates include, but are not limited
to, glass, modified and/or functionalized glass, hydrogels, films,
membranes, plastics (including e.g., acrylics, polystyrene,
copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polyurethanes, Teflon'', cyclic
olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica
or silica-based materials including silicon and modified silicon,
carbon, metals, inorganic glasses, optical fiber bundles, and
polymers, such as polystyrene, cyclic olefin copolymers (COCs),
cyclic olefin polymers (COPs), polypropylene, polyethylene
polycarbonate, or combinations thereof.
[0584] Among the examples of substrate materials discussed above,
polystyrene is a hydrophobic material suitable for binding
negatively charged macromolecules because it normally contains few
hydrophilic groups. For nucleic acids immobilized on glass slides,
by increasing the hydrophobicity of the glass surface the nucleic
acid immobilization can be increased. Such an enhancement can
permit a relatively more densely packed formation (e.g., provide
improved specificity and resolution).
[0585] In another example, a substrate can be a flow cell. Flow
cells can be formed of any of the foregoing materials, and can
include channels that permit reagents, solvents, features, and
analytes to pass through the flow cell. In some embodiments, a
hydrogel embedded biological sample is assembled in a flow cell
(e.g., the flow cell is utilized to introduce the hydrogel to the
biological sample). In some embodiments, a hydrogel embedded
biological sample is not assembled in a flow cell. In some
embodiments, the hydrogel embedded biological sample can then be
prepared and/or isometrically expanded as described herein.
[0586] (ii) Conductive Substrates
[0587] Conductive substrates (e.g., electrophoretic compatible
arrays) generated as described herein can be used in the spatial
detection of analytes. For example, an electrophoretic field can be
applied to facilitate migration of analytes towards the barcoded
oligonucleotides (e.g., capture probes) on the array (e.g., capture
probes immobilized on paper, capture probes immobilized in a
hydrogel film, or capture probes immobilized on a glass slide
having a conductive coating). In some embodiments, an
electrophoresis assembly can be arranged. For example, an anode and
a cathode can be arranged such that an array of capture probes
(e.g., capture probes immobilized on paper, capture probes
immobilized in a hydrogel film, or capture probes immobilized on a
glass slide having a conductive coating) and a biological sample
are positioned between the anode and the cathode. In such
embodiments, analytes in the biological sample are actively
migrated toward the capture probes on the conductive substrate and
captured. The biological sample can be prepared (e.g.,
permeabilized) according to any method described herein. In some
embodiments, after electrophoretic-assisted capture of the
analytes, the barcoded oligonucleotides (e.g., capture probes) and
captured analytes can be collected, processed, and/or analyzed
(e.g., sequenced) using any of the methods described herein.
[0588] In some embodiments, a conductive substrate can include
glass (e.g., a glass slide) that has been coated with a substance
or otherwise modified to confer conductive properties to the glass.
In some embodiments, a glass slide can be coated with a conductive
coating. In some embodiments, a conductive coating includes tin
oxide (TO) or indium tin oxide (ITO). In some embodiments, a
conductive coating includes a transparent conductive oxide (TCO).
In some embodiments, a conductive coating includes aluminum doped
zinc oxide (AZO). In some embodiments, a conductive coating
includes fluorine doped tin oxide (FTO).
[0589] In some embodiments, arrays that are spotted or printed with
oligonucleotides (e.g., capture probes, e.g., any of the variety of
capture probes described herein) can be generated on a conductive
substrate (e.g., any of the conductive substrates described
herein). For example, the arrays described herein can be compatible
with active analyte capture methods (e.g., any of the analyte
capture methods described herein, including without limitation,
electrophoretic capture methods). In some embodiments, a conductive
substrate is a porous medium. Non-limiting examples of porous media
that can be used in methods described herein that employ active
analyte capture include a nitrocellulose or nylon membrane. In some
embodiments, a porous medium that can be used in methods described
herein that employ active analyte capture includes paper. In some
embodiments, the oligonucleotides can be printed on a paper
substrate. In some embodiments, the printed oligonucleotides can
interact with the substrate (e.g., interact with fibers of the
paper). In some embodiments, printed oligonucleotides can
covalently bind the substrate (e.g., to fibers of the paper). In
some embodiments, oligonucleotides in a molecular precursor
solution can be printed on a conductive substrate (e.g., paper). In
some embodiments, a molecular precursor solution can polymerize,
thereby generating gel pads on the conductive substrate (e.g.,
paper). In some embodiments, a molecular precursor solution can be
polymerized by light (e.g., photocured). In some embodiments, gel
beads (e.g., any of the variety of gel beads described herein)
containing oligonucleotides (e.g., barcoded oligonucleotides such
as capture probes) can be printed on a conductive substrate (e.g.,
paper). In some embodiments, the printed oligonucleotides can be
covalently attached into the gel matrix.
[0590] (iii) Coatings
[0591] In some embodiments, a surface of a substrate can be coated
with a cell-permissive coating to allow adherence of live cells. A
"cell-permissive coating" is a coating that allows or helps cells
to maintain cell viability (e.g., remain viable) on the substrate.
For example, a cell-permissive coating can enhance cell attachment,
cell growth, and/or cell differentiation, e.g., a cell-permissive
coating can provide nutrients to the live cells. A cell-permissive
coating can include a biological material and/or a synthetic
material. Non-limiting examples of a cell-permissive coating
include coatings that feature one or more extracellular matrix
(ECM) components (e.g., proteoglycans and fibrous proteins such as
collagen, elastin, fibronectin and laminin), poly-lysine,
poly(L)-ornithine, and/or a biocompatible silicone (e.g.,
CYTOSOFT.RTM.). For example, a cell-permissive coating that
includes one or more extracellular matrix components can include
collagen Type I, collagen Type II, collagen Type IV, elastin,
fibronectin, laminin, and/or vitronectin. In some embodiments, the
cell-permissive coating includes a solubilized basement membrane
preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse
sarcoma (e.g., MATRIGEL.RTM.). In some embodiments, the
cell-permissive coating includes collagen. A cell-permissive
coating can be used to culture adherent cells on a
spatially-barcoded array, or to maintain cell viability of a tissue
sample or section while in contact with a spatially-barcoded
array.
[0592] In some embodiments, a substrate is coated with a surface
treatment such as poly(L)-lysine. Additionally or alternatively,
the substrate can be treated by silanation, e.g., with
epoxy-silane, amino-silane, and/or by a treatment with
polyacrylamide.
[0593] In some embodiments, a substrate is treated in order to
minimize or reduce non-specific analyte hybridization within or
between features. For example, treatment can include coating the
substrate with a hydrogel, film, and/or membrane that creates a
physical barrier to non-specific hybridization. Any suitable
hydrogel can be used. For example, hydrogel matrices prepared
according to the methods set forth in U.S. Pat. Nos. 6,391,937,
9,512,422, and 9,889,422, and U.S. Patent Application Publication
Nos. U.S. 2017/0253918 and U.S. 2018/0052081, can be used. The
entire contents of each of the foregoing documents is incorporated
herein by reference.
[0594] Treatment can include adding a functional group that is
reactive or capable of being activated such that it becomes
reactive after application of a stimulus (e.g., photoreactive
functional groups). Treatment can include treating with polymers
having one or more physical properties (e.g., mechanical,
electrical, magnetic, and/or thermal) that minimize non-specific
binding (e.g., that activate a substrate at certain locations to
allow analyte hybridization at those locations).
[0595] A "conditionally removable coating" is a coating that can be
removed from the surface of a substrate upon application of a
releasing agent. In some embodiments, a conditionally removable
coating includes a hydrogel as described herein, e.g., a hydrogel
including a polypeptide-based material. Non-limiting examples of a
hydrogel featuring a polypeptide-based material include a synthetic
peptide-based material featuring a combination of spider silk and a
trans-membrane segment of human muscle L-type calcium channel
(e.g., PEPGEL.RTM.), an amphiphilic 16 residue peptide containing a
repeating arginine-alanine-aspartate-alanine sequence
(RADARADARADARADA) (e.g., PURAMATRIX.RTM.), EAK16
(AEAEAKAKAEAEAKAK), KLD12 (KLDLKLDLKLDL), and PGMATRIX.TM..
[0596] In some embodiments, the hydrogel in the conditionally
removable coating is a stimulus-responsive hydrogel. A
stimulus-responsive hydrogel can undergo a gel-to-solution and/or
gel-to-solid transition upon application of one or more external
triggers (e.g., a releasing agent). See, e.g., Willner, Acc. Chem.
Res. 50:657-658, 2017, which is incorporated herein by reference in
its entirety. Non-limiting examples of a stimulus-responsive
hydrogel include a thermoresponsive hydrogel, a pH-responsive
hydrogel, a light-responsive hydrogel, a redox-responsive hydrogel,
an analyte-responsive hydrogel, or a combination thereof. In some
embodiments, a stimulus-responsive hydrogel can be a
multi-stimuli-responsive hydrogel.
[0597] A "releasing agent" or "external trigger" is an agent that
allows for the removal of a conditionally removable coating from a
substrate when the releasing agent is applied to the conditionally
removable coating. An external trigger or releasing agent can
include physical triggers such as thermal, magnetic, ultrasonic,
electrochemical, and/or light stimuli as well as chemical triggers
such as pH, redox reactions, supramolecular complexes, and/or
biocatalytically driven reactions. See e.g., Echeverria, et al.,
Gels (2018), 4, 54; doi:10.3390/gels4020054, which is incorporated
herein by reference in its entirety. The type of "releasing agent"
or "external trigger" can depend on the type of conditionally
removable coating. For example, a conditionally removable coating
featuring a redox-responsive hydrogel can be removed upon
application of a releasing agent that includes a reducing agent
such as dithiothreitol (DTT). As another example, a pH-responsive
hydrogel can be removed upon the application of a releasing agent
that changes the pH.
[0598] (iv) Gel Substrates
[0599] In some embodiments, a hydrogel can form a substrate. The
term "hydrogel" herein refers to a macromolecular polymer gel
including a network. Within the network, some polymer chains can
optionally be cross-linked, although cross-linking does not always
occur. In some embodiments, the substrate includes a hydrogel and
one or more second materials. In some embodiments, the hydrogel is
placed on top of one or more second materials. For example, the
hydrogel can be pre-formed and then placed on top of, underneath,
or in any other configuration with one or more second materials. In
some embodiments, hydrogel formation occurs after contacting one or
more second materials during formation of the substrate. Hydrogel
formation can also occur within a structure (e.g., wells, ridges,
features, projections, and/or markings) located on a substrate.
Where the substrate includes a gel (e.g., a hydrogel or gel
matrix), oligonucleotides within the gel can attach to the
substrate.
[0600] In some embodiments, a hydrogel can include hydrogel
subunits. The hydrogel subunits can include any convenient hydrogel
subunits, such as, but not limited to, acrylamide, bis-acrylamide,
polyacrylamide and derivatives thereof, poly(ethylene glycol) and
derivatives thereof (e.g., PEG-acrylate (PEG-DA), PEG-RGD),
gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA),
polyaliphatic polyurethanes, polyether polyurethanes, polyester
polyurethanes, polyethylene copolymers, polyamides, polyvinyl
alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl
pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and
poly(hydroxyethyl methacrylate), collagen, hyaluronic acid,
chitosan, dextran, agarose, gelatin, alginate, protein polymers,
methylcellulose, and the like, or combinations thereof.
[0601] In some embodiments, a hydrogel includes a hybrid material,
e.g., the hydrogel material includes elements of both synthetic and
natural polymers. Examples of suitable hydrogels are described, for
example, in U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and
in U.S. Patent Application Publication Nos. 2017/0253918,
2018/0052081 and 2010/0055733, the entire contents of each of which
is incorporated herein by reference.
[0602] In some embodiments, cross-linkers and/or initiators are
added to hydrogel subunits. Examples of cross-linkers include,
without limitation, bis-acrylamide and diazirine. Examples of
initiators include, without limitation, azobisisobutyronitrile
(AIBN), riboflavin, and L-arginine. Inclusion of cross-linkers
and/or initiators can lead to increased covalent bonding between
interacting biological macromolecules in later polymerization
steps.
[0603] In some embodiments, hydrogels can have a colloidal
structure, such as agarose, or a polymer mesh structure, such as
gelatin. In some embodiments, the hydrogel is a homopolymeric
hydrogel. In some embodiments, the hydrogel is a copolymeric
hydrogel. In some embodiments, the hydrogel is a multipolymer
interpenetrating polymeric hydrogel.
[0604] In some embodiments, some hydrogel subunits are polymerized
(e.g., undergo "formation") covalently or physically cross-linked,
to form a hydrogel network. For example, hydrogel subunits can be
polymerized by any method including, but not limited to, thermal
crosslinking, chemical crosslinking, physical crosslinking, ionic
crosslinking, photo-crosslinking, free-radical initiation
crosslinking, an addition reaction, condensation reaction,
water-soluble crosslinking reactions, irradiative crosslinking
(e.g., x-ray, electron beam), or combinations thereof. Techniques
such as lithographic photopolymerization can also be used to form
hydrogels.
[0605] In some embodiments, gel beads containing oligonucleotides
(e.g., barcoded oligonucleotides such as capture probes) can be
deposited on a substrate (e.g., a glass slide). In some
embodiments, gel pads can be deposited on a substrate (e.g., a
glass slide). In some embodiments, gel pads or gel beads are
deposited on a substrate in an arrayed format. In some embodiments
in which gel pads or gel beads are deposited on a substrate in an
arrayed format, a hydrogel molecular precursor solution can be
applied on top of the array (e.g., the array of gel pads or gel
beads on a glass slide). In some embodiments, a hydrogel molecular
precursor solution can be polymerized such that the deposited gel
pads or gel beads are immobilized within the polymerized hydrogel.
Any suitable method of polymerization can be used or (e.g., any of
the variety of methods described herein). In some embodiments, a
polymerized hydrogel that includes the gel pads or gel beads can be
removed (e.g., peeled) from the substrate (e.g., glass slide) such
that the gel beads or gel pads are secured in the hydrogel. In some
embodiments, a polymerized hydrogel that includes the gel pads or
gel beads is a conductive substrate (as described herein) that can
be used in accordance with any of the variety of analyte capture
methods described herein (e.g., electrophoretic migration of
analytes for capture).
[0606] Arrays can be prepared by depositing features (e.g.,
droplets, beads) on a substrate surface to produce a
spatially-barcoded array. Methods of depositing (e.g., droplet
manipulation) features are known in the art (see, U.S. Patent
Application Publication No. 2008/0132429, Rubina, A. Y., et al.,
Biotechniques. 2003 May; 34(5):1008-14, 1016-20, 1022 and
Vasiliskov et al. Biotechniques. 1999 September; 27(3):592-4,
596-8, 600 passim. each herein incorporated by reference in its
entirety). A feature can be printed or deposited at a specific
location on the substrate (e.g., inkjet printing). In some
embodiments, each feature can have a unique oligonucleotide that
functions as a spatial barcode. In some embodiments, each feature
can have capture probes for multiplexing (e.g., capturing multiple
analytes or multiple types of analytes, e.g., proteins and nucleic
acids). In some embodiments, a feature can be printed or deposited
at the specific location using an electric field. A feature can
contain a photo-crosslinkable polymer precursor and an
oligonucleotide. In some embodiments, the photo-crosslinkable
polymer precursor can be deposited into a patterned feature on the
substrate (e.g., well).
[0607] A "photo-crosslinkable polymer precursor" refers to a
compound that cross-links and/or polymerizes upon exposure to
light. In some embodiments, one or more photoinitiators may also be
included to induce and/or promote polymerization and/or
cross-linking. See, e.g., Choi et al. Biotechniques. 2019 January;
66(1):40-53, which is incorporated herein by reference in its
entirety.
[0608] Non-limiting examples of photo-crosslinkable polymer
precursors include polyethylene (glycol) diacrylate (PEGDA),
gelatin-methacryloyl (GelMA), and methacrylated hyaluronic acid
(MeHA). In some embodiments, a photo-crosslinkable polymer
precursor comprises polyethylene (glycol) diacrylate (PEGDA),
gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA),
or a combination thereof. In some embodiments, a
photo-crosslinkable polymer precursor (e.g., PAZAM) can be
covalently linked (e.g., cross-linked) to a substrate. In some
embodiments, a photo-crosslinkable polymer precursor is not
covalently linked to a substrate surface. For example, a
silane-free acrylamide can be used (See U.S. Patent Application
Publication No. 2011/0059865, herein incorporated by reference in
its entirety). The photo-crosslinkable polymer precursor in a
feature (e.g., droplet or bead) can be polymerized by any known
method. The oligonucleotides can be polymerized in a cross-linked
gel matrix (e.g., copolymerized or simultaneously polymerized). In
some embodiments, the features containing the photo-crosslinkable
polymer precursor deposited on the substrate surface can be exposed
to UV light. The UV light can induce polymerization of the
photo-crosslinkable polymer precursor and result in the features
becoming a gel matrix (e.g., gel pads) on the substrate surface
(e.g., array).
[0609] Polymerization methods for hydrogel subunits can be selected
to form hydrogels with different properties (e.g., pore volume,
swelling properties, biodegradability, conduction, transparency,
and/or permeability of the hydrogel). For example, a hydrogel can
include pores of sufficient volume to allow the passage of
macromolecules, (e.g., nucleic acids, proteins, chromatin,
metabolites, gRNA, antibodies, carbohydrates, peptides,
metabolites, and/or small molecules) to/from the sample (e.g.,
tissue section). It is known that pore volume generally decreases
with increasing concentration of hydrogel subunits and generally
increases with an increasing ratio of hydrogel subunits to
cross-linker. Therefore, a hydrogel composition can be prepared
that includes a concentration of hydrogel subunits that allows the
passage of such biological macromolecules.
[0610] In some embodiments, hydrogel formation on a substrate
occurs before, contemporaneously with, or after features (e.g.,
beads) are attached to the substrate. For example, when a capture
probe is attached (e.g., directly or indirectly) to a substrate,
hydrogel formation can be performed on the substrate already
containing the capture probes.
[0611] (d) Arrays
[0612] In many of the methods described herein, features (as
described further below) are collectively positioned on a
substrate. An "array" is a specific arrangement of a plurality of
features that is either irregular or forms a regular pattern.
Individual features in the array differ from one another based on
their relative spatial locations. In general, at least two of the
plurality of features in the array include a distinct capture probe
(e.g., any of the examples of capture probes described herein).
[0613] Arrays can be used to measure large numbers of analytes
simultaneously. In some embodiments, oligonucleotides are used, at
least in part, to create an array. For example, one or more copies
of a single species of oligonucleotide (e.g., capture probe) can
correspond to or be directly or indirectly attached to a given
feature in the array. In some embodiments, a given feature in the
array includes two or more species of oligonucleotides (e.g.,
capture probes). In some embodiments, the two or more species of
oligonucleotides (e.g., capture probes) attached directly or
indirectly to a given feature on the array include a common (e.g.,
identical) spatial barcode.
[0614] (i) Arrays for Analyte Capture
[0615] In some embodiments, an array can include a capture probe
attached directly or indirectly to the substrate. The capture probe
can include a capture domain (e.g., a nucleotide sequence) that can
specifically bind (e.g., hybridize) to a target analyte (e.g.,
mRNA, DNA, or protein) within a sample. In some embodiments, the
binding of the capture probe to the target (e.g., hybridization)
can be detected and quantified by detection of a visual signal,
e.g., a fluorophore, a heavy metal (e.g., silver ion), or
chemiluminescent label, which has been incorporated into the
target. In some embodiments, the intensity of the visual signal
correlates with the relative abundance of each analyte in the
biological sample. Since an array can contain thousands or millions
of capture probes (or more), an array can interrogate many analytes
in parallel.
[0616] In some embodiments, a substrate includes one or more
capture probes that are designed to capture analytes from one or
more organisms. In a non-limiting example, a substrate can contain
one or more capture probes designed to capture mRNA from one
organism (e.g., a human) and one or more capture probes designed to
capture DNA from a second organism (e.g., a bacterium).
[0617] The capture probes can be attached to a substrate or feature
using a variety of techniques. In some embodiments, the capture
probe is directly attached to a feature that is fixed on an array.
In some embodiments, the capture probes are immobilized to a
substrate by chemical immobilization. For example, a chemical
immobilization can take place between functional groups on the
substrate and corresponding functional elements on the capture
probes. Exemplary corresponding functional elements in the capture
probes can either be an inherent chemical group of the capture
probe, e.g., a hydroxyl group, or a functional element can be
introduced on to the capture probe. An example of a functional
group on the substrate is an amine group. In some embodiments, the
capture probe to be immobilized includes a functional amine group
or is chemically modified in order to include a functional amine
group. Means and methods for such a chemical modification are well
known in the art.
[0618] In some embodiments, the capture probe is a nucleic acid. In
some embodiments, the capture probe is immobilized on a substrate
or feature via its 5' end. In some embodiments, the capture probe
is immobilized on a substrate or feature via its 5' end and
includes from the 5' to 3' end: one or more barcodes (e.g., a
spatial barcode and/or a UMI) and one or more capture domains. In
some embodiments, the capture probe is immobilized on a substrate
or feature via its 5' end and includes from the 5' to 3' end: one
barcode (e.g., a spatial barcode or a UMI) and one capture domain.
In some embodiments, the capture probe is immobilized on a
substrate or feature via its 5' end and includes from the 5' to 3'
end: a cleavage domain, a functional domain, one or more barcodes
(e.g., a spatial barcode and/or a UMI), and a capture domain.
[0619] In some embodiments, the capture probe is immobilized on a
substrate or feature via its 5' end and includes from the 5' to 3'
end: a cleavage domain, a functional domain, one or more barcodes
(e.g., a spatial barcode and/or a UMI), a second functional domain,
and a capture domain. In some embodiments, the capture probe is
immobilized on a substrate or feature via its 5' end and includes
from the 5' to 3' end: a cleavage domain, a functional domain, a
spatial barcode, a UMI, and a capture domain. In some embodiments,
the capture probe is immobilized on a substrate or feature via its
5' end and does not include a spatial barcode. In some embodiments,
the capture probe is immobilized on a substrate or feature via its
5' end and does not include a UMI. In some embodiments, the capture
probe includes a sequence for initiating a sequencing reaction.
[0620] In some embodiments, the capture probe is immobilized on a
substrate or feature via its 3' end. In some embodiments, the
capture probe is immobilized on a substrate or feature via its 3'
end and includes from the 3' to 5' end: one or more barcodes (e.g.,
a spatial barcode and/or a UMI) and one or more capture domains. In
some embodiments, the capture probe is immobilized on a substrate
or feature via its 3' end and includes from the 3' to 5' end: one
barcode (e.g., a spatial barcode or a UMI) and one capture domain.
In some embodiments, the capture probe is immobilized on a
substrate or feature via its 3' end and includes from the 3' to 5'
end: a cleavage domain, a functional domain, one or more barcodes
(e.g., a spatial barcode and/or a UMI), and a capture domain. In
some embodiments, the capture probe is immobilized on a substrate
or feature via its 3' end and includes from the 3' to 5' end: a
cleavage domain, a functional domain, a spatial barcode, a UMI, and
a capture domain.
[0621] The localization of the functional group within the capture
probe to be immobilized can be used to control and shape the
binding behavior and/or orientation of the capture probe, e.g., the
functional group can be placed at the 5' or 3' end of the capture
probe or within the sequence of the capture probe. In some
embodiments, a capture probe can further include a substrate. A
typical substrate for a capture probe to be immobilized includes
moieties which are capable of binding to such capture probes, e.g.,
to amine-functionalized nucleic acids. Examples of such substrates
are carboxy, aldehyde, or epoxy substrates.
[0622] In some embodiments, the substrates on which capture probes
can be immobilized can be chemically activated, e.g., by the
activation of functional groups available on the substrate. The
term "activated substrate" relates to a material in which
interacting or reactive chemical functional groups are established
or enabled by chemical modification procedures. For example, a
substrate including carboxyl groups can be activated before use.
Furthermore, certain substrates contain functional groups that can
react with specific moieties already present in the capture
probes.
[0623] In some embodiments, a covalent linkage is used to directly
couple a capture probe to a substrate. In some embodiments a
capture probe is indirectly coupled to a substrate through a linker
separating the "first" nucleotide of the capture probe from the
substrate, e.g., a chemical linker. In some embodiments, a capture
probe does not bind directly to the substrate, but interacts
indirectly, for example by binding to a molecule which itself binds
directly or indirectly to the substrate. In some embodiments, the
capture probe is indirectly attached to a substrate (e.g., attached
to a substrate via a solution including a polymer).
[0624] In some embodiments where the capture probe is immobilized
on a feature of the array indirectly, e.g., via hybridization to a
surface probe capable of binding the capture probe, the capture
probe can further include an upstream sequence (5' to the sequence
that hybridizes to the nucleic acid, e.g., RNA of the tissue
sample) that is capable of hybridizing to 5' end of a surface
probe. Alone, the capture domain of the capture probe can be seen
as a capture domain oligonucleotide, which can be used in the
synthesis of the capture probe in embodiments where the capture
probe is immobilized on the array indirectly.
[0625] In some embodiments, a substrate is comprised of an inert
material or matrix (e.g., glass slides) that has been
functionalized by, for example, treating the substrate with a
material comprising reactive groups which enable immobilization of
capture probes. See, for example, WO 2017/019456, the entire
contents of which is herein incorporated by reference. Non-limiting
examples include polyacrylamide hydrogels supported on an inert
substrate (e.g., glass slide; see WO 2005/065814 and U.S. Patent
Application No. 2008/0280773, the entire contents of which is
incorporated herein by reference).
[0626] In some embodiments, functionalized biomolecules (e.g.,
capture probes) are immobilized on a functionalized substrate using
covalent methods. Methods for covalent attachment include, for
example, condensation of amines and activated carboxylic esters
(e.g., N-hydroxysuccinimide esters); condensation of amine and
aldehydes under reductive amination conditions; and cycloaddition
reactions such as the Diels-Alder [4+2] reaction, 1,3-dipolar
cycloaddition reactions, and [2+2] cycloaddition reactions. Methods
for covalent attachment also include, for example, click chemistry
reactions, including [3+2] cycloaddition reactions (e.g., Huisgen
1,3-dipolar cycloaddition reaction and copper(I)-catalyzed
azide-alkyne cycloaddition (CuAAC)); thiol-ene reactions; the
Diels-Alder reaction and inverse electron demand Diels-Alder
reaction; [4+1] cycloaddition of isonitriles and tetrazines; and
nucleophilic ring-opening of small carbocycles (e.g., epoxide
opening with amino oligonucleotides). Methods for covalent
attachment also include, for example, maleimides and thiols; and
para-nitrophenyl ester-functionalized oligonucleotides and
polylysine-functionalized substrate. Methods for covalent
attachment also include, for example, disulfide reactions; radical
reactions (see, e.g., U.S. Pat. No. 5,919,626, the entire contents
of which are herein incorporated by reference); and
hydrazide-functionalized substrate (e.g., wherein the hydrazide
functional group is directly or indirectly attached to the
substrate) and aldehyde-functionalized oligonucleotides (see, e.g.,
Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918, the
entire contents of which are herein incorporated by reference).
[0627] In some embodiments, functionalized biomolecules (e.g.,
capture probes) are immobilized on a functionalized substrate using
photochemical covalent methods. Methods for photochemical covalent
attachment include, for example, immobilization of
antraquinone-conjugated oligonucleotides (see, e.g., Koch et al.
(2000) Bioconjugate Chem. 11, 474-483, the entire contents of which
is herein incorporated by reference).
[0628] In some embodiments, functionalized biomolecules (e.g.,
capture probes) are immobilized on a functionalized substrate using
non-covalent methods. Methods for non-covalent attachment include,
for example, biotin-functionalized oligonucleotides and
streptavidin-treated substrates (see, e.g., Holmstrom et al. (1993)
Analytical Biochemistry 209, 278-283 and Gilles et al. (1999)
Nature Biotechnology 17, 365-370, the entire contents of which are
herein incorporated by reference).
[0629] In some embodiments, an oligonucleotide (e.g., a capture
probe) can be attached to a substrate or feature according to the
methods set forth in U.S. Pat. Nos. 6,737,236, 7,259,258,
7,375,234, 7,427,678, 5,610,287, 5,807,522, 5,837,860, and
5,472,881; U.S. Patent Application Publication Nos. 2008/0280773
and 2011/0059865; Shalon et al. (1996) Genome Research, 639-645;
Rogers et al. (1999) Analytical Biochemistry 266, 23-30; Stimpson
et al. (1995) Proc. Natl. Acad. Sci. USA 92, 6379-6383; Beattie et
al. (1995) Clin. Chem. 45, 700-706; Lamture et al. (1994) Nucleic
Acids Research 22, 2121-2125; Beier et al. (1999) Nucleic Acids
Research 27, 1970-1977; Joos et al. (1997) Analytical Biochemistry
247, 96-101; Nikiforov et al. (1995) Analytical Biochemistry 227,
201-209; Timofeev et al. (1996) Nucleic Acids Research 24,
3142-3148; Chrisey et al. (1996) Nucleic Acids Research 24,
3031-3039; Guo et al. (1994) Nucleic Acids Research 22, 5456-5465;
Running and Urdea (1990) BioTechniques 8, 276-279; Fahy et al.
(1993) Nucleic Acids Research 21, 1819-1826; Zhang et al. (1991)
19, 3929-3933; and Rogers et al. (1997) Gene Therapy 4, 1387-1392.
The entire contents of each of the foregoing documents is
incorporated herein by reference.
[0630] (ii) Generation of Capture Probes in an Array Format
[0631] Arrays can be prepared by a variety of methods. In some
embodiments, arrays are prepared through the synthesis (e.g., in
situ synthesis) of oligonucleotides on the array, or by jet
printing or lithography. For example, light-directed synthesis of
high-density DNA oligonucleotides can be achieved by
photolithography or solid-phase DNA synthesis. To implement
photolithographic synthesis, synthetic linkers modified with
photochemical protecting groups can be attached to a substrate and
the photochemical protecting groups can be modified using a
photolithographic mask (applied to specific areas of the substrate)
and light, thereby producing an array having localized
photo-deprotection. Many of these methods are known in the art, and
are described e.g., in Miller et al., "Basic concepts of
microarrays and potential applications in clinical microbiology."
Clinical Microbiology Reviews 22.4 (2009): 611-633;
US201314111482A; U.S. Pat. No. 9,593,365B2; US2019203275; and
WO2018091676, which are each incorporated herein by reference in
its entirety.
[0632] (1) Spotting or Printing
[0633] In some embodiments, oligonucleotides (e.g., capture probes)
can be "spotted" or "printed" onto a substrate to form an array.
The oligonucleotides can be applied by either noncontact or contact
printing. A noncontact printer can use the same method as computer
printers (e.g., bubble jet or inkjet) to expel small droplets of
probe solution onto the substrate. The specialized inkjet-like
printer can expel nanoliter to picoliter volume droplets of
oligonucleotide solution, instead of ink, onto the substrate. In
contact printing, each print pin directly applies the
oligonucleotide solution onto a specific location on the surface.
The oligonucleotides can be attached to the substrate surface by
the electrostatic interaction of the negative charge of the
phosphate backbone of the DNA with a positively charged coating of
the substrate surface or by UV-cross-linked covalent bonds between
the thymidine bases in the DNA and amine groups on the treated
substrate surface. In some embodiments, the substrate is a glass
slide. In some embodiments, the oligonucleotides (e.g., capture
probes) are attached to a substrate by a covalent bond to a
chemical matrix, e.g., epoxy-silane, amino-silane, lysine,
polyacrylamide, etc.
[0634] (2) In Situ Synthesis
[0635] Capture probes arrays can be prepared by in situ synthesis.
In some embodiments, capture probe arrays can be prepared using
photolithography. Photolithography typically relies on UV masking
and light-directed combinatorial chemical synthesis on a substrate
to selectively synthesize probes directly on the surface of an
array, one nucleotide at a time per spot, for many spots
simultaneously. In some embodiments, a substrate contains covalent
linker molecules that have a protecting group on the free end that
can be removed by light. UV light is directed through a
photolithographic mask to deprotect and activate selected sites
with hydroxyl groups that initiate coupling with incoming protected
nucleotides that attach to the activated sites. The mask is
designed in such a way that the exposure sites can be selected, and
thus specify the coordinates on the array where each nucleotide can
be attached. The process can be repeated, a new mask is applied
activating different sets of sites and coupling different bases,
allowing different oligonucleotides to be constructed at each site.
This process can be used to synthesize hundreds of thousands of
different oligonucleotides. In some embodiments, maskless array
synthesizer technology can be used. Programmable micromirrors can
create digital masks that reflect the desired pattern of UV light
to deprotect the features.
[0636] In some embodiments, the inkjet spotting process can also be
used for in situ oligonucleotide synthesis. The different
nucleotide precursors plus catalyst can be printed on the
substrate, and are then combined with coupling and deprotection
steps. This method relies on printing picoliter volumes of
nucleotides on the array surface in repeated rounds of base-by-base
printing that extends the length of the oligonucleotide probes on
the array.
[0637] (3) Electric Fields
[0638] Arrays can also be prepared by active hybridization via
electric fields to control nucleic acid transport. Negatively
charged nucleic acids can be transported to specific sites, or
features, when a positive current is applied to one or more test
sites on the array. The surface of the array can contain a binding
molecule, e.g., streptavidin, which allows for the formation of
bonds (e.g., streptavidin-biotin bonds) once electrically addressed
biotinylated probes reach their targeted location. The positive
current is then removed from the active features, and new test
sites can be activated by the targeted application of a positive
current. The process are repeated until all sites on the array are
covered.
[0639] (4) Ligation
[0640] In some embodiments, an array comprising barcoded probes can
be generated through ligation of a plurality of oligonucleotides.
In some instances, an oligonucleotide of the plurality contains a
portion of a barcode, and the complete barcode is generated upon
ligation of the plurality of oligonucleotides. For example, a first
oligonucleotide containing a first portion of a barcode can be
attached to a substrate (e.g., using any of the methods of
attaching an oligonucleotide to a substrate described herein), and
a second oligonucleotide containing a second portion of the barcode
can then be ligated onto the first oligonucleotide to generate a
complete barcode. Different combinations of the first, second and
any additional portions of a barcode can be used to increase the
diversity of the barcodes. In instances where the second
oligonucleotide is also attached to the substrate prior to
ligation, the first and/or the second oligonucleotide can be
attached to the substrate via a surface linker which contains a
cleavage site. Upon ligation, the ligated oligonucleotide can be
linearized by cleaving at the cleavage site.
[0641] To increase the diversity of the barcodes, a plurality of
second oligonucleotides comprising two or more different barcode
sequences can be ligated onto a plurality of first oligonucleotides
that comprise the same barcode sequence, thereby generating two or
more different species of barcodes. To achieve selective ligation,
a first oligonucleotide attached to a substrate containing a first
portion of a barcode can initially be protected with a protective
group (e.g., a photocleavable protective group), and the protective
group can be removed prior to ligation between the first and second
oligonucleotide. In instances where the barcoded probes on an array
are generated through ligation of two or more oligonucleotides, a
concentration gradient of the oligonucleotides can be applied to a
substrate such that different combinations of the oligonucleotides
are incorporated into a barcoded probe depending on its location on
the substrate.
[0642] Probes can be generated by directly ligating additional
oligonucleotides onto existing oligonucleotides via a splint
oligonucleotide. In some embodiments, oligonucleotides on an
existing array can include a recognition sequence that can
hybridize with a splint oligonucleotide. The recognition sequence
can be at the free 5' end or the free 3' end of an oligonucleotide
on the existing array. Recognition sequences useful for the methods
of the present disclosure may not contain restriction enzyme
recognition sites or secondary structures (e.g., hairpins), and may
include high contents of Guanine and Cytosine nucleotides.
[0643] (5) Polymerases
[0644] Barcoded probes on an array can also be generated by adding
single nucleotides to existing oligonucleotides on an array, for
example, using polymerases that function in a template-independent
manner. Single nucleotides can be added to existing
oligonucleotides in a concentration gradient, thereby generating
probes with varying length, depending on the location of the probes
on the array.
[0645] (6) Modification of Existing Capture Probes
[0646] Arrays can also be prepared by modifying existing arrays,
for example, by modifying oligonucleotides already attached to an
arrays. For instance, capture probes can be generated on an array
that already comprises oligonucleotides that are attached to the
array (or features on the array) at the 3' end and have a free 5'
end. In some instances, an array is any commercially available
array (e.g., any of the arrays available commercially as described
herein). The oligonucleotides can be in situ synthesized using any
of the in situ synthesis methods described herein. The
oligonucleotide can include a barcode and one or more constant
sequences. In some instances, the constant sequences are cleavable
sequences. The length of the oligonucleotides attached to the
substrate (e.g., array) can be less than 100 nucleotides (e.g.,
less than 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, or 10
nucleotides). To generate probes using oligonucleotides, a primer
complementary to a portion of an oligonucleotide (e.g., a constant
sequence shared by the oligonucleotides) can hybridize to the
oligonucleotide and extend the oligonucleotide (using the
oligonucleotide as a template) to form a duplex and to create a 3'
overhang. The 3' overhang can be created by template-independent
ligases (e.g., terminal deoxynucleotidyl transferase (TdT) or
poly(A) polymerase). The 3' overhang allows additional nucleotides
or oligonucleotides to be added to the duplex, for example, by an
enzyme. For instance, a capture probe can be generated by adding
additional oligonucleotides to the end of the 3' overhang (e.g.,
via splint oligonucleotide mediated ligation), where the additional
oligonucleotides can include a sequence or a portion of sequence of
one or more capture domains, or a complement thereof.
[0647] The additional oligonucleotide (e.g., a sequence or a
portion of sequence of a capture domain) can include a degenerate
sequence (e.g., any of the degenerate sequences as described
herein). The additional oligonucleotide (e.g., a sequence or a
portion of sequence of a capture domain) can include a sequence
compatible for hybridizing or ligating with an analyte of interest
in a biological sample. An analyte of interest can also be used as
a splint oligonucleotide to ligate additional oligonucleotides onto
a probe. When using a splint oligonucleotide to assist in the
ligation of additional oligonucleotides, an additional
oligonucleotide can include a sequence that is complementary to the
sequence of the splint oligonucleotide. Ligation of the
oligonucleotides can involve the use of an enzyme, such as, but not
limited to, a ligase. Non-limiting examples of suitable ligases
include Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain
9oN) DNA ligase (9oN.TM. DNA ligase, New England Biolabs),
Ampligase.TM. (available from Epicentre Biotechnologies, Madison,
Wis.), and SplintR (available from New England Biolabs, Ipswich,
Mass.). An array generated as described above is useful for spatial
analysis of a biological sample. For example, the one or more
capture domains can be used to hybridize with the poly(A) tail of
an mRNA molecule. Reverse transcription can be carried out using a
reverse transcriptase to generate cDNA complementary to the
captured mRNA. The sequence and location of the captured mRNA can
then be determined (e.g., by sequencing the capture probe that
contains the barcode as well as the complementary cDNA).
[0648] An array for spatial analysis can be generated by various
methods as described herein. In some embodiments, the array has a
plurality of capture probes comprising spatial barcodes. These
spatial barcodes and their relationship to the locations on the
array can be determined. In some cases, such information is readily
available, because the oligonucleotides are spotted, printed, or
synthesized on the array with a pre-determined pattern. In some
cases, the spatial barcode can be decoded by methods described
herein, e.g., by in situ sequencing, by various labels associated
with the spatial barcodes etc. In some embodiments, an array can be
used as a template to generate a daughter array. Thus, the spatial
barcode can be transferred to the daughter array with a known
pattern.
[0649] (iii) Features
[0650] A "feature" is an entity that acts as a support or
repository for various molecular entities used in sample analysis.
In some embodiments, some or all of the features in an array are
functionalized for analyte capture. In some embodiments,
functionalized features include one or more capture probe(s).
Examples of features include, but are not limited to, a bead, a
spot of any two- or three-dimensional geometry (e.g., an ink jet
spot, a masked spot, a square on a grid), a well, and a hydrogel
pad. In some embodiments, features are directly or indirectly
attached or fixed to a substrate. In some embodiments, the features
are not directly or indirectly attached or fixed to a substrate,
but instead, for example, are disposed within an enclosed or
partially enclosed three dimensional space (e.g., wells or
divots).
[0651] In addition to those above, a wide variety of other features
can be used to form the arrays described herein. For example, in
some embodiments, features that are formed from polymers and/or
biopolymers that are jet printed, screen printed, or
electrostatically deposited on a substrate can be used to form
arrays. Jet printing of biopolymers is described, for example, in
PCT Patent Application Publication No. WO 2014/085725. Jet printing
of polymers is described, for example, in de Gans et al., Adv
Mater. 16(3): 203-213 (2004). Methods for electrostatic deposition
of polymers and biopolymers are described, for example, in Hoyer et
al., Anal. Chem. 68(21): 3840-3844 (1996). The entire contents of
each of the foregoing references are incorporated herein by
reference.
[0652] As another example, in some embodiments, features are formed
by metallic micro- or nanoparticles. Suitable methods for
depositing such particles to form arrays are described, for
example, in Lee et al., Beilstein J. Nanotechnol. 8: 1049-1055
(2017), the entire contents of which are incorporated herein by
reference.
[0653] As a further example, in some embodiments, features are
formed by magnetic particles that are assembled on a substrate.
Examples of such particles and methods for assembling arrays are
described in Ye et al., Scientific Reports 6: 23145 (2016), the
entire contents of which are incorporated herein by reference.
[0654] As another example, in some embodiments, features correspond
to regions of a substrate in which one or more optical labels have
been incorporated, and/or which have been altered by a process such
as permanent photobleaching. Suitable substrates to implement
features in this manner include a wide variety of polymers, for
example. Methods for forming such features are described, for
example, in Moshrefzadeh et al., Appl. Phys. Lett. 62: 16 (1993),
the entire contents of which are incorporated herein by
reference.
[0655] As yet another example, in some embodiments, features can
correspond to colloidal particles assembled (e.g., via
self-assembly) to form an array. Suitable colloidal particles are
described for example in Sharma, Resonance 23(3): 263-275 (2018),
the entire contents of which are incorporated herein by
reference.
[0656] As a further example, in some embodiments, features can be
formed via spot-array photopolymerization of a monomer solution on
a substrate. In particular, two-photon and three-photon
polymerization can be used to fabricate features of relatively
small (e.g., sub-micron) dimensions. Suitable methods for preparing
features on a substrate in this manner are described for example in
Nguyen et al., Materials Today 20(6): 314-322 (2017), the entire
contents of which are incorporated herein by reference.
[0657] In some embodiments, features are directly or indirectly
attached or fixed to a substrate that is liquid permeable. In some
embodiments, features are directly or indirectly attached or fixed
to a substrate that is biocompatible. In some embodiments, features
are directly or indirectly attached or fixed to a substrate that is
a hydrogel.
[0658] FIG. 12 depicts an exemplary arrangement of barcoded
features within an array. From left to right, FIG. 12 shows (L) a
slide including six spatially-barcoded arrays, (C) an enlarged
schematic of one of the six spatially-barcoded arrays, showing a
grid of barcoded features in relation to a biological sample, and
(R) an enlarged schematic of one section of an array, showing the
specific identification of multiple features within the array
(labelled as ID578, ID579, ID560, etc.).
[0659] (1) Beads
[0660] A "bead" can be a particle. A bead can be porous,
non-porous, solid, semi-solid, and/or a combination thereof. In
some embodiments, a bead can be dissolvable, disruptable, and/or
degradable, whereas in certain embodiments, a bead is not
degradable. A semi-solid bead can be a liposomal bead. Solid beads
can include metals including, without limitation, iron oxide, gold,
and silver. In some embodiments, the bead can be a silica bead. In
some embodiments, the bead can be rigid. In some embodiments, the
bead can be flexible and/or compressible.
[0661] The bead can be a macromolecule. The bead can be formed of
nucleic acid molecules bound together. The bead can be formed via
covalent or non-covalent assembly of molecules (e.g.,
macromolecules), such as monomers or polymers. Polymers or monomers
can be natural or synthetic. Polymers or monomers can be or
include, for example, nucleic acid molecules (e.g., DNA or
RNA).
[0662] A bead can be rigid, or flexible and/or compressible. A bead
can include a coating including one or more polymers. Such a
coating can be disruptable or dissolvable. In some embodiments, a
bead includes a spectral or optical label (e.g., dye) attached
directly or indirectly (e.g., through a linker) to the bead. For
example, a bead can be prepared as a colored preparation (e.g., a
bead exhibiting a distinct color within the visible spectrum) that
can change color (e.g., colorimetric beads) upon application of a
desired stimulus (e.g., heat and/or chemical reaction) to form
differently colored beads (e.g., opaque and/or clear beads).
[0663] A bead can include natural and/or synthetic materials. For
example, a bead can include a natural polymer, a synthetic polymer
or both natural and synthetic polymers. Examples of natural
polymers include, without limitation, proteins, sugars such as
deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose,
amylopectin), enzymes, polysaccharides, silks,
polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,
ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan
gum, corn sugar gum, guar gum, gum karaya, agarose, alginic acid,
alginate, or natural polymers thereof. Examples of synthetic
polymers include, without limitation, acrylics, nylons, silicones,
spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate,
polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes,
polylactic acid, silica, polystyrene, polyacrylonitrile,
polybutadiene, poly carbonate, polyethylene, polyethylene
terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide),
poly(ethylene terephthalate), polyethylene, polyisobutylene,
poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,
polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl
acetate), poly(vinyl alcohol), poly(vinyl chloride),
poly(vinylidene dichloride), poly(vinylidene difluoride),
poly(vinyl fluoride) and/or combinations (e.g., co-polymers)
thereof. Beads can also be formed from materials other than
polymers, including for example, lipids, micelles, ceramics,
glass-ceramics, material composites, metals, and/or other inorganic
materials.
[0664] In some embodiments, a bead is a degradable bead. A
degradable bead can include one or more species (e.g., disulfide
linkers, primers, other oligonucleotides, etc.) with a labile bond
such that, when the bead/species is exposed to the appropriate
stimuli, the labile bond is broken and the bead degrades. The
labile bond can be a chemical bond (e.g., covalent bond, ionic
bond) or can be another type of physical interaction (e.g., van der
Waals interactions, dipole-dipole interactions, etc.). In some
embodiments, a cross-linker used to generate a bead can include a
labile bond. Upon exposure to the appropriate conditions, the
labile bond can be broken and the bead degraded. For example, upon
exposure of a polyacrylamide gel bead including cystamine
cross-linkers to a reducing agent, the disulfide bonds of the
cystamine can be broken and the bead degraded.
[0665] Degradation can refer to the disassociation of a bound or
entrained species (e.g., disulfide linkers, primers, other
oligonucleotides, etc.) from a bead, both with and without
structurally degrading the physical bead itself. For example,
entrained species can be released from beads through osmotic
pressure differences due to, for example, changing chemical
environments. By way of example, alteration of bead pore volumes
due to osmotic pressure differences can generally occur without
structural degradation of the bead itself. In some embodiments, an
increase in pore volume due to osmotic swelling of a bead can
permit the release of entrained species within the bead. In some
embodiments, osmotic shrinking of a bead can cause a bead to better
retain an entrained species due to pore volume contraction.
[0666] Any suitable agent that can degrade beads can be used. In
some embodiments, changes in temperature or pH can be used to
degrade thermo-sensitive or pH-sensitive bonds within beads. In
some embodiments, chemical degrading agents can be used to degrade
chemical bonds within beads by oxidation, reduction or other
chemical changes. For example, a chemical degrading agent can be a
reducing agent, such as DTT, where DTT can degrade the disulfide
bonds formed between a cross-linker and gel precursors, thus
degrading the bead. In some embodiments, a reducing agent can be
added to degrade the bead, which can cause the bead to release its
contents. Examples of reducing agents can include, without
limitation, dithiothreitol (DTT), .beta.-mercaptoethanol,
(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA),
tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.
[0667] Any of a variety of chemical agents can be used to trigger
the degradation of beads. Examples of chemical agents include, but
are not limited to, pH-mediated changes to the integrity of a
component within the bead, degradation of a component of a bead via
cleavage of cross-linked bonds, and depolymerization of a component
of a bead.
[0668] In some embodiments, a bead can be formed from materials
that include degradable chemical cross-linkers, such as
N,N'-bis-(acryloyl)cystamine (BAC) or cystamine. Degradation of
such degradable cross-linkers can be accomplished through any
variety of mechanisms. In some examples, a bead can be contacted
with a chemical degrading agent that can induce oxidation,
reduction or other chemical changes. For example, a chemical
degrading agent can be a reducing agent, such as dithiothreitol
(DTT). Additional examples of reducing agents can include
.beta.-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane
(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP),
or combinations thereof.
[0669] In some embodiments, exposure to an aqueous solution, such
as water, can trigger hydrolytic degradation, and thus degradation
of the bead. Beads can also be induced to release their contents
upon the application of a thermal stimulus. A change in temperature
can cause a variety of changes to a bead. For example, heat can
cause a solid bead to liquefy. A change in heat can cause melting
of a bead such that a portion of the bead degrades. In some
embodiments, heat can increase the internal pressure of the bead
components such that the bead ruptures or explodes. Heat can also
act upon heat-sensitive polymers used as materials to construct
beads.
[0670] Where degradable beads are used, it can be beneficial to
avoid exposing such beads to the stimulus or stimuli that cause
such degradation prior to a given time, in order to, for example,
avoid premature bead degradation and issues that arise from such
degradation, including for example poor flow characteristics and
aggregation. By way of example, where beads include reducible
cross-linking groups, such as disulfide groups, it will be
desirable to avoid contacting such beads with reducing agents,
e.g., DTT or other disulfide cleaving reagents. In such
embodiments, treatment of the beads described herein will, in some
embodiments be provided free of reducing agents, such as DTT.
Because reducing agents are often provided in commercial enzyme
preparations, it can be desirable to provide reducing agent free
(or DTT free) enzyme preparations in treating the beads described
herein. Examples of such enzymes include, e.g., polymerase enzyme
preparations, reverse transcriptase enzyme preparations, ligase
enzyme preparations, as well as many other enzyme preparations that
can be used to treat the beads described herein. The terms
"reducing agent free" or "DTT free" preparations refer to a
preparation having less than about 1/10th, less than about 1/50th,
or less than about 1/100th of the lower ranges for such materials
used in degrading the beads. For example, for DTT, the reducing
agent free preparation can have less than about 0.01 millimolar
(mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about
0.0001 mM DTT. In some embodiments, the amount of DTT can be
undetectable.
[0671] A degradable bead can be useful to more quickly release an
attached capture probe (e.g., a nucleic acid molecule, a spatial
barcode sequence, and/or a primer) from the bead when the
appropriate stimulus is applied to the bead as compared to a bead
that does not degrade. For example, for a species bound to an inner
surface of a porous bead or in the case of an encapsulated species,
the species can have greater mobility and accessibility to other
species in solution upon degradation of the bead. In some
embodiments, a species can also be attached to a degradable bead
via a degradable linker (e.g., disulfide linker). The degradable
linker can respond to the same stimuli as the degradable bead or
the two degradable species can respond to different stimuli. For
example, a capture probe having one or more spatial barcodes can be
attached, via a disulfide bond, to a polyacrylamide bead including
cystamine. Upon exposure of the spatially-barcoded bead to a
reducing agent, the bead degrades and the capture probe having the
one or more spatial barcode sequences is released upon breakage of
both the disulfide linkage between the capture probe and the bead
and the disulfide linkages of the cystamine in the bead.
[0672] The addition of multiple types of labile bonds to a bead can
result in the generation of a bead capable of responding to varied
stimuli. Each type of labile bond can be sensitive to an associated
stimulus (e.g., chemical stimulus, light, temperature, pH, enzymes,
etc.) such that release of reagents attached to a bead via each
labile bond can be controlled by the application of the appropriate
stimulus. Some non-limiting examples of labile bonds that can be
coupled to a precursor or bead include an ester linkage (e.g.,
cleavable with an acid, a base, or hydroxylamine), a vicinal diol
linkage (e.g., cleavable via sodium periodate), a Diels-Alder
linkage (e.g., cleavable via heat), a sulfone linkage (e.g.,
cleavable via a base), a silyl ether linkage (e.g., cleavable via
an acid), a glycosidic linkage (e.g., cleavable via an amylase), a
peptide linkage (e.g., cleavable via a protease), or a
phosphodiester linkage (e.g., cleavable via a nuclease (e.g.,
DNAase)). A bond can be cleavable via other nucleic acid molecule
targeting enzymes, such as restriction enzymes (e.g., restriction
endonucleases). Such functionality can be useful in controlled
release of reagents from a bead. In some embodiments, another
reagent including a labile bond can be linked to a bead after gel
bead formation via, for example, an activated functional group of
the bead as described above. In some embodiments, a gel bead
including a labile bond is reversible. In some embodiments, a gel
bead with a reversible labile bond is used to capture one or more
regions of interest of a biological sample. For example, without
limitation, a bead including a thermolabile bond can be heated by a
light source (e.g., a laser) that causes a change in the gel bead
that facilitates capture of a biological sample in contact with the
gel bead. Capture probes having one or more spatial barcodes that
are releasably, cleavably, or reversibly attached to the beads
described herein include capture probes that are released or
releasable through cleavage of a linkage between the capture probe
and the bead, or that are released through degradation of the
underlying bead itself, allowing the capture probes having the one
or more spatial barcodes to be accessed or become accessible by
other reagents, or both.
[0673] Beads can have different physical properties. Physical
properties of beads can be used to characterize the beads.
Non-limiting examples of physical properties of beads that can
differ include volume, shape, circularity, density, symmetry, and
hardness. For example, beads can be of different volumes. Beads of
different diameters can be obtained by using microfluidic channel
networks configured to provide beads of a specific volume (e.g.,
based on channel sizes, flow rates, etc.). In some embodiments,
beads have different hardness values that can be obtained by
varying the concentration of polymer used to generate the beads. In
some embodiments, a spatial barcode attached to a bead can be made
optically detectable using a physical property of the capture
probe. For example, a nucleic acid origami, such as a
deoxyribonucleic acid (DNA) origami, can be used to generate an
optically detectable spatial barcode. To do so, a nucleic acid
molecule, or a plurality of nucleic acid molecules, can be folded
to create two- and/or three-dimensional geometric shapes. The
different geometric shapes can be optically detected.
[0674] In some embodiments, special types of nanoparticles with
more than one distinct physical property can be used to make the
beads physically distinguishable. For example, Janus particles with
both hydrophilic and hydrophobic surfaces can be used to provide
unique physical properties.
[0675] A bead can generally be of any suitable shape. Examples of
bead shapes include, but are not limited to, spherical,
non-spherical, oval, oblong, amorphous, circular, cylindrical,
cuboidal, hexagonal, and variations thereof. In some embodiments,
non-spherical (e.g., hexagonal, cuboidal, shaped beads can assemble
more closely (e.g., tighter) than spherical shaped beads. In some
embodiments, beads can self-assemble into a monolayer. A cross
section (e.g., a first cross-section) can correspond to a diameter
or maximum cross-sectional dimension of the bead. In some
embodiments, the bead can be approximately spherical. In such
embodiments, the first cross-section can correspond to the diameter
of the bead. In some embodiments, the bead can be approximately
cylindrical. In such embodiments, the first cross-section can
correspond to a diameter, length, or width along the approximately
cylindrical bead.
[0676] Beads can be of uniform size or heterogeneous size.
"Polydispersity" generally refers to heterogeneity of sizes of
molecules or particles. The polydispersity index (PDI) of a bead
can be calculated using the equation PDI=Mw/Mn, where Mw is the
weight-average molar mass and Mn is the number-average molar mass.
In certain embodiments, beads can be provided as a population or
plurality of beads having a relatively monodisperse size
distribution. Where it can be desirable to provide relatively
consistent amounts of reagents, maintaining relatively consistent
bead characteristics, such as size, can contribute to the overall
consistency.
[0677] In some embodiments, the beads provided herein can have size
distributions that have a coefficient of variation in their
cross-sectional dimensions of less than 50%, less than 40%, less
than 30%, less than 20%, less than 15%, less than 10%, less than
5%, or lower. In some embodiments, a plurality of beads provided
herein has a polydispersity index of less than 50%, less than 45%,
less than 40%, less than 35%, less than 30%, less than 25%, less
than 20%, less than 15%, less than 10%, less than 5%, or lower.
[0678] In some embodiments, the bead can have a diameter or maximum
dimension no larger than 100 .mu.m (e.g., no larger than 95 .mu.m,
90 .mu.m, 85 .mu.m, 80 .mu.m, 75 .mu.m, 70 .mu.m, 65 .mu.m, 60
.mu.m, 55 .mu.m, 50 .mu.m, 45 .mu.m, 40 .mu.m, 35 .mu.m, 30 .mu.m,
25 .mu.m, 20 .mu.m, 15 .mu.m, 14 .mu.m, 13 .mu.m, 12 .mu.m, 11
.mu.m, 10 .mu.m, 9 .mu.m, 8 .mu.m, 7 .mu.m, 6 .mu.m, 5 .mu.m, 4
.mu.m, 3 .mu.m, 2 .mu.m, or 1 .mu.m.)
[0679] In some embodiments, a plurality of beads has an average
diameter no larger than 100 .mu.m. In some embodiments, a plurality
of beads has an average diameter or maximum dimension no larger
than 95 .mu.m, 90 .mu.m, 85 .mu.m, 80 .mu.m, 75 .mu.m, 70 .mu.m, 65
.mu.m, 60 .mu.m, 55 .mu.m, 50 .mu.m, 45 .mu.m, 40 .mu.m, 35 .mu.m,
30 .mu.m, 25 .mu.m, 20 .mu.m, 15 .mu.m, 14 .mu.m, 13 .mu.m, 12
.mu.m, 11 .mu.m, 10 .mu.m, 9 .mu.m, 8 .mu.m, 7 .mu.m, 6 .mu.m, 5
.mu.m, 4 .mu.m, 3 .mu.m, 2 .mu.m, or 1 .mu.m.
[0680] In some embodiments, the volume of the bead can be at least
about 1 .mu.m.sup.3, e.g., at least 1 .mu.m.sup.3, 2 .mu.m.sup.3, 3
.mu.m.sup.3, 4 .mu.m.sup.3, 5 .mu.m.sup.3, 6 .mu.m.sup.3, 7
.mu.m.sup.3, 8 .mu.m.sup.3, 9 .mu.m.sup.3, 10 .mu.m.sup.3, 12
.mu.m.sup.3, 14 .mu.m.sup.3, 16 .mu.m.sup.3, 18 .mu.m.sup.3, 20
.mu.m.sup.3, 25 .mu.m.sup.3, 30 .mu.m.sup.3, 35 .mu.m.sup.3, 40
.mu.m.sup.3, 45 .mu.m.sup.3, 50 .mu.m.sup.3, 55 .mu.m.sup.3, 60
.mu.m.sup.3, 65 .mu.m.sup.3, 70 .mu.m.sup.3, 75 .mu.m.sup.3, 80
.mu.m.sup.3, 85 .mu.m.sup.3, 90 .mu.m.sup.3, 95 .mu.m.sup.3, 100
.mu.m.sup.3, 125 .mu.m.sup.3, 150 .mu.m.sup.3, 175 .mu.m.sup.3, 200
.mu.m.sup.3, 250 .mu.m.sup.3, 300 .mu.m.sup.3, 350 .mu.m.sup.3, 400
.mu.m.sup.3, 450 .mu.m.sup.3, .mu.m.sup.3, 500 .mu.m.sup.3, 550
.mu.m.sup.3, 600 .mu.m.sup.3, 650 .mu.m.sup.3, 700 .mu.m.sup.3, 750
.mu.m.sup.3, 800 .mu.m.sup.3, 850 .mu.m.sup.3, 900 .mu.m.sup.3, 950
.mu.m.sup.3, 1000 .mu.m.sup.3, 1200 .mu.m.sup.3, 1400 .mu.m.sup.3,
1600 .mu.m.sup.3, 1800 .mu.m.sup.3, 2000 .mu.m.sup.3, 2200
.mu.m.sup.3, 2400 .mu.m.sup.3, 2600 .mu.m.sup.3, 2800 .mu.m.sup.3,
3000 .mu.m.sup.3, or greater.
[0681] In some embodiments, the bead can have a volume of between
about 1 .mu.m.sup.3 and 100 .mu.m.sup.3, such as between about 1
.mu.m.sup.3 and 10 .mu.m.sup.3, between about 10 .mu.m.sup.3 and 50
.mu.m.sup.3, or between about 50 .mu.m.sup.3 and 100 .mu.m.sup.3.
In some embodiments, the bead can include a volume of between about
100 .mu.m.sup.3 and 1000 .mu.m.sup.3, such as between about 100
.mu.m.sup.3 and 500 .mu.m.sup.3 or between about 500 .mu.m.sup.3
and 1000 .mu.m.sup.3. In some embodiments, the bead can include a
volume between about 1000 .mu.m.sup.3 and 3000 .mu.m.sup.3, such as
between about 1000 .mu.m.sup.3 and 2000 .mu.m.sup.3 or between
about 2000 .mu.m.sup.3 and 3000 .mu.m.sup.3. In some embodiments,
the bead can include a volume between about 1 .mu.m.sup.3 and 3000
.mu.m.sup.3, such as between about 1 .mu.m.sup.3 and 2000
.mu.m.sup.3, between about 1 .mu.m.sup.3 and 1000 .mu.m.sup.3,
between about 1 .mu.m.sup.3 and 500 .mu.m.sup.3, or between about 1
.mu.m.sup.3 and 250 .mu.m.sup.3.
[0682] The bead can include one or more cross-sections that can be
the same or different. In some embodiments, the bead can have a
first cross-section that is different from a second cross-section.
The bead can have a first cross-section that is at least about
0.0001 micrometer, 0.001 micrometer, 0.01 micrometer, 0.1
micrometer, or 1 micrometer. In some embodiments, the bead can
include a cross-section (e.g., a first cross-section) of at least
about 1 micrometer (.mu.m), 2 .mu.m, 3 .mu.m, 4 .mu.m, 5 .mu.m, 6
.mu.m, 7 .mu.m, 8 .mu.m, 9 .mu.m, 10 .mu.m, 11 .mu.m, 12 .mu.m, 13
.mu.m, 14 .mu.m, 15 .mu.m, 16 .mu.m, 17 .mu.m, 18 .mu.m, 19 .mu.m,
20 .mu.m, 25 .mu.m, 30 .mu.m, 35 .mu.m, 40 .mu.m, 45 .mu.m, 50
.mu.m, 55 .mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m,
85 .mu.m, 90 .mu.m, 100 .mu.m, 120 .mu.m, 140 .mu.m, 160 .mu.m, 180
.mu.m, 200 .mu.m, 250 .mu.m, 300 .mu.m, 350 .mu.m, 400 .mu.m, 450
.mu.m, 500 .mu.m, 550 .mu.m, 600 .mu.m, 650 .mu.m, 700 .mu.m, 750
.mu.m, 800 .mu.m, 850 .mu.m, 900 .mu.m, 950 .mu.m, 1 millimeter
(mm), or greater. In some embodiments, the bead can include a
cross-section (e.g., a first cross-section) of between about 1
.mu.m and 500 .mu.m, such as between about 1 .mu.m and 100 .mu.m,
between about 100 .mu.m and 200 .mu.m, between about 200 .mu.m and
300 .mu.m, between about 300 .mu.m and 400 .mu.m, or between about
400 .mu.m and 500 .mu.m. For example, the bead can include a
cross-section (e.g., a first cross-section) of between about 1
.mu.m and 100 .mu.m. In some embodiments, the bead can have a
second cross-section that is at least about 1 .mu.m. For example,
the bead can include a second cross-section of at least about 1
micrometer (.mu.m), 2 .mu.m, 3 .mu.m, 4 .mu.m, 5 .mu.m, 6 .mu.m, 7
.mu.m, 8 .mu.m, 9 .mu.m, 10 .mu.m, 11 .mu.m, 12 .mu.m, 13 .mu.m, 14
.mu.m, 15 .mu.m, 16 .mu.m, 17 .mu.m, 18 .mu.m, 19 .mu.m, 20 .mu.m,
25 .mu.m, 30 .mu.m, 35 .mu.m, 40 .mu.m, 45 .mu.m, 50 .mu.m, 55
.mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m, 85 .mu.m,
90 .mu.m, 100 .mu.m, 120 .mu.m, 140 .mu.m, 160 .mu.m, 180 .mu.m,
200 .mu.m, 250 .mu.m, 300 .mu.m, 350 .mu.m, 400 .mu.m, 450 .mu.m,
500 .mu.m, 550 .mu.m, 600 .mu.m, 650 .mu.m, 700 .mu.m, 750 .mu.m,
800 .mu.m, 850 .mu.m, 900 .mu.m, 950 .mu.m, 1 millimeter (mm), or
greater. In some embodiments, the bead can include a second
cross-section of between about 1 .mu.m and 500 .mu.m, such as
between about 1 .mu.m and 100 .mu.m, between about 100 .mu.m and
200 .mu.m, between about 200 .mu.m and 300 .mu.m, between about 300
.mu.m and 400 .mu.m, or between about 400 .mu.m and 500 .mu.m. For
example, the bead can include a second cross-section of between
about 1 .mu.m and 100 .mu.m.
[0683] In some embodiments, beads can be of a nanometer scale
(e.g., beads can have a diameter or maximum cross-sectional
dimension of about 100 nanometers (nm) to about 900 nanometers (nm)
(e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or
less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or
less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or
less, 250 nm or less, 200 nm or less, 150 nm or less). A plurality
of beads can have an average diameter or average maximum
cross-sectional dimension of about 100 nanometers (nm) to about 900
nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm or
less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or
less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or
less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or
less). In some embodiments, a bead has a diameter or volume that is
about the diameter of a single cell (e.g., a single cell under
evaluation).
[0684] In some embodiments, a bead is able to identify multiple
analytes (e.g., nucleic acids, proteins, chromatin, metabolites,
drugs, gRNA, and lipids) from a single cell. In some embodiments, a
bead is able to identify a single analyte from a single cell (e.g.,
mRNA).
[0685] A bead can have a tunable pore volume. The pore volume can
be chosen to, for instance, retain denatured nucleic acids. The
pore volume can be chosen to maintain diffusive permeability to
exogenous chemicals such as sodium hydroxide (NaOH) and/or
endogenous chemicals such as inhibitors. A bead can be formed of a
biocompatible and/or biochemically compatible material, and/or a
material that maintains or enhances cell viability. A bead can be
formed from a material that can be depolymerized thermally,
chemically, enzymatically, and/or optically.
[0686] In some embodiments, beads can be non-covalently loaded with
one or more reagents. The beads can be non-covalently loaded by,
for instance, subjecting the beads to conditions sufficient to
swell the beads, allowing sufficient time for the reagents to
diffuse into the interiors of the beads, and subjecting the beads
to conditions sufficient to de-swell the beads. Swelling of the
beads can be accomplished, for instance, by placing the beads in a
thermodynamically favorable solvent, subjecting the beads to a
higher or lower temperature, subjecting the beads to a higher or
lower ion concentration, and/or subjecting the beads to an electric
field.
[0687] The swelling of the beads can be accomplished by various
swelling methods. In some embodiments, swelling is reversible
(e.g., by subjecting beads to conditions that promote de-swelling).
In some embodiments, the de-swelling of the beads is accomplished,
for instance, by transferring the beads in a thermodynamically
unfavorable solvent, subjecting the beads to lower or higher
temperatures, subjecting the beads to a lower or higher ion
concentration, and/or adding or removing an electric field. The
de-swelling of the beads can be accomplished by various de-swelling
methods. In some embodiments, de-swelling is reversible (e.g.,
subject beads to conditions that promote swelling). In some
embodiments, the de-swelling of beads can include transferring the
beads to cause pores in the bead to shrink. The shrinking can then
hinder reagents within the beads from diffusing out of the
interiors of the beads. The hindrance created can be due to steric
interactions between the reagents and the interiors of the beads.
The transfer can be accomplished microfluidically. For instance,
the transfer can be achieved by moving the beads from one
co-flowing solvent stream to a different co-flowing solvent stream.
The swellability and/or pore volume of the beads can be adjusted by
changing the polymer composition of the bead.
[0688] A bead can include a polymer that is responsive to
temperature so that when the bead is heated or cooled, the
characteristics or dimensions of the bead can change. For example,
a polymer can include poly(N-isopropylacrylamide). A gel bead can
include poly(N-isopropylacrylamide) and when heated the gel bead
can decrease in one or more dimensions (e.g., a cross-sectional
diameter, multiple cross-sectional diameters). A temperature
sufficient for changing one or more characteristics of the gel bead
can be, for example, at least about 0 degrees Celsius (.degree.
C.), 1.degree. C., 2.degree. C., 3.degree. C., 4.degree. C.,
5.degree. C., 10.degree. C., or higher. For example, the
temperature can be about 4.degree. C. In some embodiments, a
temperature sufficient for changing one or more characteristics of
the gel bead can be, for example, at least about 25.degree. C.,
30.degree. C., 35.degree. C., 37.degree. C., 40.degree. C.,
45.degree. C., 50.degree. C., or higher. For example, the
temperature can be about 37.degree. C.
[0689] Functionalization of beads for attachment of capture probes
can be achieved through a wide range of different approaches,
including, without limitation, activation of chemical groups within
a polymer, incorporation of active or activatable functional groups
in the polymer structure, or attachment at the pre-polymer or
monomer stage in bead production. The bead can be functionalized to
bind to targeted analytes, such as nucleic acids, proteins,
carbohydrates, lipids, metabolites, peptides, or other
analytes.
[0690] In some embodiments, a bead can contain molecular precursors
(e.g., monomers or polymers), which can form a polymer network via
polymerization of the molecular precursors. In some embodiments, a
precursor can be an already polymerized species capable of
undergoing further polymerization via, for example, a chemical
cross-linkage. In some embodiments, a precursor can include one or
more of an acrylamide or a methacrylamide monomer, oligomer, or
polymer. In some embodiments, the bead can include prepolymers,
which are oligomers capable of further polymerization. For example,
polyurethane beads can be prepared using prepolymers. In some
embodiments, a bead can contain individual polymers that can be
further polymerized together (e.g., to form a co-polymer). In some
embodiments, a bead can be generated via polymerization of
different precursors, such that they include mixed polymers,
co-polymers, and/or block co-polymers. In some embodiments, a bead
can include covalent or ionic bonds between polymeric precursors
(e.g., monomers, oligomers, and linear polymers), nucleic acid
molecules (e.g., oligonucleotides), primers, and other entities. In
some embodiments, covalent bonds can be carbon-carbon bonds or
thioether bonds.
[0691] Cross-linking of polymers can be permanent or reversible,
depending upon the particular cross-linker used. Reversible
cross-linking can allow the polymer to linearize or dissociate
under appropriate conditions. In some embodiments, reversible
cross-linking can also allow for reversible attachment of a
material bound to the surface of a bead. In some embodiments, a
cross-linker can form a disulfide linkage. In some embodiments, a
chemical cross-linker forming a disulfide linkage can be cystamine
or a modified cystamine.
[0692] For example, where the polymer precursor material includes a
linear polymer material, such as a linear polyacrylamide, PEG, or
other linear polymeric material, the activation agent can include a
cross-linking agent, or a chemical that activates a cross-linking
agent within formed droplets. Likewise, for polymer precursors that
include polymerizable monomers, the activation agent can include a
polymerization initiator. For example, in certain embodiments,
where the polymer precursor includes a mixture of acrylamide
monomer with a N,N'-bis-(acryloyl)cystamine (BAC) comonomer, an
agent such as tetraethylmethylenediamine (TEMED) can be provided,
which can initiate the copolymerization of the acrylamide and BAC
into a cross-linked polymer network, or other conditions sufficient
to polymerize or gel the precursors. The conditions sufficient to
polymerize or gel the precursors can include exposure to heating,
cooling, electromagnetic radiation, and/or light.
[0693] Following polymerization or gelling, a polymer or gel can be
formed. The polymer or gel can be diffusively permeable to chemical
or biochemical reagents. The polymer or gel can be diffusively
impermeable to macromolecular constituents. The polymer or gel can
include one or more of disulfide cross-linked polyacrylamide,
agarose, alginate, polyvinyl alcohol, polyethylene glycol
(PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne,
other acrylates, chitosan, hyaluronic acid, collagen, fibrin,
gelatin, or elastin. The polymer or gel can include any other
polymer or gel.
[0694] In some embodiments, disulfide linkages can be formed
between molecular precursor units (e.g., monomers, oligomers, or
linear polymers) or precursors incorporated into a bead and nucleic
acid molecules (e.g., oligonucleotides, capture probes). Cystamine
(including modified cystamines), for example, is an organic agent
including a disulfide bond that can be used as a cross-linker agent
between individual monomeric or polymeric precursors of a bead.
Polyacrylamide can be polymerized in the presence of cystamine or a
species including cystamine (e.g., a modified cystamine) to
generate polyacrylamide gel beads including disulfide linkages
(e.g., chemically degradable beads including chemically-reducible
cross-linkers). The disulfide linkages can permit the bead to be
degraded (or dissolved) upon exposure of the bead to a reducing
agent.
[0695] In some embodiments, chitosan, a linear polysaccharide
polymer, can be cross-linked with glutaraldehyde via hydrophilic
chains to form a bead. Crosslinking of chitosan polymers can be
achieved by chemical reactions that are initiated by heat,
pressure, change in pH, and/or radiation.
[0696] In some embodiments, a bead can include an acrydite moiety,
which in certain aspects can be used to attach one or more capture
probes to the bead. In some embodiments, an acrydite moiety can
refer to an acrydite analogue generated from the reaction of
acrydite with one or more species (e.g., disulfide linkers,
primers, other oligonucleotides, etc.), such as, without
limitation, the reaction of acrydite with other monomers and
cross-linkers during a polymerization reaction. Acrydite moieties
can be modified to form chemical bonds with a species to be
attached, such as a capture probe. Acrydite moieties can be
modified with thiol groups capable of forming a disulfide bond or
can be modified with groups already including a disulfide bond. The
thiol or disulfide (via disulfide exchange) can be used as an
anchor point for a species to be attached or another part of the
acrydite moiety can be used for attachment. In some embodiments,
attachment can be reversible, such that when the disulfide bond is
broken (e.g., in the presence of a reducing agent), the attached
species is released from the bead. In some embodiments, an acrydite
moiety can include a reactive hydroxyl group that can be used for
attachment of species.
[0697] In some embodiments, precursors (e.g., monomers or
cross-linkers) that are polymerized to form a bead can include
acrydite moieties, such that when a bead is generated, the bead
also includes acrydite moieties. The acrydite moieties can be
attached to a nucleic acid molecule (e.g., an oligonucleotide),
which can include a priming sequence (e.g., a primer for amplifying
target nucleic acids, random primer, primer sequence for messenger
RNA) and/or one or more capture probes. The one or more capture
probes can include sequences that are the same for all capture
probes coupled to a given bead and/or sequences that are different
across all capture probes coupled to the given bead. The capture
probe can be incorporated into the bead. In some embodiments, the
capture probe can be incorporated or attached to the bead such that
the capture probe retains a free 3' end. In some embodiments, the
capture probe can be incorporated or attached to the bead such that
the capture probe retains a free 5' end. In some embodiments, beads
can be functionalized such that each bead contains a plurality of
different capture probes. For example, a bead can include a
plurality of capture probes e.g., Capture Probe 1, Capture Probe 2,
and Capture Probe 3, and each of Capture Probes 1, Capture Probes
2, and Capture Probes 3 contain a distinct capture domain (e.g.,
capture domain of Capture Probe 1 includes a poly(dT) capture
domain, capture domain of Capture Probe 2 includes a gene-specific
capture domain, and capture domain of Capture Probe 3 includes a
CRISPR-specific capture domain). By functionalizing beads to
contain a plurality of different capture domains per bead, the
level of multiplex capability for analyte detection can be
improved.
[0698] In some embodiments, precursors (e.g., monomers or
cross-linkers) that are polymerized to form a bead can include a
functional group that is reactive or capable of being activated
such that when it becomes reactive it can be polymerized with other
precursors to generate beads including the activated or activatable
functional group. The functional group can then be used to attach
additional species (e.g., disulfide linkers, primers, other
oligonucleotides, etc.) to the beads. For example, some precursors
including a carboxylic acid (COOH) group can co-polymerize with
other precursors to form a bead that also includes a COOH
functional group. In some embodiments, acrylic acid (a species
including free COOH groups), acrylamide, and bis(acryloyl)cystamine
can be co-polymerized together to generate a bead including free
COOH groups. The COOH groups of the bead can be activated (e.g.,
via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and
N-Hydroxysuccinimide (NHS) or
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM)) such that they are reactive (e.g., reactive to amine
functional groups where EDC/NHS or DMTMM are used for activation).
The activated COOH groups can then react with an appropriate
species (e.g., a species including an amine functional group where
the carboxylic acid groups are activated to be reactive with an
amine functional group) as a functional group on a moiety to be
linked to the bead.
[0699] Beads including disulfide linkages in their polymeric
network can be functionalized with additional species (e.g.,
disulfide linkers, primers, other oligonucleotides, etc.) via
reduction of some of the disulfide linkages to free thiols. The
disulfide linkages can be reduced via, for example, the action of a
reducing agent (e.g., DTT, TCEP, etc.) to generate free thiol
groups, without dissolution of the bead. Free thiols of the beads
can then react with free thiols of a species or a species including
another disulfide bond (e.g., via thiol-disulfide exchange) such
that the species can be linked to the beads (e.g., via a generated
disulfide bond). In some embodiments, free thiols of the beads can
react with any other suitable group. For example, free thiols of
the beads can react with species including an acrydite moiety. The
free thiol groups of the beads can react with the acrydite via
Michael addition chemistry, such that the species including the
acrydite is linked to the bead. In some embodiments, uncontrolled
reactions can be prevented by inclusion of a thiol capping agent
such as N-ethylmalieamide or iodoacetate.
[0700] Activation of disulfide linkages within a bead can be
controlled such that only a small number of disulfide linkages are
activated. Control can be exerted, for example, by controlling the
concentration of a reducing agent used to generate free thiol
groups and/or concentration of reagents used to form disulfide
bonds in bead polymerization. In some embodiments, a low
concentration of reducing agent (e.g., molecules of reducing
agent:gel bead ratios) of less than or equal to about
1:100,000,000,000, less than or equal to about 1:10,000,000,000,
less than or equal to about 1:1,000,000,000, less than or equal to
about 1:100,000,000, less than or equal to about 1:10,000,000, less
than or equal to about 1:1,000,000, less than or equal to about
1:100,000, or less than or equal to about 1:10,000) can be used for
reduction. Controlling the number of disulfide linkages that are
reduced to free thiols can be useful in ensuring bead structural
integrity during functionalization. In some embodiments,
optically-active agents, such as fluorescent dyes can be coupled to
beads via free thiol groups of the beads and used to quantify the
number of free thiols present in a bead and/or track a bead.
[0701] In some embodiments, addition of moieties to a bead after
bead formation can be advantageous. For example, addition of a
capture probe after bead formation can avoid loss of the species
(e.g., disulfide linkers, primers, other oligonucleotides, etc.)
during chain transfer termination that can occur during
polymerization. In some embodiments, smaller precursors (e.g.,
monomers or cross linkers that do not include side chain groups and
linked moieties) can be used for polymerization and can be
minimally hindered from growing chain ends due to viscous effects.
In some embodiments, functionalization after bead synthesis can
minimize exposure of species (e.g., oligonucleotides) to be loaded
with potentially damaging agents (e.g., free radicals) and/or
chemical environments. In some embodiments, the generated hydrogel
can possess an upper critical solution temperature (UCST) that can
permit temperature driven swelling and collapse of a bead. Such
functionality can aid in oligonucleotide (e.g., a primer)
infiltration into the bead during subsequent functionalization of
the bead with the oligonucleotide. Post-production
functionalization can also be useful in controlling loading ratios
of species in beads, such that, for example, the variability in
loading ratio is minimized. Species loading can also be performed
in a batch process such that a plurality of beads can be
functionalized with the species in a single batch.
[0702] Reagents can be encapsulated in beads during bead generation
(e.g., during polymerization of precursors). Such reagents can or
cannot participate in polymerization. Such reagents can be entered
into polymerization reaction mixtures such that generated beads
include the reagents upon bead formation. In some embodiments, such
reagents can be added to the beads after formation. Such reagents
can include, for example, capture probes (e.g., oligonucleotides),
reagents for a nucleic acid amplification reaction (e.g., primers,
polymerases, dNTPs, co-factors (e.g., ionic co-factors), buffers)
including those described herein, reagents for enzymatic reactions
(e.g., enzymes, co-factors, chemical substrates, buffers), reagents
for nucleic acid modification reactions such as polymerization,
ligation, or digestion, and/or reagents for template preparation
(e.g., tagmentation) for one or more sequencing platforms (e.g.,
Nextera.RTM. for Illumina.RTM.). Such reagents can include one or
more enzymes described herein, including without limitation,
polymerase, reverse transcriptase, restriction enzymes (e.g.,
endonuclease), transposase, ligase, proteinase K, DNAse, etc. Such
reagents can also or alternatively include one or more reagents
such as lysis agents, inhibitors, inactivating agents, chelating
agents, stimulus agents. Trapping of such reagents can be
controlled by the polymer network density generated during
polymerization of precursors, control of ionic charge within the
bead (e.g., via ionic species linked to polymerized species), or by
the release of other species. Encapsulated reagents can be released
from a bead upon bead degradation and/or by application of a
stimulus capable of releasing the reagents from the bead. In some
embodiments, the beads or bead arrangements can be incubated in
permeabilization reagents as described herein.
[0703] In some embodiments, the beads can also include (e.g.,
encapsulate or have attached thereto) a plurality of capture probes
that include spatial barcodes, and the optical properties of the
spatial barcodes can be used for optical detection of the beads.
For example, the absorbance of light by the spatial barcodes can be
used to distinguish the beads from one another. In some
embodiments, a detectable label can directly or indirectly attach
to a spatial barcode and provide optical detection of the bead. In
some embodiments, each bead in a group of one or more beads has a
unique detectable label, and detection of the unique detectable
label determines the location of the spatial barcode sequence
associated with the bead.
[0704] Optical properties giving rise to optical detection of beads
can be due to optical properties of the bead surface (e.g., a
detectable label attached to the bead), or optical properties from
the bulk region of the bead (e.g., a detectable label incorporated
during bead formation or an optical property of the bead itself).
In some embodiments, a detectable label can be associated with a
bead or one or more moieties coupled to the bead.
[0705] In some embodiments, the beads include a plurality of
detectable labels. For example, a fluorescent dye can be attached
to the surface of the beads and/or can be incorporated into the
beads. Different intensities of the different fluorescent dyes can
be used to increase the number of optical combinations that can be
used to differentiate between beads. For example, if N is the
number of fluorescent dyes (e.g., between 2 and 10 fluorescent
dyes, such as 4 fluorescent dyes) and M is the possible intensities
for the dyes (e.g., between 2 and 50 intensities, such as 20
intensities), then MN are the possible distinct optical
combinations. In one example, 4 fluorescent dyes with 20 possible
intensities can be used to generate 160,000 distinct optical
combinations.
[0706] One or more optical properties of the beads or biological
contents, such as cells or nuclei, can be used to distinguish the
individual beads or biological contents from other beads or
biological contents. In some embodiments, the beads are made
optically detectable by including a detectable label having optical
properties to distinguish the beads from one another.
[0707] In some embodiments, optical properties of the beads can be
used for optical detection of the beads. For example, without
limitation, optical properties can include absorbance,
birefringence, color, fluorescence, luminosity, photosensitivity,
reflectivity, refractive index, scattering, or transmittance. For
example, beads can have different birefringence values based on
degree of polymerization, chain length, or monomer chemistry.
[0708] In some embodiments, nanobeads, such as quantum dots or
Janus beads, can be used as optical labels or components thereof.
For example, a quantum dot can be attached to a spatial barcode of
a bead.
[0709] Optical labels of beads can provide enhanced spectral
resolution to distinguish (e.g., identify) between beads with
unique spatial barcodes (e.g., beads including unique spatial
barcode sequences). That is, the beads are manufactured in a way
that the optical labels and the barcodes on the beads (e.g.,
spatial barcodes) are correlated with each other. In some aspects,
the beads can be loaded into a flowcell such that beads are arrayed
in a closely packed manner (e.g., single-cell resolution). Imaging
can be performed, and the spatial location of the barcodes can be
determined (e.g., based on information from a look-up table (LUT)).
The optical labels for spatial profiling allow for quick
deconvolution of bead-barcode (e.g., spatial barcode) identify.
[0710] In some examples, a lookup table (LUT) can be used to
associate a property (e.g., an optical label, such as a color
and/or intensity) of the bead with the barcode sequence. The
property may derive from the particle (e.g., bead) or an optical
label associated with the bead. The beads can be imaged to obtain
optical information of the bead, including, for example, the
property (e.g., color and/or intensity) of the bead or the optical
label associated with the bead, and optical information of the
biological sample. For example, an image can include optical
information in the visible spectrum, non-visible spectrum, or both.
In some embodiments, multiple images can be obtained across various
optical frequencies.
[0711] In some embodiments, a first bead includes a first optical
label and spatial barcodes each having a first spatial barcode
sequence. A second bead includes a second optical label and spatial
barcodes each having a second spatial barcode sequence. The first
optical label and second optical label can be different (e.g.,
provided by two different fluorescent dyes or the same fluorescent
dye at two different intensities). The first and second spatial
barcode sequences can be different nucleic acid sequences. In some
embodiments, the beads can be imaged to identify the first and
second optical labels, and the first and second optical labels can
then be used to associate the first and second optical labels with
the first and second spatial barcode sequences, respectively. In
some embodiments, the nucleic acid containing the spatial barcode
can further have a capture domain for analytes (e.g., mRNA). In
some embodiments, the nucleic acid (e.g., nucleic acid containing
the spatial barcode) can have a unique molecular identifier, a
cleavage domain, a functional domain, or combinations thereof.
[0712] In some embodiments, the optical label has a characteristic
electromagnetic spectrum. As used herein, the "electromagnetic
spectrum" refers to the range of frequencies of electromagnetic
radiation. In some embodiments, the optical label has a
characteristic absorption spectrum. As used herein, the "absorption
spectrum" refers to the range of frequencies of electromagnetic
radiation that are absorbed. The "electromagnetic spectrum" or
"absorption spectrum" can lead to different characteristic
spectrum. In some embodiments, the peak radiation or the peak
absorption occurs at 380-450 nm (Violet), 450-485 nm (Blue),
485-500 nm (Cyan), 500-565 nm (Green), 565-590 nm (Yellow), 590-625
nm (Orange), or 625-740 nm (Red). In some embodiments, the peak
radiation or the peak absorption occurs around 400 nm, 460 nm, or
520 nm.
[0713] Optical labels included on the beads can identify the
associated spatial barcode on the bead. Due to the relative limited
diversity of optical labels it can be advantageous to limit the
size of the spatial array for deconvolution. For example, the
substrate can be partitioned into two or more partitions (e.g.,
bins). In some embodiments, the substrate can be partitioned into
three or more partitions. In some embodiments, the substrate can be
partitioned into four or more partitions (e.g., bins). In some
embodiments, a set of beads are deposited to the partition. Within
each set of beads, one or more beads (e.g., equal to or more than
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,
400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000
beads) can have an unique optical label.
[0714] In some cases, beads within the same partition can have
different coordinates on the substrate. These coordinates can be
determined e.g., by various imaging techniques, such as observation
through microscope under an appropriate condition. In some
embodiments, the beads within the same partition can share the same
spatial barcode. In some embodiments, the beads (e.g., beads having
capture probes with barcodes, e.g., spatial barcodes or UMI) are
different from each other for different partition bins. In some
embodiments, the beads having capture probes with barcodes (e.g.,
spatial barcodes or UMI) can have different barcodes. For example,
in some cases, within each set of beads, which beads are associated
with a capture probe, the capture probes on individual beads can
have a unique barcode. In some cases, among all beads (e.g., within
two or more sets of beads), individual beads can have capture
probes with a unique barcode.
[0715] In some aspects, the present disclosure provides a
substrate. The substrate can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or
more than 1000 partitions (e.g., bins, or pre-defined area). The
partitions can have the same shape or different shapes. In some
embodiments, the substrate has only one partition (e.g., bin or
pre-defined area). In some embodiments, the first partition (e.g.,
the first pre-defined area, or the only bin on the substrate) can
have a first set of beads. In some embodiments, at least one bead
from the first set of beads comprises an optical label, and a
capture probe (e.g., an oligonucleotide capture probe) comprising a
barcode and a capture domain. At least one of the beads can have a
unique optical label among the first set of beads. In some
embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the beads in the first
set of beads have a unique optical label. In some embodiments, each
bead in the first set of beads has a unique optical label.
[0716] In some embodiments, the substrate can have a second
partition (e.g., the second pre-defined area, or the second bin).
The second partition can have a second set of beads. In some
embodiments, at least one bead from the second set of beads
comprises an optical label, and a capture probe (e.g., an
oligonucleotide capture probe) comprising a barcode and a capture
domain. At least one of the beads can have a unique optical label
among the second set of beads. In some embodiments, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,
99.7%, 99.8%, 99.9% of the beads in the second set of beads have a
unique optical label. In some embodiments, each bead in the second
set of beads has a unique optical label.
[0717] In some embodiments, the substrate can have a third
partition, a fourth partition, a fifth partition, a sixth
partition, a seventh partition, an eighth partition, a ninth
partition, or a tenth partition, etc. In some embodiments, the
substrate can have multiple partitions. In some cases, each of
these partitions has properties that are similar to the first or
the second partitions described herein. For example, at least one
bead from each set of beads comprises an optical label, and a
capture probe (e.g., an oligonucleotide capture probe) comprising a
barcode and a capture domain. At least one of these beads can have
a unique optical label among each set of beads. In some
embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the beads in each set
of beads have a unique optical label. In some embodiments, each
bead in each set of beads has a unique optical label.
[0718] In some embodiments, the beads are deposited on the
substrate. In some embodiments, the beads can be deposited directly
on or into a biological sample. Thus, in some cases, the biological
sample can be fixed or attached on the substrate before beads are
deposited onto the substrate.
[0719] In some embodiments, the beads are only deposited to areas
of interest (e.g., specific locations on the substrate, specific
cell types, and specific tissue structures). Thus, the deposited
beads do not necessarily cover the entire biological sample. In
some embodiments, one or more regions of a substrate can be masked
or modified (e.g., capped capture domains) such that the masked
regions do not interact with a corresponding region of the
biological sample.
[0720] In some embodiments, two or more than two sets of beads
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 sets) are
deposited at two or more than two partitions (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, or more than 10 partitions). These partitions do not
need to be adjacent to each other. As long as the location of the
partitions on the substrate is recorded, the identity of the beads
can be determined from the optical labels.
[0721] In some embodiments, a set of beads can have equal to or
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1000, 2000, 3000, 4000, or 5000 beads. In some embodiments, a
set 25 of beads can have less than 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,
600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 beads.
[0722] Optical labels can be included while generating the beads.
For example, optical labels can be included in the polymer
structure of a gel bead, or attached at the pre-polymer or monomer
stage in bead production. In some embodiments, the beads include
moieties that attach to one or more optical labels (e.g., at a
surface of a bead and/or within a bead). In some embodiments,
optical labels can be loaded into the beads with one or more
reagents. For example, reagents and optical labels can be loaded
into the beads by diffusion of the reagents (e.g., a solution of
reagents including the optical labels). In some embodiments,
optical labels can be included while preparing spatial barcodes.
For example, spatial barcodes can be prepared by synthesizing
molecules including barcode sequences (e.g., using a split pool or
combinatorial approach). Optical labels can be attached to spatial
barcodes prior to attaching the spatial barcodes to a bead. In some
embodiments, optical labels can be included after attaching spatial
barcodes to a bead. For example, optical labels can be attached to
spatial barcodes coupled to the bead. In some embodiments, spatial
barcodes or sequences thereof can be releasably or cleavably
attached to the bead. Optical labels can be releasably or
non-releasably attached to the bead. In some embodiments, a first
bead (e.g., a bead including a plurality of spatial barcodes) can
be coupled to a second bead including one or more optical labels.
For example, the first bead can be covalently coupled to the second
bead via a chemical bond. In some embodiments, the first bead can
be non-covalently associated with the second bead.
[0723] The first and/or second bead can include a plurality of
spatial barcodes. The plurality of spatial barcodes coupled to a
given bead can include the same barcode sequences. Where both the
first and second beads include spatial barcodes, the first and
second beads can include spatial barcodes including the same
barcode sequences or different barcode sequences.
[0724] Bead arrays containing captured analytes can be processed in
bulk or partitioned into droplet emulsions for preparing sequencing
libraries. In some embodiments, next generation sequencing reads
are clustered and correlated to the spatial position of the spatial
barcode on the bead array. For example, the information can be
computationally superimposed over a high-resolution image of the
tissue section to identify the location(s), where the analytes were
detected.
[0725] In some embodiments, de-cross linking can be performed to
account for de-crosslinking chemistries that may be incompatible
with certain barcoding/library prep biochemistry (e.g., presence of
proteases). For example, a two-step process is possible. In the
first step, beads can be provided in droplets such that DNA binds
to the beads after the conventional de-crosslinking chemistry is
performed. In the second step, the emulsion is broken and beads
collected and then re-encapsulated after washing for further
processing.
[0726] In some embodiments, beads can be affixed or attached to a
substrate using photochemical methods. For example, a bead can be
functionalized with perfluorophenylazide silane (PFPA silane),
contacted with a substrate, and then exposed to irradiation (see,
e.g., Liu et al. (2006) Journal of the American Chemical Society
128, 14067-14072). For example, immobilization of
antraquinone-functionalized substrates (see, e.g., Koch et al.
(2000) Bioconjugate Chem. 11, 474-483, the entire contents of which
are herein incorporated by reference).
[0727] The arrays can also be prepared by bead self-assembly. Each
bead can be covered with hundreds of thousands of copies of a
specific oligonucleotide. In some embodiments, each bead can be
covered with about 1,000 to about 1,000,000 oligonucleotides. In
some embodiments, each bead can be covered with about 1,000,000 to
about 10,000,000 oligonucleotides. In some embodiments, each bead
can covered with about 2,000,000 to about 3,000,000, about
3,000,000 to about 4,000,000, about 4,000,000 to about 5,000,000,
about 5,000,000 to about 6,000,000, about 6,000,000 to about
7,000,000, about 7,000,000 to about 8,000,000, about 8,000,000 to
about 9,000,000, or about 9,000,000 to about 10,000,000
oligonucleotides. In some embodiments, each bead can be covered
with about 10,000,000 to about 100,000,000 oligonucleotides. In
some embodiments, each bead can be covered with about 100,000,000
to about 1,000,000,000 oligonucleotides. In some embodiments, each
bead can be covered with about 1,000,000,000 to about
10,000,000,000 oligonucleotides. The beads can be irregularly
distributed across etched substrates during the array production
process. During this process, the beads can be self-assembled into
arrays (e.g., on a fiber-optic bundle substrate or a silica slide
substrate). In some embodiments, the beads irregularly arrive at
their final location on the array. Thus, the bead location may need
to be mapped or the oligonucleotides may need to be synthesized
based on a predetermined pattern.
[0728] Beads can be affixed or attached to a substrate covalently,
non-covalently, with adhesive, or a combination thereof. The
attached beads can be, for example, layered in a monolayer, a
bilayer, a trilayer, or as a cluster. As defined herein, a
"monolayer" generally refers to an arrayed series of probes, beads,
spots, dots, features, micro-locations, or islands that are affixed
or attached to a substrate, such that the beads are arranged as one
layer of single beads. In some embodiments, the beads are closely
packed.
[0729] As defined herein, the phrase "substantial monolayer" or
"substantially form(s) a monolayer" generally refers to (the
formation of) an arrayed series of probes, beads, microspheres,
spots, dots, features, micro-locations, or islands that are affixed
or attached to a substrate, such that about 50% to about 99% (e.g.,
about 50% to about 98%) of the beads are arranged as one layer of
single beads. This arrangement can be determined using a variety of
methods, including microscopic imaging.
[0730] In some embodiments, the monolayer of beads is a located in
a predefined area on the substrate. For example, the predefined
area can be partitioned with physical barriers, a photomask, divots
in the substrate, or wells in the substrate.
[0731] As used herein, the term "reactive element" generally refers
to a molecule or molecular moiety that can react with another
molecule or molecular moiety to form a covalent bond. Reactive
elements include, for example, amines, aldehydes, alkynes, azides,
thiols, haloacetyls, pyridyl disulfides, hydrazides, carboxylic
acids, alkoxyamines, sulfhydryls, maleimides, Michael acceptors,
hydroxyls, and active esters. Some reactive elements, for example,
carboxylic acids, can be treated with one or more activating agents
(e.g., acylating agents, isourea-forming agents) to increase
susceptibility of the reactive element to nucleophilic attack.
Non-limiting examples of activating agents include
N-hydroxysuccinimide, N-hydroxysulfosuccinimide,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,
dicyclohexylcarbodiimide, diisopropylcarbodiimide,
1-hydroxybenzotriazole,
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexfluorophosphate,
(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 4-(N,N-dimethylamino)pyridine, and
carbonyldiimidazole.
[0732] In some embodiments, the reactive element is bound directly
to a bead. For example, hydrogel beads can be treated with an
acrylic acid monomer to form acrylic acid-functionalized hydrogel
beads. In some cases, the reactive element is bound indirectly to
the bead via one or more linkers. As used herein, a "linker"
generally refers to a multifunctional (e.g., bifunctional,
trifunctional) reagent used for conjugating two or more chemical
moieties. A linker can be a cleavable linker that can undergo
induced dissociation. For example, the dissociation can be induced
by a solvent (e.g., hydrolysis and solvolysis); by irradiation
(e.g., photolysis); by an enzyme (e.g., enzymolysis); or by
treatment with a solution of specific pH (e.g., pH 4, 5, 6, 7, or
8).
[0733] In some embodiments, the reactive element is bound directly
to a substrate. For example, a glass slide can be coated with
(3-aminopropyl)triethoxysilane. In some embodiments, the reactive
element is bound indirectly to a substrate via one or more
linkers.
[0734] Gel/Hydrogel Beads
[0735] In some embodiments, the bead can be a gel bead. A "gel" is
a semi-rigid material permeable to liquids and gases. Exemplary
gels include, but are not limited to, those having a colloidal
structure, such as agarose; polymer mesh structures, such as
gelatin; hydrogels; and cross-linked polymer structures, such as
polyacrylamide, SFA (see, for example, U.S. Patent Application
Publication No. 2011/0059865, which is incorporated herein by
reference in its entirety) and PAZAM (see, for example, U.S. Patent
Application Publication No. 2014/0079923, which is incorporated
herein by reference in its entirety).
[0736] A gel can be formulated into various shapes and dimensions
depending on the context of intended use. In some embodiments, a
gel is prepared and formulated as a gel bead (e.g., a gel bead
including capture probes attached or associated with the gel bead).
A gel bead can be a hydrogel bead. A hydrogel bead can be formed
from molecular precursors, such as a polymeric or monomeric
species.
[0737] In some cases, a bead comprises a polymer or hydrogel. The
polymer or hydrogel may determine one or more characteristics of
the hydrogel bead, such as the volume, fluidity, porosity,
rigidity, organization, or one or more other features of the
hydrogel bead. In some embodiments, a hydrogel bead can include a
polymer matrix (e.g., a matrix formed by polymerization or
cross-linking). A polymer matrix can include one or more polymers
(e.g., polymers having different functional groups or repeat
units). Cross-linking can be via covalent, ionic, and/or inductive
interactions, and/or physical entanglement.
[0738] A polymer or hydrogel may be formed, for example, upon
cross-linking one or more cross-linkable molecules within the
hydrogel bead. For example, a hydrogel may be formed upon
cross-linking one or more molecules within the hydrogel bead. The
hydrogel may be formed upon polymerizing a plurality of monomers
within the hydrogel bead. The hydrogel may be formed upon
polymerizing a plurality of polymers within the hydrogel bead.
Polymeric or hydrogel precursors may be provided to the hydrogel
bead and may not form a polymer or hydrogel without application of
a stimulus (e.g., as described herein). In some cases, the hydrogel
bead may be encapsulated within the polymer or hydrogel. Formation
of a hydrogel bead may take place following one or more other
changes to the cell that may be brought about by one or more other
conditions.
[0739] The methods described herein may be applied to a single
hydrogel bead or a plurality of hydrogel beads. A method of
processing a plurality of hydrogel beads may comprise providing the
plurality of hydrogel beads within a vessel and subjecting the
plurality of hydrogel beads to conditions sufficient to change one
or more characteristics of the hydrogel bead. For example,
plurality of hydrogel beads may be subjected to a first condition
or set of conditions comprising a chemical species, and a
cross-section of the hydrogel beads of the plurality of hydrogel
beads may change from a first cross-section to a second
cross-section, which second cross-section is less than the first
cross-section. The chemical species may comprise, for example, an
organic solvent such as ethanol, methanol, or acetone. The
plurality of hydrogel beads may then be subjected to a second
condition or set of conditions comprising a chemical species, and
crosslinks may form within each of the hydrogel beads. The chemical
species may comprise, for example, a cross-linking agent. The
plurality of processed hydrogel beads may be provided in an aqueous
fluid. In some instances, the second cross-section of the plurality
of hydrogel beads is substantially maintained in the aqueous fluid.
The plurality of processed hydrogel beads may be partitioned within
a plurality of partitions. The partitions may be, for example,
aqueous droplets included in a water-in-oil emulsion. The
partitions may be, for example, a plurality of wells. The plurality
of fixed hydrogel beads may be co-partitioned with one or more
reagents. In some cases, the plurality of fixed hydrogel beads may
be co-partitioned with one or more beads, where each bead comprises
a plurality of nucleic acid barcode molecules attached thereto. The
nucleic acid barcode molecules attached to a given bead may
comprise a common barcode sequence, and the nucleic acid barcode
molecules attached to each different bead may comprise a sequence
comprising a different common barcode sequence. The nucleic acid
barcode molecules, or portions thereof, may then be used in
reactions with target molecules associated with hydrogel beads of
the plurality of hydrogel beads.
[0740] Core/Shell Beads
[0741] In some embodiments, the bead is a core/shell bead that
comprises an inner core (e.g., a nanosphere or microsphere) and an
outer shell (e.g., a hydrogel coating the nanosphere or
microsphere). In some embodiments, the inner core can be a solid
nanoparticle or solid microparticle. In some embodiments, the inner
core can be a silica inner core (e.g., a silica nanoparticle or
silica microparticle). In some embodiments, the inner core of the
core/shell bead can have an average diameter of about 1 micron. In
some embodiments, the inner core can have an average diameter of
about 2 microns. In some embodiments, the inner core can have an
average diameter of about 3 microns. In some embodiments, the inner
core can have an average diameter of about 4 microns. In some
embodiments, the inner core can have an average diameter of about 5
microns. In some embodiments, the inner core can have an average
diameter of about 6 microns. In some embodiments, the inner core
can have an average diameter of about 7 microns. In some
embodiments, the inner core can have an average diameter of about 8
microns. In some embodiments, the inner core can have an average
diameter of about 9 microns. In some embodiments, the inner core
can have an average diameter of about 10 microns. In some
embodiments, the inner core can have an average diameter of about
100 nanometers to about 10 microns.
[0742] In some embodiments, the core/shell bead can decrease its
outer shell volume by removing solvents, salts, or water (e.g.,
dehydrated, desiccated, dried, exsiccated) from the outer shell to
form a shrunken core/shell bead. In another example, the core/shell
bead can decrease its outer shell volume by adjusting temperature
or pH, as described above. In some embodiments, the core/shell bead
can expand its outer shell volume, for example by the addition of
solvents, salts, or water (e.g., rehydration) to form an expanded
core/shell bead. In some embodiments, the outer shell (e.g.,
coating the inner core) can have an average thickness of about 1
micron. In some embodiments, the outer shell can have an average
thickness of about 2 microns. In some embodiments, the outer shell
can have an average thickness of about 3 microns. In some
embodiments, the outer shell can have an average thickness of about
4 microns. In some embodiments, the outer shell can have an average
thickness of about 5 microns.
[0743] In some embodiments, the core/shell bead can have an average
diameter of about 1 micron to about 10 microns. In some
embodiments, the core/shell bead can have an average diameter of
about 1 micron. In some embodiments, the core/shell bead can have
an average diameter of about 2 microns. In some embodiments, the
core/shell bead can have an average diameter of about 3 microns. In
some embodiments, the core/shell bead can have an average diameter
of about 4 microns. In some embodiments, the core/shell bead can
have an average diameter of about 5 microns. In some embodiments,
the core/shell bead can have an average diameter of about 6
microns. In some embodiments, the core/shell bead can have an
average diameter of about 7 microns. In some embodiments, the
core/shell bead can have an average diameter of about 8 microns. In
some embodiments, the core/shell bead can have an average diameter
of about 9 microns. In some embodiments, the core/shell bead can
have an average diameter of about 10 microns.
[0744] (2) Methods for Covalently Bonding Features to a
Substrate
[0745] Provided herein are methods for the covalent bonding of
features (e.g., optically labeled beads, hydrogel beads,
microsphere beads) to a substrate.
[0746] In some embodiments, the features (e.g., beads) are coupled
to a substrate via a covalent bond between a first reactive element
and a second reactive element. In some embodiments, the
covalently-bound beads substantially form a monolayer of features
(e.g., hydrogel beads, microsphere beads) on the substrate.
[0747] In some embodiments, the features (e.g., beads) are
functionalized with a first reactive element, which is directly
bound to the features. In some embodiments, the features are
functionalized with a first reactive element, which is indirectly
bound to the beads via a linker. In some embodiments, the linker is
a benzophenone. In some embodiments, the linker is an amino
methacrylamide. For example, the linker can be 3-aminopropyl
methacrylamide. In some embodiments, the linker is a PEG linker. In
some embodiments, the linker is a cleavable linker.
[0748] In some embodiments, the substrate is functionalized with a
second reactive element, which is directly bound to the substrate.
In some embodiments, the substrate is functionalized with a second
reactive element, which is indirectly bound to the beads via a
linker. In some embodiments, the linker is a benzophenone. For
example, the linker can be benzophenone. In some embodiments, the
linker is an amino methacrylamide. For example, the linker can be
3-aminopropyl methacrylamide. In some embodiments, the linker is a
PEG linker. In some embodiments, the linker is a cleavable
linker.
[0749] In some embodiments, the substrate is a glass slide. In some
embodiments, the substrate is a pre-functionalized glass slide.
[0750] In some embodiments, about 99% of the covalently-bound beads
form a monolayer of beads on the substrate. In some embodiments,
about 50% to about 98% form a monolayer of beads on the substrate.
For example, about 50% to about 95%, about 50% to about 90%, about
50% to about 85%, about 50% to about 80%, about 50% to about 75%,
about 50% to about 70%, about 50% to about 65%, about 50% to about
60%, or about 50% to about 55% of the covalently-bound beads form a
monolayer of beads on the substrate. In some embodiments, about 55%
to about 98%, about 60% to about 98%, about 65% to about 98%, about
70% to about 98%, about 75% to about 98%, about 80% to about 98%,
about 85% to about 98%, about 90% to about 95%, or about 95% to
about 98% of the covalently-bound beads form a monolayer of beads
on the substrate. In some embodiments, about 55% to about 95%,
about 60% to about 90%, about 65% to about 95%, about 70% to about
95%, about 75% to about 90%, about 75% to about 95%, about 80% to
about 90%, about 80% to about 95%, about 85% to about 90%, or about
85% to about 95% of the covalently-bound beads for a monolayer of
beads on the substrate.
[0751] In some embodiments, at least one of the first reactive
element and the second reactive element is selected from the group
consisting of:
##STR00011##
wherein
[0752] R.sup.1 is selected from H, C.sub.1-C.sub.6 alkyl, or
--SO.sub.3;
[0753] R.sup.2 is C.sub.1-C.sub.6 alkyl; and
[0754] X is a halo moiety.
[0755] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00012##
[0756] wherein the indicates the point of attachment of the first
reactive element or the second reactive element to the bead (e.g.,
hydrogel bead or microsphere bead) or to the substrate.
[0757] In some embodiments, at least one of the first reactive
element or the second reactive element is selected from the group
consisting of:
##STR00013##
wherein
[0758] R.sup.1 is selected from H, C.sub.1-C.sub.6 alkyl, or
--SO.sub.3;
[0759] R.sup.2 is C.sub.1-C.sub.6 alkyl; and
[0760] X is a halo moiety.
[0761] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00014##
wherein R.sup.1 is selected from H, C.sub.1-C.sub.6 alkyl, or
--SO.sub.3. In some embodiments, R.sup.1 is H. In some embodiments,
R.sup.1 is C.sub.1-C.sub.6 alkyl. In some embodiments, R.sup.1 is
--SO.sub.3.
[0762] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00015##
wherein R.sup.2 is C.sub.1-C.sub.6 alkyl. In some embodiments,
R.sup.2 is methyl.
[0763] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00016##
Is some embodiments,
##STR00017##
can be reacted with an activating agent to form an active ester. In
some embodiments, the active ester is
##STR00018##
In some embodiments, the activating agent is an acylating agent
(e.g., N-hydroxysuccinimide and N-hydroxysulfosuccinimide). In some
embodiments, the activating agent is an O-acylisourea-forming agent
(e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
dicyclohexylcarbodiimide, and diisopropylcarbodiimide). In some
embodiments, the activating agent is a combination of at least one
acylating agent and at least one O-isourea-forming agents (e.g.,
N-hydroxysuccinimide (NHS),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
N-hydroxysulfosuccinimide (sulfo-NHS), and a combination
thereof).
[0764] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00019##
[0765] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00020##
wherein X is a halo moiety. For example, X is chloro, bromo, or
iodo.
[0766] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00021##
[0767] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00022##
[0768] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00023##
[0769] In some embodiments, at least one of the first reactive
element or the second reactive element is selected from the group
consisting of:
##STR00024##
wherein
[0770] R.sup.3 is H or C.sub.1-C.sub.6 alkyl; and
[0771] R.sup.4 is H or trimethylsilyl.
[0772] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00025##
wherein R.sup.4 is H or trimethylsilyl. In some embodiments,
R.sup.4 is H.
[0773] In some embodiments, at least one of the first reactive
element or the second reactive element is selected from the group
consisting of:
##STR00026##
wherein R.sup.3 is H or C.sub.1-C.sub.6 alkyl. In some embodiments,
R.sup.3 is H. In some embodiments, R.sup.3 is C.sub.1-C.sub.6
alkyl.
[0774] In some embodiments, at least one of the first reactive
element or the second reactive element comprises
##STR00027##
wherein R.sup.3 is H or C.sub.1-C.sub.6 alkyl. In some embodiments,
R.sup.3 is H. In some embodiments, R.sup.3 is C.sub.1-C.sub.6
alkyl.
[0775] In some embodiments, at least one of the first reactive
elements or the second reactive elements comprises
##STR00028##
[0776] In some embodiments, at least one of the first reactive
elements or the second reactive elements comprises
##STR00029##
[0777] In some embodiments, one of the first reactive elements or
the second reactive elements is selected from the group consisting
of:
##STR00030##
wherein
[0778] R.sup.1 is selected from H, C.sub.1-C.sub.6 alkyl, or
--SO.sub.3;
[0779] R.sup.2 is C.sub.1-C.sub.6 alkyl;
[0780] X is a halo moiety;
and the other of the first reactive element or the second reactive
element is selected from the group consisting of:
##STR00031##
wherein
[0781] R.sup.3 is H or C.sub.1-C.sub.6 alkyl; and
[0782] R.sup.4 is H or trimethylsilyl.
[0783] In some embodiments, one of the first reactive elements or
the second reactive elements is selected from the group consisting
of
##STR00032##
wherein R.sup.3 is H or C.sub.1-C.sub.6 alkyl; and the other of the
first reactive element or the second reactive element is
##STR00033##
wherein R.sup.4 is H or trimethylsilyl. In some embodiments,
R.sup.3 is H. In some embodiments, R.sup.3 is C.sub.1-C.sub.6
alkyl. In some embodiments, R.sup.4 is H. In some embodiments,
R.sup.4 is trimethylsilyl.
[0784] In some embodiments, one of the first reactive element or
the second reactive element is selected from the group consisting
of:
##STR00034##
wherein
[0785] R.sup.1 is selected from H, C.sub.1-C.sub.6 alkyl, or
--SO.sub.3;
[0786] R.sup.2 is C.sub.1-C.sub.6 alkyl;
[0787] X is a halo moiety;
and the other of the first reactive element or the second reactive
element is selected from the group consisting of:
##STR00035##
wherein R.sup.3 is H or C.sub.1-C.sub.6 alkyl. In some embodiments,
R.sup.1 is H. In some embodiments, R.sup.1 is C.sub.1-C.sub.6
alkyl. In some embodiments, R.sup.1 is --SO.sub.3. In some
embodiments, R.sup.2 is methyl. In some embodiments, X is iodo. In
some embodiments, R.sup.3 is H. In some embodiments, R.sup.3 is
C.sub.1-C.sub.6 alkyl.
[0788] In some embodiments, one of the first reactive elements or
the second reactive elements is selected from the group consisting
of:
##STR00036##
wherein
[0789] R.sup.1 is selected from H, C.sub.1-C.sub.6 alkyl, or
--SO.sub.3;
[0790] R.sup.2 is C.sub.1-C.sub.6 alkyl;
and the other of the first reactive elements or the second reactive
elements comprises
##STR00037##
wherein R.sup.3 is H or C.sub.1-C.sub.6 alkyl. In some embodiments,
R.sup.1 is H. In some embodiments, R.sup.1 is C.sub.1-C.sub.6
alkyl. In some embodiments, R.sup.1 is --SO.sub.3. In some
embodiments, R.sup.2 is methyl. In some embodiments, R.sup.3 is H.
In some embodiments, R.sup.3 is C.sub.1-C.sub.6 alkyl.
[0791] In some embodiments, one of the first reactive element or
the second reactive element is selected from the group consisting
of:
##STR00038##
wherein X is a halo moiety; and the other of the first reactive
element or the second reactive element comprises
##STR00039##
In some embodiments, X is bromo. In some embodiments, X is
iodo.
[0792] In some embodiments, one of the first reactive element or
the second reactive element is selected from the group consisting
of
##STR00040##
and the other of the first reactive element or the second reactive
element comprises
##STR00041##
[0793] The term "halo" refers to fluoro (F), chloro (Cl), bromo
(Br), or iodo (I).
[0794] The term "alkyl" refers to a hydrocarbon chain that may be a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-10 indicates that the group
may have from 1 to 10 (inclusive) carbon atoms in it. Non-limiting
examples include methyl, ethyl, iso-propyl, tert-butyl,
n-hexyl.
[0795] The term "haloalkyl" refers to an alkyl, in which one or
more hydrogen atoms is/are replaced with an independently selected
halo.
[0796] The term "alkoxy" refers to an --O-alkyl radical (e.g.,
--OCH.sub.3).
[0797] The term "alkylene" refers to a divalent alkyl (e.g.,
--CH.sub.2--).
[0798] The term "alkenyl" refers to a hydrocarbon chain that may be
a straight chain or branched chain having one or more carbon-carbon
double bonds. The alkenyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-6 indicates that the group may
have from 2 to 6 (inclusive) carbon atoms in it.
[0799] The term "alkynyl" refers to a hydrocarbon chain that may be
a straight chain or branched chain having one or more carbon-carbon
triple bonds. The alkynyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-6 indicates that the group may
have from 2 to 6 (inclusive) carbon atoms in it.
[0800] The term "aryl" refers to a 6-20 carbon mono-, bi-, tri- or
polycyclic group wherein at least one ring in the system is
aromatic (e.g., 6-carbon monocyclic, 10-carbon bicyclic, or
14-carbon tricyclic aromatic ring system); and wherein 0, 1, 2, 3,
or 4 atoms of each ring may be substituted by a substituent.
Examples of aryl groups include phenyl, naphthyl,
tetrahydronaphthyl, and the like.
[0801] (3) Methods for Non-Covalently Bonding Features to a
Substrate
[0802] Provided herein are methods for the non-covalent bonding of
features (e.g., optically-labeled beads, hydrogel beads, or
microsphere beads) to a substrate.
[0803] In some embodiments, features (e.g., beads) are coupled to a
substrate via a non-covalent bond between a first affinity group
and a second affinity group. In some embodiments, the
non-covalently-bound features (e.g., beads) substantially form a
monolayer of beads (e.g., hydrogel beads, microsphere beads) on the
substrate.
[0804] In some embodiments, the features are functionalized with a
first affinity group, which is directly bound to the features. In
some embodiments, the features are functionalized with a first
affinity group, which is indirectly bound to the beads via a
linker. In some embodiments, the linker is a benzophenone. In some
embodiments, the linker is an amino methacrylamide. For example,
the linker can be 3-aminopropyl methacrylamide. In some
embodiments, the linker is a PEG linker. In some embodiments, the
linker is a cleavable linker.
[0805] In some embodiments, the substrate is functionalized with a
second affinity group, which is directly bound to the substrate. In
some embodiments, the substrate is functionalized with a second
affinity group, which is indirectly bound to the beads via a
linker. In some embodiments, the linker is a benzophenone. In some
embodiments, the linker is an amino methacrylamide. For example,
the linker can be 3-aminopropyl methacrylamide. In some
embodiments, the linker is a PEG linker. In some embodiments, the
linker is a cleavable linker.
[0806] In some embodiments the first affinity group or the second
affinity group is biotin, and the other of the first affinity group
or the second affinity group is streptavidin.
[0807] In some embodiments, about 99% of the non-covalently-bound
beads form a monolayer of beads on the substrate. In some
embodiments, about 50% to about 98% form a monolayer of beads on
the substrate. For example, about 50% to about 95%, about 50% to
about 90%, about 50% to about 85%, about 50% to about 80%, about
50% to about 75%, about 50% to about 70%, about 50% to about 65%,
about 50% to about 60%, or about 50% to about 55% of the
non-covalently-bound beads form a monolayer of beads on the
substrate. In some embodiments, about 55% to about 98%, about 60%
to about 98%, about 65% to about 98%, about 70% to about 98%, about
75% to about 98%, about 80% to about 98%, about 85% to about 98%,
about 90% to about 95%, or about 95% to about 98% of the
non-covalently-bound beads form a monolayer of beads on the
substrate. In some embodiments, about 55% to about 95%, about 60%
to about 90%, about 65% to about 95%, about 70% to about 95%, about
75% to about 90%, about 75% to about 95%, about 80% to about 90%,
about 80% to about 95%, about 85% to about 90%, or about 85% to
about 95% of the non-covalently-bound beads for a monolayer of
beads on the substrate.
[0808] In some embodiments, the monolayer of beads is a formed in a
predefined area on the substrate. In some embodiments, the
predefined area is partitioned with physical barriers. For example,
divots or wells in the substrate. In some embodiments, the
predefined area is partitioned using a photomask. For example, the
substrate is coated with a photo-activated solution, dried, and
then irradiated under a photomask. In some embodiments, the
photo-activated solution is UV-activated.
[0809] As used herein, an "adhesive" generally refers to a
substance used for sticking objects or materials together.
Adhesives include, for example, glues, pastes, liquid tapes, epoxy,
bioadhesives, gels, and mucilage. In some embodiments, an adhesive
is liquid tape. In some embodiments, the adhesive is glue.
[0810] In some embodiments, beads are adhered to a substrate using
an adhesive (e.g., liquid tape, glue, paste). In some embodiments,
the adhered beads substantially form a monolayer of beads on the
substrate (e.g., a glass slide). In some embodiments, the beads are
hydrogel beads. In some embodiments, the beads are microsphere
beads. In some embodiments, the beads are coated with the adhesive,
and then the beads are contacted with the substrate. In some
embodiments, the substrate is coated with the adhesive, and then
the substrate is contacted with the beads. In some embodiments,
both the substrate is coated with the adhesive and the beads are
coated with the adhesive, and then the beads and substrate are
contacted with one another.
[0811] In some embodiments, about 99% of the adhered beads form a
monolayer of beads on the substrate. In some embodiments, about 50%
to about 98% form a monolayer of beads on the substrate. For
example, about 50% to about 95%, about 50% to about 90%, about 50%
to about 85%, about 50% to about 80%, about 50% to about 75%, about
50% to about 70%, about 50% to about 65%, about 50% to about 60%,
or about 50% to about 55% of the adhered beads form a monolayer of
beads on the substrate. In some embodiments, about 55% to about
98%, about 60% to about 98%, about 65% to about 98%, about 70% to
about 98%, about 75% to about 98%, about 80% to about 98%, about
85% to about 98%, about 90% to about 95%, or about 95% to about 98%
of the adhered beads form a monolayer of beads on the substrate. In
some embodiments, about 55% to about 95%, about 60% to about 90%,
about 65% to about 95%, about 70% to about 95%, about 75% to about
90%, about 75% to about 95%, about 80% to about 90%, about 80% to
about 95%, about 85% to about 90%, or about 85% to about 95% of the
adhered beads for a monolayer of beads on the substrate.
[0812] In some embodiments, beads can be deposited onto a
biological sample such that the deposited beads form a monolayer of
beads on the biological sample (e.g., over or under the biological
sample). In some embodiments, beads deposited on the substrate can
self-assemble into a monolayer of beads that saturate the intended
surface area of the biological sample under investigation. In this
approach, bead arrays can be designed, formulated, and prepared to
evaluate a plurality of analytes from a biological sample of any
size or dimension. In some embodiments, the concentration or
density of beads (e.g., gel beads) applied to the biological sample
is such that the area as a whole, or one or more regions of
interest in the biological sample, is saturated with a monolayer of
beads. In some embodiments, the beads are contacted with the
biological sample by pouring, pipetting, spraying, and the like,
onto the biological sample. Any suitable form of bead deposition
can be used.
[0813] In some embodiments, the biological sample can be confined
to a specific region or area of the array. For example, a
biological sample can be affixed to a glass slide and a chamber,
gasket, or cage positioned over the biological sample to act as a
containment region or frame within which the beads are deposited.
As will be apparent, the density or concentration of beads needed
to saturate an area or biological sample can be readily determined
by one of ordinary skill in the art (e.g., through microscopic
visualization of the beads on the biological sample). In some
embodiments, the bead array contains microfluidic channels to
direct reagents to the spots or beads of the array.
[0814] (4) Feature Geometric Attributes
[0815] Features on an array can have a variety of sizes. In some
embodiments, a feature of an array can have an average diameter or
maximum dimension between 500 nm .mu.m to 100 .mu.m. For example,
between 500 nm to 2 .mu.m, 1 .mu.m to 3 .mu.m, 1 .mu.m to 5 .mu.m,
1 .mu.m to 10 .mu.m, 1 .mu.m to 20 .mu.m, 1 .mu.m to 30 .mu.m, 1
.mu.m to 40 .mu.m, 1 .mu.m to 50 .mu.m, 1 .mu.m to 60 .mu.m, 1
.mu.m to 70 .mu.m, 1 .mu.m to 80 .mu.m, 1 .mu.m to 90 .mu.m, 90
.mu.m to 100 .mu.m, 80 .mu.m to 100 .mu.m, 70 .mu.m to 100 .mu.m,
60 .mu.m to 100 .mu.m, 50 .mu.m to 100 .mu.m, 40 .mu.m to 100
.mu.m, 30 .mu.m to 100 .mu.m, 20 .mu.m to 100 .mu.m, 10 .mu.m to
100 .mu.m, about 40 .mu.m to about 70 .mu.m, or about 50 .mu.m to
about 60 .mu.m. In some embodiments, the feature has an average
diameter or maximum dimension between 30 .mu.m to 100 .mu.m, 40
.mu.m to 90 .mu.m, 50 .mu.m to 80 .mu.m, 60 .mu.m to 70 .mu.m, or
any range within the disclosed sub-ranges. In some embodiments, the
feature has an average diameter or maximum dimension no larger than
95 .mu.m, 90 .mu.m, 85 .mu.m, 80 .mu.m, 75 .mu.m, 70 .mu.m, 65
.mu.m, 60 .mu.m, 55 .mu.m, 50 .mu.m, 45 .mu.m, 40 .mu.m, 35 .mu.m,
30 .mu.m, 25 .mu.m, 20 .mu.m, 15 .mu.m, 14 .mu.m, 13 .mu.m, 12
.mu.m, 11 .mu.m, 10 .mu.m, 9 .mu.m, 8 .mu.m, 7 .mu.m, 6 .mu.m, 5
.mu.m, 4 .mu.m, 3 .mu.m, 2 .mu.m, or 1 .mu.m. In some embodiments,
the feature has an average diameter or maximum dimension of
approximately 65 .mu.m. In some embodiments, the feature has an
average diameter or maximum distance of approximately 55 .mu.m.
[0816] In some embodiments, the size and/or shape of a plurality of
features of an array are approximately uniform. In some
embodiments, the size and/or shape of a plurality of features of an
array is not uniform. For example, in some embodiments, features in
an array can have an average cross-sectional dimension, and a
distribution of cross-sectional dimensions among the features can
have a full-width and half-maximum value of 0% or more (e.g., 5% or
more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or
more, 70% or more, or 100% or more) of the average cross-sectional
dimension for the distribution.
[0817] In certain embodiments, features in an array can have an
average cross-sectional dimension of between about 1 .mu.m and
about 10 .mu.m. This range in average feature cross-sectional
dimension corresponds to the approximate diameter of a single
mammalian cell. Thus, an array of such features can be used to
detect analytes at, or below, mammalian single-cell resolution.
[0818] In some embodiments, a plurality of features has a mean
diameter or mean maximum dimension of about 0.1 .mu.m to about 100
.mu.m (e.g., about 0.1 .mu.m to about 5 .mu.m, about 1 .mu.m to
about 10 .mu.m, about 1 .mu.m to about 20 .mu.m, about 1 .mu.m to
about 30 .mu.m, about 1 .mu.m to about 40 .mu.m, about 1 .mu.m to
about 50 .mu.m, about 1 .mu.m to about 60 .mu.m, about 1 .mu.m to
about 70 .mu.m, about 1 .mu.m to about 80 .mu.m, about 1 .mu.m to
about 90 .mu.m, about 90 .mu.m to about 100 .mu.m, about 80 .mu.m
to about 100 .mu.m, about 70 .mu.m to about 100 .mu.m, about 60
.mu.m to about 100 .mu.m, about 50 .mu.m to about 100 .mu.m, about
40 .mu.m to about 100 .mu.m, about 30 .mu.m to about 100 .mu.m,
about 20 .mu.m to about 100 .mu.m, or about 10 .mu.m to about 100
.mu.m). In some embodiments, the plurality of features has a mean
diameter or mean maximum dimension between 30 .mu.m to 100 .mu.m,
40 .mu.m to 90 .mu.m, 50 .mu.m to 80 .mu.m, 60 .mu.m to 70 .mu.m,
or any range within the disclosed sub-ranges. In some embodiments,
the plurality of features has a mean diameter or a mean maximum
dimension no larger than 95 .mu.m, 90 .mu.m, 85 .mu.m, 80 .mu.m, 75
.mu.m, 70 .mu.m, 65 .mu.m, 60 .mu.m, 55 .mu.m, 50 .mu.m, 45 .mu.m,
40 .mu.m, 35 .mu.m, 30 .mu.m, 25 .mu.m, 20 .mu.m, 15 .mu.m, 14
.mu.m, 13 .mu.m, 12 .mu.m, 11 .mu.m, 10 .mu.m, 9 .mu.m, 8 .mu.m, 7
.mu.m, 6 .mu.m, 5 .mu.m, 4 .mu.m, 3 .mu.m, 2 .mu.m, or 1 .mu.m. In
some embodiments, the plurality of features has a mean average
diameter or a mean maximum dimension of approximately 65 .mu.m,
approximately 60 .mu.m, approximately 55 .mu.m, approximately 50
.mu.m, approximately 45 .mu.m, approximately 40 .mu.m,
approximately 35 .mu.m, approximately 30 .mu.m, approximately 25
.mu.m, approximately 20 .mu.m, approximately 15 .mu.m,
approximately 10 .mu.m, approximately 5 .mu.m, approximately 4
.mu.m, approximately 3 .mu.m, approximately 2 .mu.m, or
approximately 1 .mu.m.
[0819] (iv) Array Geometric Attributes
[0820] In some embodiments, an array includes a plurality of
features. For example, an array includes between 4,000 and 50,000
features, or any range within 4,000 to 40,000 features. For
example, an array includes between 4,000 to 35,000 features, 4,000
to 30,000 features, 4,000 to 25,000 features, 4,000 to 20,000
features, 4,000 to 15,000 features, 4,000 to 10,000 features, 4,000
to 6,000 features, or 4,400 to 6,000 features. In some embodiments,
the array includes between 4,100 and 5,900 features, between 4,200
and 5,800 features, between 4,300 and 5,700 features, between 4,400
and 5,600 features, between 4,500 and 5,500 features, between 4,600
and 5,400 features, between 4,700 and 5,300 features, between 4,800
and 5,200 features, between 4,900 and 5,100 features, or any range
within the disclosed sub-ranges. For example, the array can include
about 4,000 features, about 4,200 features, about 4,400 features,
about 4,800 features, about 5,000 features, about 5,200 features,
about 5,400 features, about 5,600 features, or about 6,000
features, about 10,000 features, about 20,000 features, about
30,000 features, about 40,000 features, or about 50,000 features.
In some embodiments, the array comprises at least 4,000 features.
In some embodiments, the array includes approximately 5,000
features.
[0821] In some embodiments, features within an array have an
irregular arrangement or relationship to one another, such that no
discernable pattern or regularity is evident in the geometrical
spacing relationships among the features. For example, features
within an array may be positioned randomly with respect to one
another. Alternatively, features within an array may be positioned
irregularly, but the spacings may be selected deterministically to
ensure that the resulting arrangement of features is irregular.
[0822] In some embodiments, features within an array are positioned
regularly with respect to one another to form a pattern. A wide
variety of different patterns of features can be implemented in
arrays. Examples of such patterns include, but are not limited to,
square arrays of features, rectangular arrays of features,
hexagonal arrays of features (including hexagonal close-packed
arrays), radial arrays of features, spiral arrays of features,
triangular arrays of features, and more generally, any array in
which adjacent features in the array are reached from one another
by regular increments in linear and/or angular coordinate
dimensions.
[0823] In some embodiments, features within an array are positioned
with a degree of regularity with respect to one another such that
the array of features is neither perfectly regular nor perfectly
irregular (i.e., the array is "partially regular"). For example, in
some embodiments, adjacent features in an array can be separated by
a displacement in one or more linear and/or angular coordinate
dimensions that is 10% or more (e.g., 20% or more, 30% or more, 40%
or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or
more, 100% or more, 110% or more, 120% or more, 130% or more, 140%
or more, 150% or more, 160% or more, 170% or more, 180% or more,
190% or more, 200% or more) of an average displacement or a nominal
displacement between adjacent features in the array. In certain
embodiments, the distribution of displacements (linear and/or
angular) between adjacent features in an array has a full-width at
half-maximum of between 0% and 200% (e.g., between 0% and 100%,
between 0% and 75%, between 0% and 50%, between 0% and 25%, between
0% and 15%, between 0% and 10%) of an average displacement or
nominal displacement between adjacent features in the array.
[0824] In some embodiments, arrays of features can have a variable
geometry. For example, a first subset of features in an array can
be arranged according to a first geometrical pattern, and a second
subset of features in the array can be arranged according to a
second geometrical pattern that is different from the first
pattern. Any of the patterns described above can correspond to the
first and/or second geometrical patterns, for example.
[0825] In general, arrays of different feature densities can be
prepared by adjusting the spacing between adjacent features in the
array. In some embodiments, the geometric center-to-center (e.g.,
pitch) spacing between adjacent features in an array is between 100
nm to 10 .mu.m, 500 nm to 2 .mu.m, 1 .mu.m to 5 .mu.m, and 20 .mu.m
to 200 .mu.m. For example, the center-to-center spacing can be
between 100 nm to 10 .mu.m, 500 nm to 2 .mu.m, 1 .mu.m, to 5 .mu.m,
20 .mu.m to 40 .mu.m, 20 .mu.m to 60 .mu.m, 20 .mu.m to 80 .mu.m,
80 .mu.m to 100 .mu.m, 100 .mu.m to 120 .mu.m, 120 .mu.m to 140
.mu.m, 140 .mu.m to 160 .mu.m, 160 .mu.m to 180 .mu.m, 180 .mu.m to
200 .mu.m, 60 .mu.m to 100 .mu.m, or 40 .mu.m to 100 .mu.m, 50
.mu.m to 150 .mu.m, 80 .mu.m to 120 .mu.m, or 90 .mu.m to 110
.mu.m. In some embodiments, the pitch between adjacent array
features is between 30 .mu.m and 100 .mu.m, 40 .mu.m and 90 .mu.m,
50 .mu.m and 80 .mu.m, 60 .mu.m and 70 .mu.m, 80 .mu.m and 120
.mu.m, or any range within the disclosed sub-ranges. In some
embodiments, the pitch between adjacent array features of an array
is approximately 65 .mu.m, approximately 60 .mu.m, approximately 55
.mu.m, approximately 50 .mu.m, approximately 45 .mu.m,
approximately 40 .mu.m, approximately 35 .mu.m, approximately 30
.mu.m, approximately 25 .mu.m, approximately 20 .mu.m,
approximately 15 .mu.m, approximately 10 .mu.m, approximately 5
.mu.m, approximately 4 .mu.m, approximately 3 approximately 2
.mu.m, or approximately 1 .mu.m. In some embodiments, the pitch
between adjacent array features of an array is less than 100
.mu.m.
[0826] An array of features can have any appropriate resolution. In
some embodiments, an array of features can have a spatially
constant (e.g., within a margin of error) resolution. In general,
an array with a spatially consistent resolution is an array in
which the pitch between adjacent features in the array is constant
(e.g., within a margin of error). Such arrays can be useful in a
variety of applications. In some embodiments, an array of features
can have a spatially varying resolution. In general, an array with
a spatially varying resolution is an array in which the
center-to-center spacing (e.g., pitch) (along linear, angular, or
both linear and angular coordinate dimensions) between adjacent
features in the array varies. Such arrays can be useful in a
variety of applications. For example, in some embodiments,
depending upon the spatial resolution at which the sample is to be
investigated, the sample can be selectively associated with the
portion of the array that corresponds approximately to the desired
spatial resolution of the measurement.
[0827] In some embodiments, it may be useful to describe the
resolution of an array of features by functional aspects, for
example, the number of reads that can be carried out per feature
(which can be a proxy for sequencing saturation), the number of
transcripts that can be detected per feature, or the number of
genes that can be detected per feature. For example, in some
embodiments, the number of reads that can be performed per feature
is between 50,000 and 1,000,000. For example, the number of reads
that can be performed per feature can be between 50,000 and
100,000, 50,000 and 150,000, 50,000 and 200,000, 50,000 and
250,000, 50,000 and 300,000, 50,000 and 350,000, 50,000 and
400,000, 50,000 and 500,000, 50,000 and 550,000, 50,000 and
600,000, 50,000 and 650,000, 50,000 and 700,000, 50,000 and
750,000, 50,000 and 800,000, 50,000 and 850,000, 50,000 and
900,000, 50,000 and 950,000, 50,000 and 1,000,000, 100,000 to
500,000, 150,000 to 500,000, 200,000 to 500,000, 250,000 to
500,000, 300,000 and 500,000, 350,000 and 500,000, 400,000 and
500,000, 450,000 and 500,000, 150,000 to 250,000, or 300,000 to
400,000. In some embodiments, the number of reads that can be
performed per feature is about 70,000. In some embodiments, the
number of reads that can be performed per feature is about 170,000.
In some embodiments, the number reads that can be performed per
feature is about 330,000. In some embodiments, the number reads
that can be performed per feature is about 500,000. In some
embodiments, the number reads that can be performed per feature is
about 800,000.
[0828] In some embodiments, the number of transcripts that can be
detected per feature is between 20,000 and 200,000. For example, in
some embodiments, the number of transcripts that can be detected
per feature can be between 20,000 and 30,000, 20,000 and 40,000,
20,000 and 50,000, 30,000 and 60,000, 40,000 and 60,000, 50,000 and
60,000, 20,000 and 100,000, 30,000 and 100,000, 40,000 and 200,000,
50,000 and 200,000, or 30,000 and 200,000. In some embodiments, the
number of transcripts that can be detected per feature is about
40,000. In some embodiments, the number of transcripts that can be
detected per feature is about 60,000. In some embodiments, the
number of transcripts that can be detected per feature is about
80,000. In some embodiments, the number of transcripts that can be
detected per feature is about 100,000.
[0829] In some embodiments, the number of genes that can be
detected per feature is between 1,000 and 5,000. For example, the
number of genes that can be detected per feature can be between
1,000 and 1,500, 1,000 and 2,000, 1,000 and 2,500, 1,000 and 3,000,
1,000 and 3,500, 1,000 and 4,000, 1,000 and 4,500, 1,500 and 5,000,
2,000 and 5,000, 2,500 and 5,000, 3,000 and 5,000, 3,500 and 5,000,
4,000 and 5,000, 4,500 and 5,000, 1,500 and 2,500, 2,500 and 3,500,
or 3,500 and 4,000. In some embodiments, the number of genes that
can be detected per feature is about 2,000. In some embodiments,
the number of genes that can be detected per feature is about
3,000. In some embodiments, the number of genes that can be
detected per feature is about 4,000.
[0830] In some embodiments, it may be useful to describe the
resolution of an array of features by functional aspects, for
example, the number of UMI counts per feature. For example, in some
embodiments, the number of UMI counts that can be performed per
feature is between 1,000 and 50,000. In some embodiments, the
number of UMI counts can be averaged to obtain a mean UMI per
feature. In some embodiments, the number of UMI counts can be
averaged to obtain a median UMI count per feature. For example, the
median UMI count per feature can be between 1,000 and 50,000, 1,000
and 40,000, 1,000 and 30,000, 1,000 and 20,000, 1,000 and 10,000,
1,000 and 5,000. In some embodiments, the median UMI count per
feature is about 5,000. In some embodiments, the median UMI count
per feature is about 10,000.
[0831] These components can be used to determine the sequencing
saturation of the array. The sequencing saturation can be a measure
of the library complexity and sequencing depth. For example,
different cell types will have different amounts of RNA, thus
different number of transcripts, influencing library complexity.
Additionally, sequencing depth is related to the number of
sequencing reads. In some embodiments, the inverse of sequencing
saturation is the number of additional reads it would take to
detect a new transcript. One way of measuring the sequencing
saturation of an array is to determine the number of reads to
detect a new UMI. For example, if a new UMI is detected every 2
reads of the feature, the sequencing saturation would be 50%. As
another example, if a new UMI is detected every 10 reads of a
feature, the sequencing saturation would be 90%.
[0832] Arrays of spatially varying resolution can be implemented in
a variety of ways. In some embodiments, for example, the pitch
between adjacent features in the array varies continuously along
one or more linear and/or angular coordinate directions. Thus, for
a rectangular array, the spacing between successive rows of
features, between successive columns of features, or between both
successive rows and successive columns of features, can vary
continuously.
[0833] In certain embodiments, arrays of spatially varying
resolution can include discrete domains with populations of
features. Within each domain, adjacent features can have a regular
pitch. Thus, for example, an array can include a first domain
within which adjacent features are spaced from one another along
linear and/or angular coordinate dimensions by a first set of
uniform coordinate displacements, and a second domain within which
adjacent features are spaced from one another along linear and/or
angular coordinate dimensions by a second set of uniform coordinate
displacements. The first and second sets of displacements differ in
at least one coordinate displacement, such that adjacent features
in the two domains are spaced differently, and the resolution of
the array in the first domain is therefore different from the
resolution of the array in the second domain.
[0834] In some embodiments, the pitch of array features can be
sufficiently small such that array features are effectively
positioned continuously or nearly continuously along one or more
array dimensions, with little or no displacement between array
features along those dimensions. For example, in a feature array
where the features correspond to regions of a substrate (i.e.,
oligonucleotides are directly bound to the substrate), the
displacement between adjacent oligonucleotides can be very
small--effectively, the molecular width of a single
oligonucleotide. In such embodiments, each oligonucleotide can
include a distinct spatial barcode such that the spatial location
of each oligonucleotide in the array can be determined during
sample analysis. Arrays of this type can have very high spatial
resolution, but may only include a single oligonucleotide
corresponding to each distinct spatial location in a sample.
[0835] In general, the size of the array (which corresponds to the
maximum dimension of the smallest boundary that encloses all
features in the array along one coordinate direction) can be
selected as desired, based on criteria such as the size of the
sample, the feature diameter, and the density of capture probes
within each feature. For example, in some embodiments, the array
can be a rectangular or square array for which the maximum array
dimension along each coordinate direction is 10 mm or less (e.g., 9
mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less,
4 mm or less, 3 mm or less). Thus, for example, a square array of
features can have dimensions of 8 mm by 8 mm, 7 mm by 7 mm, 5 mm by
5 mm, or be smaller than 5 mm by 5 mm.
[0836] (v) Bead Arrays
[0837] As used herein, the term "bead array" refers to an array
that includes a plurality of beads as the features in the array. In
some embodiments, two or more beads are dispersed onto a substrate
to create an array, where each bead is a feature on the array. In
some embodiments, the beads are attached to a substrate. For
example, the beads can optionally attach to a substrate such as a
microscope slide and in proximity to a biological sample (e.g., a
tissue section that includes cells). The beads can also be
suspended in a solution and deposited on a surface (e.g., a
membrane, a tissue section, or a substrate (e.g., a microscope
slide)). Beads can optionally be dispersed into wells on a
substrate, e.g., such that only a single bead is accommodated per
well.
[0838] Examples of arrays of beads on or within a substrate include
beads located in wells such as the BeadChip array (available from
Illumina Inc., San Diego, Calif.), arrays used in sequencing
platforms from 454 LifeSciences (a subsidiary of Roche, Basel,
Switzerland), and array used in sequencing platforms from Ion
Torrent (a subsidiary of Life Technologies, Carlsbad, Calif.).
Examples of bead arrays are described in, e.g., U.S. Pat. Nos.
6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568;
and 6,274,320; U.S. Pat. Application Publication Nos. 2009/0026082;
2009/0127589; 2010/0137143; 2019/0177777; and 2010/0282617; and PCT
Patent Application Publication Nos. WO 00/063437 and WO
2016/162309, the entire contents of each of which is incorporated
herein by reference.
[0839] In some embodiments, the bead array includes a plurality of
beads. For example, the bead array can include at least 10,000
beads (e.g., at least 100,000 beads, at least 1,000,000 beads, at
least 5,000,000 beads, at least 10,000,000 beads). In some
embodiments, the plurality of beads includes a single type of bead
(e.g., substantially uniform in volume, shape, and other physical
properties, such as translucence). In some embodiments, the
plurality of beads includes two or more types of different
beads.
[0840] Bead arrays can be generated by attaching beads (e.g.,
barcoded beads) to a substrate in a regular pattern, or an
irregular arrangement. In some embodiments, the barcode sequences
are known before attaching them to the substrate. In some
embodiments, the barcode sequences are not known before attaching
them to the substrate. Beads can be attached to selective regions
on a substrate by, e.g., selectively activating regions on the
substrate to allow for attachment of the beads. Activating
selective regions on the substrate can include activating or
degrading a coating (e.g., a conditionally removable coating as
described herein) at the selective regions where the coating has
been applied on the substrate, rendering the selective regions more
permissive to bead attachment as compared to regions outside of the
selected regions. The regions that are rendered more permissive for
bead attachment can be configured to fit only one bead or multiple
beads (e.g., limited by well size or surface patterning, such as
fabrication techniques). Beads bound to the selected regions can
form a two-dimensional array on the substrate. The substrate can be
uniformly or non-uniformly coated with the coating. The beads can
be any suitable beads described herein, including beads that are
attached to one or more spatial barcodes. Beads can be attached to
the selected regions according to any of the methods suitable for
attaching beads to substrates described herein, such as through
covalent bonds, non-covalent bonds, or chemical linkers.
[0841] Any variety of suitable patterning techniques can be used to
attach beads to a substrate surface. In some embodiments, in a
non-limiting way, physical techniques such as inkjet printing,
optical and optoelectronic cell trapping, laser-based patterning,
acoustic patterning, dielectrophoresis, or magnetic techniques can
be used to pattern the substrate. Alternatively, chemical and/or
physiochemical techniques can be used such as, in a non-limiting
way, surface chemistry methods, microcontact printing, microwells
and filtration, DUV patterning, or patterning in microfluidic
devices combined with microcontact printing (See, e.g.,
Martinez-Rivas, A., Methods of micropatterning and manipulation of
cells for biomedical applications, Micromachines (Basel) 8, (2017),
which in is incorporated herein by reference).
[0842] The coating can be photoreactive, and selectively activating
or degrading the coating involves exposing selected regions of the
coating to light or radiation. Selectivity can be achieved through
the application of photomasks. Regions of the coating that are
exposed to light can be rendered more permissive for bead
attachment (e.g., more adhesive), as compared to regions not
exposed to light (e.g., regions protected from the light by a
photomask). When applied to the substrate, the beads thus
preferentially attach to the more permissive regions on the
substrate, and un-attached beads can optionally be removed from the
substrate. The light source and/or the photomask can be adjusted to
allow further sites on the substrate to become more permissive for
bead attachment, allowing additional beads to be attached at these
sites. Alternatively, a different light source, or a different
photomask can be applied. The process of photopatterning thus
allows beads to be attached at pre-determined locations on the
substrate, thereby generating a bead array.
[0843] Beads can be attached iteratively, e.g., a subset of the
beads can be attached at one time, and the process can be repeated
to attach one or more additional subsets of beads. In some
embodiments, the size of the activated spot (e.g., spot on the
substrate) is smaller than the size of a bead. For example, a bead
can be attached to the activated substrate (e.g., spot) such that
only a single bead attaches to the activated substrate. In some
embodiments, the substrate can be washed to remove unbound beads.
In some embodiments, the substrate can be activated in a second
location and a second bead can be attached to the activated
substrate surface. This process can be done iteratively to attach
beads to the entire substrate, or a portion thereof. Alternatively,
beads can be attached to the substrate all in one step.
Furthermore, methods of attaching beads to a substrate are known in
the art. Any suitable method can be used, including, in a
non-limiting way, specific chemical bonds, non-specific chemical
bonds, linkers, physically trapping the beads (e.g., polymer,
hydrogel), or any of the methods described herein.
[0844] An exemplary workflow for generating a bead array can
include selectively rendering a first set of one or more selected
regions on a coated substrate to be more permissive for bead
attachment as compared to regions outside of the selected regions,
applying a plurality of beads to the array and allowing the beads
to attach to the first set of selected regions, optionally removing
un-attached beads, rendering a second set of one or more selected
regions more permissive to bead attachment as compared to regions
outside the second set of selected regions, applying a plurality of
beads and allowing the beads to attach to the second set of
selected regions, and optionally removing the un-attached beads.
This iterative process can be carried out for any number of times
to generate a patterned bead array.
[0845] Another exemplary process includes activating a first region
on a coated substrate and exposing the activated first region to a
plurality of barcoded beads, so that a first set of one or more
beads are bound to the first region; and activating a second region
on the coated substrate and exposing the activated second region to
a plurality of barcoded beads, so that a second set of one or more
beads are bound to the second region, wherein the first set of one
or more beads comprise an identical first oligonucleotide sequence
unique to the first region of the surface of the substrate, and the
second set of one or more beads comprise an identical second
oligonucleotide sequence unique to the second region of the surface
of the substrate, and wherein the first and second oligonucleotide
sequences are different. Additional regions on the coated substrate
may be activated and exposed to additional barcoded beads. Each set
of barcoded beads can include an oligonucleotide sequence that is
different from all other sets of barcoded beads and that is unique
to the location of the activated region. Additionally, the first
set of one or more beads and the second set of one or more beads
can be different. In other words, the first set of one or more
beads and the second set of one or more beads can have different
surface chemistries, different compositions (e.g., solid bead, gel
bead, silica bead)(e.g., nanoparticles vs microparticles), and/or
physical volumes. In some embodiments, a third set of one or more
beads, a fourth set of one or more beads, a fifth set of one or
more beads or more can have different surface chemistries,
different compositions (e.g., solid bead, gel bead, silica
bead)(e.g., nanoparticles vs microparticles), and/or physical
volumes can be attached to the substrate surface. The methods may
include removing the beads that do not bind to the first, second,
and/or any of the additional regions. In some embodiments, removing
the beads comprise washing the beads off the surface of the
substrate. The removing may be carried out after each round of or
after several rounds of activating a region (e.g., first, second or
additional regions on the surface of the substrate), and binding of
beads to the activated region. In some instances, each bead is
bound to the substrate at a single location. The beads bound to the
first, second, and additional regions can form a two-dimensional
array of beads on the substrate.
[0846] A photoreactive coating can comprise a plurality of
photoreactive molecules, which can undergo a chemical reaction
(e.g., hydrolysis, oxidation, photolysis) when exposed to light of
certain wavelengths or range of wavelengths. A photo-reactive
molecule can become reactive when exposed to light and can react
with other molecules and form chemical bonds with other
molecules.
[0847] The coating can comprise a polymer, and activating selected
regions on the substrate include modifying the polymer at the
respective regions. Modifying the polymer includes, for example,
photochemically modifying the polymer by exposing the polymer to
radiation or light. Alternatively or additionally, modifying the
polymer can include chemically modifying the polymer by contacting
the polymer with one or more chemical reagents. In some instances,
the coating is a hydrogel. In some instances, the coating comprises
a photoreactive polymer. Exemplary photo-reactive polymers include
poly(ethylene glycol) (PEG)-based polymers, poly(L-lysine)
(PLL)-based polymer, copolymer comprising functionalized or
unfunctionalized units of PEG and PLL (e.g.,
poly-L-lysine-grafted-polyethylene glycol (PLL-g-PEG)), and
methacrylated gelatin (GelMA) polymers.
[0848] Beads can also be attached to selective regions on a
substrate by selectively crosslinking beads to a coating that has
been applied on the substrate. For example, a plurality of beads
can be applied to a substrate having a photocrosslinkable coating,
and upon crosslinking of a subset of the beads to the coating, the
non-cross-linked beads can be removed, leaving only the
cross-linked beads on the substrate. The process can be repeated
multiple times. The coating can include a photo-crosslinkable
polymer. Exemplary photo-crosslinkable polymers are described,
e.g., in Shirai, Polymer Journal 46:859-865 (2014), Ravve,
Photocrosslinkable Polymers, Light-Associated Reactions of
Synthetic Polymers. Springer, New York, N.Y. (2006), and Ferreira
et al. Photocrosslinkable Polymers for Biomedical Applications,
Biomedical Engineering--Frontiers and Challenges, Prof. Reza Fazel
(Ed.), ISBN: 978-953-307-309-5 (2011), each of which are herein
incorporated by reference in its entirety.
[0849] Suitable light sources for activating, degrading or
crosslinking the coating as described herein include, but are not
limited to, Ultraviolet (UV) light (e.g., 250-350 nm or 350-460 nm
UV light) and visible light (e.g., broad spectrum visible light). A
Digital Micromirror Device (DMD) can also be used to provide the
light source.
[0850] The distance between a first pair of adjacent selected
regions according to the methods described herein can be the same
or different from a second pair of adjacent selected regions.
[0851] Barcoded beads, or beads comprising a plurality of barcoded
probes, can be generated by first preparing a plurality of barcoded
probes on a substrate, depositing a plurality of beads on the
substrate, and generating probes attached to the beads using the
probes on the substrate as a template.
[0852] Large scale commercial manufacturing methods allow for
millions of oligonucleotides to be attached to an array.
Commercially available arrays include those from Roche NimbleGen,
Inc., (Wisconsin) and Affymetrix (ThermoFisher Scientific).
[0853] In some embodiments, arrays can be prepared according to the
methods set forth in WO 2012/140224, WO 2014/060483, WO
2016/162309, WO 2017/019456, WO 2018/091676, and WO 2012/140224,
and U.S. Patent Application No. 2018/0245142. The entire contents
of each of the foregoing documents are herein incorporated by
reference.
[0854] In some embodiments, a bead array is formed when beads are
embedded in a hydrogel layer where the hydrogel polymerizes and
secures the relative bead positions. The bead-arrays can be
pre-equilibrated and combined with reaction buffers and enzymes
(e.g., reverse-transcription mix). In some embodiments, the bead
arrays can be stored (e.g., frozen) long-term (e.g., days) prior to
use.
[0855] (vi) Flexible Arrays
[0856] A "flexible array" includes a plurality of
spatially-barcoded features attached to, or embedded in, a flexible
substrate (e.g., a membrane, a hydrogel, or tape) placed onto, or
proximal to, a biological sample. In some embodiments, a flexible
array includes a plurality of spatially-barcoded features embedded
within a hydrogel.
[0857] Flexible arrays can be highly modular. In some embodiments,
spatially-barcoded features (e.g., beads) can be loaded onto a
substrate (e.g., a slide) to produce a high-density self-assembled
array. In some embodiments, the features (e.g., beads) can be
loaded onto the substrate with a flow cell. In some embodiments,
the features (e.g., beads) are embedded in a hydrogel (e.g., a
hydrogel pad or layer placed on top of the self-assembled
features). In some embodiments, the hydrogel can polymerize,
thereby securing the features in the hydrogel. In some embodiments,
the hydrogel containing the features can be removed from the
substrate and used as a flexible array. In some embodiments, the
flexible array can be deconvolved by optical sequencing or any
other method described herein. In some embodiments, the features
(e.g., beads) can be about 1 .mu.m to about 25 .mu.m in diameter.
In some embodiments, about 25 .mu.m diameter features in the
flexible array can provide for approximately 1000 DPI and about 1
megapixel resolution. In some embodiments, the features (e.g.,
beads) can be about 13.2 .mu.m in diameter. In some embodiments,
the about 13.2 .mu.m beads in the flexible array can provide for
approximately 1920.times.1080 resolution.
[0858] Flexible arrays generated according to any of the methods
described herein (e.g., beads embedded within a hydrogel) can
contain a thermolabile polymer. In some embodiments, flexible
arrays having thermolabile beads can be contacted with a biological
sample. In some embodiments, a region of interest in the biological
sample can be identified such that an infrared laser can be used to
select a region of interest. In some embodiments, the infrared
laser can cause the flexible array (e.g., thermolabile beads) to
deform and become adhesive. In some embodiments, the adhesive
portion of the flexible array can adhere (e.g., bind) to the region
of interest (e.g., cells) directly above or underneath. The process
of identifying a region of interest, applying an infrared laser to
the region of interest, and adhering the underlying biological
sample (e.g., cells) to the flexible array can iteratively
repeated. In some embodiments, the flexible array can be removed
such that only the adhered biological sample (e.g., cells) from the
one or more regions of interest can also be removed with the
flexible array. In some embodiments, the flexible array and the
adhered biological sample can be further processed (e.g.,
amplified, quantitated, and/or sequenced) according to any method
described herein.
[0859] Flexible arrays can be pre-equilibrated with reaction
buffers and enzymes at functional concentrations (e.g., a
reverse-transcription mix). In some embodiments, the flexible
arrays can be stored for extended periods (e.g., days) or frozen
until ready for use. In some embodiments, permeabilization of
biological samples (e.g., a tissue section) can be performed with
the addition of enzymes/detergents prior to contact with the
flexible array. In some embodiments, the flexible array can be
placed directly on the sample, or placed in indirect contact with
the sample (e.g., with an intervening layer or substance between
the biological sample and the flexible bead-array). In some
embodiments, the flexible array can be mechanically applied (e.g.,
pressed downward or compressed between two surfaces) on the
biological sample to enhance analyte capture. In some embodiments,
a flexible array can be applied to the side of a biological sample.
For example, a biological sample can be cut (e.g., sliced) in any
direction and a flexible array can be applied to the exposed
analytes. In some embodiments, the flexible array can be
dissolvable (e.g., via heat, chemical, or enzymatic disruption). In
some embodiments, once a flexible array is applied to the sample,
reverse transcription and targeted capture of analytes can be
performed on microspheres, or beads of a first volume and beads of
a second volume, or any of the beads described herein. In some
embodiments once a flexible array is applied to the biological
sample and allowed to capture analytes, the flexible array can be
removed (e.g., peeled) from the biological sample for further
processing (e.g., amplification, quantitation, and/or sequencing)
according to any method described herein.
[0860] Flexible arrays can also be used with any of the methods
(e.g., active capture methods such as electrophoresis) described
herein. For example, flexible arrays can be contacted with a
biological sample on a conductive substrate (e.g., an indium tin
oxide coated glass slide), such that an electric field can be
applied to the conductive substrate to facilitate migration of
analytes through, across, within, or in the direction of the
flexible array. Additionally and alternatively, flexible arrays can
be contacted to a biological sample in an electrophoretic assembly
(e.g., electrophoretic chamber), such that an electric field can be
applied to migrate analytes in the direction of the flexible array
or across, through, or within the flexible array.
[0861] In some embodiments, a flexible array can be generated with
the assistance of a substrate holder (e.g., any array alignment
device). For example, a spatially-barcoded bead array can be placed
in one placeholder of the substrate holder and second substrate
(e.g., a glass slide) can be placed in the second placeholder of
the substrate holder. In some embodiments, the array is optionally
optically decoded and a gel prepolymer solution is introduced
between the spatially-barcoded bead array and second substrate. In
some embodiments, the substrate holder is closed such that the
second substrate is on top (e.g., above, parallel to) the
spatially-barcoded bead array. The gel prepolymer solution can be
polymerized by any method described herein and result in
spatially-barcoded features cross-linked in the hydrogel, thereby
generating a flexible array. In some embodiments, the substrate
holder can be opened and the second substrate with the hydrogel and
the spatially-barcoded cross-linked features can be removed from
the substrate holder (the flexible array optionally can be removed
from the second substrate) for use in spatial analysis by any of
the methods described herein.
[0862] (vii) Shrinking Hydrogel Features/Arrays
[0863] As used herein "shrinking" or "reducing the size" of a
hydrogel refers to any process causing the hydrogel to physically
contract and/or the size of the hydrogel to decrease in volume. For
example, the scaffold of the gel may shrink or "implode" upon
solvent removal (see, e.g., Long and Williams. Science. 2018;
362(6420):1244-1245, and Oran et al. Science 2018; 362(6420):
1281-1285; each of which is incorporated herein by reference in its
entirety). As another example, the process to shrink or reduce the
volume of a hydrogel may be one that removes water (i.e., a
dehydrating process) from the hydrogel. There are many methods
known to one of skill in the art for shrinking or reducing the
volume of a hydrogel. Non-limiting examples of a method to shrink
or reduce the volume of a hydrogel include exposing the hydrogel to
one or more of: a dehydrating solvent, a salt, heat, a vacuum,
lyophilization, desiccation, filtration, air-drying, or
combinations thereof.
[0864] In some embodiments, a hydrogel bead can be decreased in
volume (e.g., shrunken hydrogel bead) before being attached to or
embedded in a hydrogel. In some embodiments, a hydrogel bead can be
decreased in volume (e.g., shrunken hydrogel bead) after being
attached to or embedded in a hydrogel. In some embodiments, one or
more hydrogel beads can be attached to or embedded in a hydrogel.
In some embodiments, one or more hydrogel beads can be decreased in
volume (e.g., one or more shrunken hydrogel beads) before being
attached to or embedded in a hydrogel. In some embodiments, one or
more hydrogel beads can be decreased in volume (e.g., one or more
shrunken hydrogel beads) after being attached to or embedded in a
hydrogel. In some embodiments, one or more hydrogel beads attached
to or embedded in a hydrogel can be decreased in volume. For
example, the one or more hydrogel beads and the hydrogel that the
hydrogel beads are attached to or embedded in are decreased in
volume at the same time (e.g., shrunken hydrogel bead-containing
hydrogel). In some embodiments, one or more hydrogel beads attached
to or embedded in a hydrogel can be isometrically decreased in
volume.
[0865] In some embodiments, one or more hydrogel beads attached to
or embedded in a hydrogel can be decreased in volume from about 3
fold to about 4 fold. For example, one or more hydrogel beads
attached to or embedded in a hydrogel can be decreased in volume by
removing or exchanging solvents, salts, or water (e.g.,
dehydration). In another example, one or more hydrogel beads
attached to or embedded in a hydrogel can be decreased in volume by
controlling temperature or pH. See e.g., Ahmed, E. M. J. of
Advanced Research. 2015 March; 6(2):105-121, which is incorporated
herein by reference in its entirety. In some embodiments, one or
more hydrogel beads attached to or embedded in a hydrogel can be
decreased in volume by removing water.
[0866] In some embodiments, decreasing the volume of one or more
hydrogel beads attached to or embedded in a hydrogel can increase
the spatial resolution of the subsequent analysis of the sample.
The increased resolution in spatial profiling can be determined by
comparison of the spatial analysis of the sample using one or more
shrunken hydrogel beads attached to or embedded in a hydrogel with
one or more non-shrunken hydrogel beads attached to or embedded in
a hydrogel.
[0867] In some embodiments, a hydrogel bead is not decreased in
volume. In some embodiments, a hydrogel bead can be decreased in
volume (e.g., shrunken hydrogel bead). In some embodiments, a
shrunken hydrogel gel bead is stabilized. For example, the hydrogel
bead can be decreased in volume by removing solvents, salts, or
water (e.g., dehydrated, desiccated, dried, exsiccated) from the
hydrogel bead to form a shrunken hydrogel bead. In another example,
the hydrogel bead can be decreased in volume by controlling
temperature or pH. See e.g., Ahmed, E. M. J. of Advanced Research.
2015 March; 6(2):105-121, which is incorporated herein by reference
in its entirety. Non-limiting examples of solvents that may be used
to form a shrunken hydrogel bead or shrunken hydrogel bead array
include a ketone, such as methyl ethyl ketone (MEK), isopropanol
(IPA), acetone, 1-butanol, methanol (MeOH), dimethyl sulfoxide
(DMSO), glycerol, propylene glycol, ethylene glycol, ethanol, (k)
1,4-dioxane, propylene carbonate, furfuryl alcohol,
N,N-dimethylformamide (DMF), acetonitrile, aldehyde, such as
formaldehyde or glutaraldehyde, or any combinations thereof.
[0868] In some embodiments, the hydrogel bead or hydrogel bead
array is shrunken or stabilized via a cross-linking agent. For
example, the cross-linking agent may comprise disuccinimidyl
suberate (DSS), dimethylsuberimidate (DMS), formalin, and
dimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate)
(DSP), disuccinimidyl tartrate (DST), and ethylene glycol
bis(succinimidyl succinate) (EGS).
[0869] In some embodiments, the hydrogel bead or hydrogel bead
array is processed with salts to form a shrunken hydrogel bead or
shrunken hydrogel bead array. Non-limiting examples of salts that
may be used to form a shrunken hydrogel bead or shrunken hydrogel
bead array are inorganic salts including aluminum, ammonium,
barium, beryllium, calcium, cesium, lithium, magnesium, potassium,
rubidium, sodium, and strontium salts. Further non-limiting
examples of inorganic salts include sodium chloride, potassium
chloride, lithium chloride, cesium chloride, sodium fluoride,
sodium bromide, sodium iodide, sodium nitrite, potassium sulfate,
potassium nitrate, potassium carbonate, potassium bicarbonate,
sodium sulfate, sodium nitrate, sodium carbonate, sodium
bicarbonate, calcium sulfate, copper oxychloride, calcium chloride,
calcium carbonate, calcium bicarbonate, magnesium sulfate,
magnesium nitrate, magnesium chloride, magnesium carbonate,
magnesium bicarbonate, ammonium sulfate, ammonium chloride,
ammonium nitrate, ammonium carbonate, ammonium bicarbonate,
trisodium phosphate, tripotassium phosphate, calcium phosphate,
copper(II) sulfate, sodium sulfide, potassium sulfide, calcium
sulfide, potassium permanganate, iron(II) chloride, iron(III)
chloride, iron (2+) sulfate, iron(III) sulfate, iron(II) nitrate,
iron(III) nitrate, manganese(II) chloride, manganese(III) chloride,
manganese(II) sulfate, manganese(II) nitrate, zinc chloride, zinc
nitrate, zinc sulfate, ammonium orthomolybdate, monopotassium
phosphate, nickel(II) sulfate, nickel(II) nitrate, sodium
metavanadate, sodium paravanadate, potassium dichromate, ammonium
dichromate, antipyonin, ammonium nitrite, potassium fluoride,
sodium fluoride, ammonium fluoride, calcium fluoride, chrome alum,
potassium alum, potassium iodide, sodium hypochlorite, tin(II)
sulfate, tin(II) nitrate, gold selenite, dicesium chromate,
potassium perchlorate, calcium perchlorate, aluminum sulphate,
lead(II) bisulfate, barium phosphate, barium hydrogen
orthophosphate, barium dihydrogen phosphate, silver dichromate,
potassium bromate, sodium bromate, sodium iodate, sodium silicate,
diammonium phosphate, ammonium phosphate, ammonium dihydrogen
phosphate, chromium orthophosphate, copper(II) chloride, copper(I)
chloride, sodium tetrametaphosphate, potassium heptafluoroniobate,
zinc phosphate, sodium sulfite, copper(I) nitrate, copper(II)
nitrate, potassium silicate, copper(II) carbonate basic, copper(II)
carbonate salts of acrylic acid and sulfopropyl acrylate.
[0870] In some embodiments, the removal of water comprises an acid.
Non-limiting examples of an acid include: HCl, HI, HBr, HClO4,
HClO3, HNO3, H2SO4, phosphoric acid, phosphorous acid, acetic acid,
oxalic acid, ascorbic acid, carbonic acid, sulfurous acid, tartaric
acid, citric acid, malonic acid, phthalic acid, barbituric acid,
cinnamic acid, glutaric acid, hexanoic acid, malic acid, folic
acid, propionic acid, stearic acid, trifluoroacetic acid,
acetylsalicylic acid, glutamic acid, azelaic acid, benzilic acid,
fumaric acid, gluconic acid, lactic acid, oleic acid, propiolic
acid, rosolic acid, tannic acid, uric acid, gallic acid, and
combinations of two or more thereof. In some embodiments, the
hydrogel is exposed to a different pH environment. For example, the
hydrogel can be exposed to an acidic pH or a basic pH. In some
embodiments, the hydrogel is exposed to a pH of less than about
6.5, e.g., a pH of about 6, about 5.5, about 5, about 4.5, about 4,
about 3.5, about 3, about 2.5, about 2, about 1.5, or about 1. In
some embodiments, the hydrogel is exposed to a pH of greater than
about 7.5, e.g., a pH of about 8, about 8.5, about 9, about 9.5,
about 10, about 10.5, about 11, about 11.5, about 12, about 12.5,
about 13, about 13.5, or about 14.
[0871] In some embodiments, the removal of water comprises a
dehydrating process such as heat, a vacuum, lyophilization,
desiccation, filtration, and air-drying. In some embodiments, the
hydrogel bead or hydrogel bead array undergoes an alteration in pH
to form a shrunken hydrogel bead or shrunken hydrogel bead array
(e.g., an alteration from about pH 7 to about pH 5, from about pH 7
to about pH 5.5, from about pH 7 to about pH 6, from about pH 7 to
about pH 6.5, from about pH 6.5 to about pH 5, from about pH 6 to
about pH 5, from about pH 6 to about pH 5.5, or any pH alteration
encompassed within these ranges).
[0872] In some embodiments, the hydrogel bead or hydrogel bead
array undergoes an alteration in temperature (e.g., an alteration
from about 37.degree. C., 38.degree. C., 39.degree. C., 40.degree.
C., 41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C. to about 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C. 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., 70.degree. C., or higher, or any temperature
alteration encompassed within these ranges) to form a shrunken
hydrogel bead or shrunken hydrogel bead array.
[0873] In some embodiments, a hydrogel bead can be decreased in
size in linear dimension by about 2 fold, about 3 fold, about 4
fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about
9 fold, or any intervals therein. In some embodiments, a hydrogel
bead can be decreased in volume by about 1 fold, about 5 fold,
about 10 fold, about 15 fold, about 20 fold, about 25 fold, about
30 fold, about 35 fold, about 40 fold, about 45 fold, about 50
fold, about 55 fold, about 60 fold, about 65 fold about 70 fold,
about 75 fold, about 80 fold, or any intervals therein. In some
embodiments, a hydrogel bead can be decreased in size such that the
hydrogel bead has an average diameter of about 1 .mu.m to about 15
.mu.m.
[0874] In some embodiments, a plurality of hydrogel beads can be
decreased in size in linear dimension by about 2 fold, about 3
fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about
8 fold, about 9 fold, or any intervals therein. In some
embodiments, a plurality of hydrogel beads can be decreased in size
such that the average diameter of a hydrogel bead is about 1 .mu.m
to about 15 .mu.m. In some embodiments, a plurality of hydrogel
beads can be decreased in volume by about 1 fold, about 5 fold,
about 10 fold, about 15 fold, about 20 fold, about 25 fold, about
30 fold, about 35 fold, about 40 fold, about 45 fold, about 50
fold, about 55 fold, about 60 fold, about 65 fold about 70 fold,
about 75 fold, about 80 fold, or any intervals therein.
[0875] In some embodiments, a plurality of hydrogel beads can be
decreased in volume such that the hydrogel bead has an average
diameter of about 1 .mu.m to about 15 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 15 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 14 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 13 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 12 .mu.m.
In some embodiments, a plurality of shrunken hydrogel beads has an
average diameter of about 11 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 10 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 9 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 8 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 7 .mu.m.
In some embodiments, a plurality of shrunken hydrogel beads has an
average diameter of about 6 .mu.m. In some embodiments, a plurality
of shrunken hydrogel beads has an average diameter of about 5
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 4 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 3 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 2 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 1 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 14-15
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 13-15 .mu.m. In some embodiments,
a plurality of shrunken hydrogel beads has an average diameter of
about 12-15 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 11-15 .mu.m. In
some embodiments, a plurality of shrunken hydrogel beads has an
average diameter of about 10-15 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 9-15 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 8-15 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 7-15 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 6-15
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 1-10 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 1-5 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 1-3 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 13-14 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 12-14
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 11-14 .mu.m. In some embodiments,
a plurality of shrunken hydrogel beads has an average diameter of
about 10-14 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 9-14 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 8-14 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 7-14
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 6-14 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 5-14 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 12-13 .mu.m. In
some embodiments, a plurality of shrunken hydrogel beads has an
average diameter of about 11-13 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 10-13 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 9-13 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 8-13 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 7-13
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 6-13 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 5-13 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 11-12 .mu.m. In
some embodiments, a plurality of shrunken hydrogel beads has an
average diameter of about 10-12 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 9-12 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 8-12 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 7-12 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 6-12
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 5-12 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 10-11 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 9-11 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 8-11 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 7-11
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 6-11 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 5-11 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 9-10 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 8-10 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 7-10
.mu.m. In some embodiments, a plurality of shrunken hydrogel beads
has an average diameter of about 6-10 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 5-10 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 8-9 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 7-9 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 6-9 .mu.m.
In some embodiments, a plurality of shrunken hydrogel beads has an
average diameter of about 5-9 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 7-8 .mu.m. In some embodiments, a plurality of shrunken
hydrogel beads has an average diameter of about 6-8 .mu.m. In some
embodiments, a plurality of shrunken hydrogel beads has an average
diameter of about 5-8 .mu.m. In some embodiments, a plurality of
shrunken hydrogel beads has an average diameter of about 6-7 .mu.m.
In some embodiments, a plurality of shrunken hydrogel beads has an
average diameter of about 5-7 .mu.m. In some embodiments, a
plurality of shrunken hydrogel beads has an average diameter of
about 5-6 .mu.m.
[0876] In some embodiments, one or more hydrogel beads can be
decreased in volume at the same time. In some embodiments, one or
more hydrogel beads can be decreased in volume at different times.
In some embodiments, one or more hydrogel beads can be assembled
into an array before decreasing the volume of the one or more
hydrogel beads. In some embodiments, one or more hydrogel beads can
be assembled into an array after decreasing the volume of the one
or more hydrogel beads. In some embodiments, the one or more
shrunken hydrogel beads can be reversibly attached to a substrate.
In some embodiments, the one or more shrunken hydrogel beads can be
irreversibly attached to a substrate. In some embodiments, the one
or more shrunken hydrogel beads can be re-expanded. In some
embodiments, the one or more shrunken hydrogel beads can be
isometrically re-expanded. In some embodiments, the one or more
shrunken hydrogel beads can be re-expanded primarily in the
z-dimension. In some embodiments, the one or more shrunken hydrogel
beads attached to a substrate (e.g., reversibly or irreversibly)
can be re-expanded primarily in the z-dimension. In some
embodiments, the one or more shrunken hydrogel beads attached to a
substrate (e.g., reversibly or irreversibly) can be isometrically
re-expanded primarily in the z-dimension.
[0877] In some embodiments, decreasing the volume of the hydrogel
bead (e.g., shrunken hydrogel bead) can increase the spatial
resolution of the subsequent analysis of the sample. The increased
resolution in spatial profiling can be determined by comparison of
spatial analysis of the sample using a shrunken hydrogel bead with
a non-shrunken hydrogel bead. For example, in some embodiments, the
subsequent analysis of the sample can include any array-based
spatial analysis method disclosed herein.
[0878] In some embodiments, one or more physical parameters or
dimensions and/or one or more other characteristics of the hydrogel
bead may be changed. For example, a cross-section of the hydrogel
bead may be changed from a first cross-section to a second
cross-section. The first cross-section may be smaller or larger
than the second cross-section. Alternatively, or in addition, one
or more other characteristics of the hydrogel bead may be changed.
For example, the fluidity, density, rigidity, porosity, refractive
index, polarity, and/or other characteristic of the hydrogel bead
or one or more components thereof may be changed. In a non-limiting
example, the hydrogel bead includes a hydrogel. In another example,
the hydrogel bead hydrogel may form crosslinks within the bead. The
same or different conditions may be used to change or affect
different characteristics of the hydrogel bead at the same or
different times. In some cases, a first condition or set of
conditions may be used to change a first characteristic or set of
characteristics of the hydrogel bead (e.g., a cross-section) and a
second condition or set of conditions may be used to change a
second characteristic or set of characteristics of the hydrogel
bead. The first condition or set of conditions may be applied at
the same or a different time as the second condition or set of
conditions. For example, a first characteristic or set of
characteristics may be changed under a first condition or set of
conditions, after which a second characteristic or set of
characteristics may be changed under a second condition or set of
conditions.
[0879] A characteristic or set of characteristics of the hydrogel
bead may be changed by one or more conditions. A condition suitable
for changing a characteristic or set of characteristics of the
hydrogel bead may be, for example, a temperature, a pH, an ion or
salt concentration, a pressure, chemical species, any combinations
thereof, or another condition. For example, hydrogel bead may be
exposed to a chemical species that may bring about a change in one
or more characteristics of the hydrogel bead. In some cases, a
stimulus may be used to change one or more characteristics of the
hydrogel bead. For example, upon application of the stimulus, one
or more characteristics of the hydrogel bead may be changed. The
stimulus may be, for example, a thermal stimulus, a photo stimulus,
a chemical stimulus, or another stimulus. A temperature sufficient
for changing one or more characteristics of the hydrogel bead may
be, for example, at least about 0 degrees Celsius (.degree. C.),
1.degree. C., 2.degree. C., 3.degree. C., 4.degree. C., 5.degree.
C., 10.degree. C., or higher. For example, the temperature may be
about 4.degree. C. In other cases, a temperature sufficient for
changing one or more characteristics of the hydrogel bead may be,
for example, at least about 25.degree. C., 30.degree. C.,
35.degree. C., 37.degree. C., 40.degree. C., 45.degree. C.,
50.degree. C., or higher. For example, the temperature may be about
37.degree. C. A pH sufficient for changing one or more
characteristics of the hydrogel bead may be, for example, between
about 5 and 8, such as between about 6 and 7.
[0880] In some cases, a chemical species or a chemical stimulus may
be used to change one or more characteristics of the hydrogel bead.
For example, a chemical species or a chemical stimulus may be used
to change a dimension of a hydrogel bead (e.g., a cross-section,
diameter, or volume). In some cases, a chemical species or a
chemical stimulus may be used to change a dimension of a hydrogel
bead (e.g., a cross-sectional diameter) from a first dimension to a
second dimension (e.g., a second cross-sectional dimeter), where
the second dimension is reduced compared to the first dimension.
The chemical species may comprise an organic solvent, such as an
alcohol, ketone, or aldehyde. For example, the chemical species may
comprise acetone, methanol, ethanol, formaldehyde, or
glutaraldehyde. The chemical species may comprise a cross-linking
agent. For example, the cross-linking agent may comprise
disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS),
formalin, and dimethyladipimidate (DMA), dithio-bis(-succinimidyl
propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene
glycol bis(succinimidyl succinate) (EGS), and any combinations
thereof. In some cases, a cross-linking agent may be a
photo-cleavable cross-linking agent. In some cases, a chemical
stimulus may be used to change one or more characteristics of the
hydrogel bead (e.g., a dimension of a hydrogel bead), where the
chemical stimulus comprises one or more chemical species. For
example, the chemical stimulus may comprise a first chemical
species and a second chemical species, where the first chemical
species is an organic solvent and the second chemical species is a
cross-linking agent. In some cases, a chemical stimulus may
comprise a chemical species that is a fixation agent that is
capable of fixing or preserving a hydrogel bead. For example, an
organic solvent such as an alcohol (e.g., ethanol or methanol),
ketone (e.g., acetone), or aldehyde (e.g., formaldehyde or
glutaraldehyde), or any combinations thereof may act as a fixation
agent. Alternatively, or in addition, a cross-linking agent may act
as a fixation agent. In some cases, a fixation agent may comprise
disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS),
formalin, and dimethyladipimidate (DMA), dithio-bis(-succinimidyl
propionate) (DSP), disuccinimidyl tartrate (DST), and/or ethylene
glycol bis(succinimidyl succinate) (EGS), and any combinations
thereof. In some cases, a first chemical species and/or fixation
agent may be provided to or brought into contact with the hydrogel
bead to bring about a change in a first characteristic or set of
characteristics of the hydrogel bead, and a second chemical species
and/or fixation agent may be provided to or brought into contact
with the hydrogel bead to bring about a change in a second
characteristic or set of characteristics of the hydrogel bead. For
example, a first chemical species and/or fixation agent may be
provided to or brought into contact with the hydrogel bead to bring
about a change in a dimension of a hydrogel bead (e.g., a reduction
in cross-sectional diameter), and a second chemical species and/or
fixation agent may be provided to or brought into contact with the
hydrogel bead to bring about a change in a second characteristic or
set of characteristics of the hydrogel bead (e.g., forming
crosslinks within and/or surrounding the hydrogel bead). The first
and second chemical species and/or fixation agents may be provided
to or brought into contact with the hydrogel bead at the same or
different times.
[0881] In some embodiments, fixation may affect one or more
parameters or characteristics of the hydrogel bead. For example,
fixation may result in shrinkage or volumetric reduction of the
hydrogel bead. Fixation may include dehydration of the hydrogel
bead. Providing a fixation agent to the hydrogel bead may result in
a change in a dimension of the hydrogel bead. For example,
providing a fixation agent to the hydrogel bead may result in a
change in the volume or diameter of the hydrogel bead. Providing a
fixation agent to the hydrogel bead may result in a change in a
cross-section of the hydrogel bead (e.g., a cross-sectional
diameter). For example, a first cross-section of the hydrogel bead
prior to fixation may be different (e.g., larger) than a second
cross-section of the hydrogel bead following fixation. In an
example, an approximately spherical hydrogel bead may comprise a
first cross section (e.g., a cross-sectional diameter) prior to
fixation that is reduced in size to a second cross-section
following fixation. Providing a fixation agent to the hydrogel bead
may result in a second cross-section that is reduced by at least
about 5% compared to the first cross-section. In some cases, the
second cross-section may be reduced by at least 6%, 8%, 10%, 15%,
25%, 30%, 35%, 40%, 45%, 50%, or more relative to the first
cross-section. For example, the second cross-section may be reduced
by at least about 10%, 15%, 25%, or 50% relative to the first
cross-section. Fixation may also affect other features of the
hydrogel bead. For example, fixation may result in a change in the
porosity of a membrane or wall of a hydrogel bead, reorganization
of components of the hydrogel bead, a change in hydrogel bead
fluidity or rigidity, or other changes. In an example, a first
fixation agent that is an organic solvent is provided to the
hydrogel bead to change a first characteristic (e.g., hydrogel bead
volume) and a second fixation agent that is a cross-linking agent
is provided to the hydrogel bead to change a second characteristic
(e.g., hydrogel bead fluidity or rigidity). The first fixation
agent may be provided to the hydrogel bead before the second
fixation agent.
[0882] In some instances, an approximately spherical hydrogel bead
may comprise a first diameter prior to fixation (e.g., by an
organic solvent) that is reduced in volume compared to a second
diameter following fixation when maintained in a non-aqueous
environment. Following fixation and reduction in volume to said
second diameter, when maintained in an aqueous environment, the
hydrogel bead may increase in volume to have a diameter
substantially similar to the first diameter. In some cases, an
approximately spherical hydrogel bead may include a first diameter
prior to fixation (e.g., by an organic solvent) that is reduced in
volume compared to a second diameter following fixation. Following
fixation and reduction in volume to said second diameter, the
hydrogel bead may be cross-linked by a second fixative, wherein the
second diameter is substantially maintained in an aqueous
environment following cross-linking by the second fixative.
[0883] A change to a characteristic or set of characteristics of
the hydrogel bead may be reversible or irreversible. In some cases,
a change to a characteristic or set of characteristics of the
hydrogel bead may be irreversible, such that the change cannot be
readily undone. For example, the volume, morphology, or other
feature of the hydrogel bead may be altered in a way that cannot be
readily reversed. In an example, the change from a first
cross-section of the hydrogel bead to a second cross-section of the
hydrogel bead is irreversible. In some cases, an irreversible
change may be at least partially reversed upon the application of
appropriate conditions and/or over a period of time. In other
cases, a change to a characteristic or set of characteristics of
the hydrogel bead may be reversible. For example, the volume of a
hydrogel bead may be reduced upon being subjected to a first
condition or set of conditions, and the volume of a hydrogel bead
may be increased to approximately the original volume upon being
subjected to a second condition or set of conditions. Thus, the
change from a first cross-section of the hydrogel bead to the
second cross-section may be reversible. A reversible change (e.g.,
a reversible volume reduction) may be useful in, for example,
providing a hydrogel bead of a given volume to a given location,
such as a partition. In some cases, a change to a characteristic or
set of characteristics of the hydrogel bead may be only partially
reversible. For example, the volume of a hydrogel bead may be
reduced (e.g., by dehydration), and the reduction in hydrogel bead
volume may be accompanied by reorganization of components within
the hydrogel bead. Upon reversal of the volume of the hydrogel bead
(e.g., by rehydration), the arrangement of one or more components
may not revert to the original arrangement of the hydrogel bead
prior to the volume reduction. A change to a characteristic or set
of characteristics of the hydrogel bead, such as a cross-section of
the hydrogel bead, may be reversible upon application of a
stimulus. The stimulus may be, for example, a thermal stimulus, a
photo stimulus, or a chemical stimulus. In some cases, the stimulus
may comprise a change in pH and/or application of a reducing agent
such as dithiothreitol. Application of the stimulus may reverse,
wholly or in part, a change from, for example, a first
cross-section to a second cross-section.
[0884] In some embodiments, a plurality of hydrogel beads can be
shrunken hydrogel beads generated by removing water from a
plurality of first hydrogel beads. In some embodiments, the
plurality of shrunken hydrogel beads has an average diameter no
larger than about 15 microns. For example, the plurality of
shrunken hydrogel beads has an average diameter no larger than
about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron.
In some embodiments, each member of the plurality of shrunken
hydrogel beads has a diameter no larger than about 15 microns. For
example, each member of the plurality of shrunken hydrogel beads
can have a diameter no larger than about 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, 5, 4, 3, 2, or 1 micron. In some embodiments, the
plurality of shrunken hydrogel beads has an average diameter no
larger than 10 microns. In some embodiments, each member of the
plurality of shrunken hydrogel beads has a diameter no larger than
10 microns. In some embodiments, the plurality of shrunken hydrogel
beads has an average diameter no larger than 5 microns. In some
embodiments, each member of the plurality of shrunken hydrogel
beads has a diameter no larger than 5 microns. In some embodiments,
the plurality of shrunken hydrogel beads has an average diameter no
larger than 1 micron. In some embodiments, each member of the
plurality of shrunken hydrogel beads has a diameter no larger than
1 micron. In some embodiments, the plurality of shrunken hydrogel
beads has an average diameter no larger than the diameter of a cell
(e.g., a mammalian cell, a plant cell, or a fungal cell). In some
embodiments, each member of the plurality of shrunken hydrogel
beads has a diameter no larger than the diameter of a cell (e.g., a
mammalian cell, a plant cell, or a fungal cell).
[0885] In some embodiments, the plurality of shrunken hydrogel
beads has a polydispersity index of less than about 25%. For
example, the plurality of shrunken hydrogel beads can have a
polydispersity index of less than about 25%, 20%, 15%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the plurality
of shrunken hydrogel beads has a polydispersity index of less than
15%. In some embodiments, the plurality of shrunken hydrogel beads
has an average diameter of about 8 to about 13 microns. In some
embodiments, the plurality of shrunken hydrogel beads has an
average diameter of about 10 to about 12 microns. In some
embodiments, the plurality of shrunken hydrogel beads has an
average diameter of about the diameter of a cell (e.g., a mammalian
cell, a plant cell, or a fungal cell). In some embodiments, the
plurality of shrunken hydrogel beads has an average diameter of
less than the diameter of a cell (e.g., a mammalian cell, a plant
cell, or a fungal cell). In some embodiments, the plurality of
capture probes on the plurality of shrunken gel beads bind cellular
analytes at single-cell resolution. In some embodiments, the
plurality of capture probes on the plurality of shrunken gel beads
bind cellular analytes at higher than single-cell resolution (e.g.,
at a resolution that is at a higher density than the diameter of a
cell).
[0886] In some embodiments, bead arrays having a plurality of
hydrogel beads disposed on a substrate are generated by patterning
or self-assembly of larger gel beads, after which the array of
larger gel beads is shrunken (e.g., by any of the variety of
methods provided herein). In some embodiments, the larger gel beads
are not small enough for single-cell resolution, while the shrunken
gel beads are small enough for single-cell resolution. In some
embodiments, bead arrays having a plurality of hydrogel beads
disposed on a substrate are generated by patterning or
self-assembly of shrunken gel beads that have previously been
generated by shrinking larger gel beads (e.g., by any of the
variety of methods provided herein). Beads can be spatially
confined by any of a variety of methods, including without
limitation, reversible or irreversible crosslinking.
[0887] In some embodiments, bead arrays include spatially-confined
gel beads with high aspect ratios (e.g., pillared arrays). For
example, bead arrays having a plurality of hydrogel beads disposed
on a substrate can be generated by any of the variety of methods
described herein (e.g., by patterning or self-assembly of shrunken
gel beads or by patterning or self-assembly of larger gel beads
followed by shrinking), after which the high-density bead array is
expanded (or re-expanded). When expanding, spatial constraints
direct the beads to expand primarily in the Z dimension (away from
the substrate), resulting in pillar arrays. In some embodiments,
the gel beads of the pillar arrays have high aspect ratios. In some
embodiments, aspect ratio of the expanded plurality of
spatially-confined shrunken hydrogel beads is at least 2. In other
embodiments, the aspect ratio of the expanded plurality of
spatially-confined shrunken hydrogel beads is at least 3. In some
embodiments, the plurality of spatially-confined shrunken hydrogel
beads has an average aspect ratio of at least 4, 5, 6, 7, 8 or
more.
[0888] In some embodiments, the method for the removal of water
from a hydrogel is the same for each hydrogel (e.g., the first
hydrogel, the second hydrogel, or the third hydrogel). In some
embodiments, the method for the removal of water from one hydrogel
(e.g., the first hydrogel) is different from the method for the
removal of water for at least one other hydrogel (e.g., a second
hydrogel, a third hydrogel, or a fourth hydrogel). For example, the
method for the removal of water from one hydrogel can be different
from the method for the removal of water for the other hydrogels
(e.g., a second hydrogel, a third hydrogel, or a fourth hydrogel).
In some embodiments, the method for the removal of water is
different for each hydrogel (e.g., the first hydrogel, the second
hydrogel, the third hydrogel, and the fourth hydrogel).
[0889] In some embodiments, the shrunken hydrogel is at least about
2-fold smaller in a linear dimension (e.g., along one axis) than
the pre-shrunk hydrogel. For example, at least about 2.5, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10, or
more fold smaller in a linear dimension than the pre-shrunk
hydrogel.
[0890] In some embodiments, the size of the hydrogel is reduced
along more than one axes, e.g., along 2 or 3 axes. In some
embodiments, each axis intersects each other axis at 90 degrees. In
some embodiments, the size of the hydrogel along the first axis is
about 2 to about 10 or more fold smaller than the pre-shrunk
hydrogel, e.g., about 2, about 3, about 4, about 5, about 6, about
7, about 8, about 9, about 10 or more fold smaller than the
pre-shrunk hydrogel. In some embodiments, the size of the hydrogel
along the second axis is about 2 to about 10 or more fold smaller
than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10 or more fold
smaller than the pre-shrunk hydrogel. In some embodiments, the size
of the hydrogel along the third axis is about 2 to about 10 or more
fold smaller than the pre-shrunk hydrogel, e.g., about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10 or
more fold smaller than the pre-shrunk hydrogel. In some
embodiments, the reduction in the volume of the hydrogel is
isometric.
[0891] In some embodiments, the volume of each hydrogel (e.g., a
first hydrogel, a second hydrogel, a third hydrogel, or a fourth
hydrogel) is the same. In some embodiments, the volume of at least
one hydrogel is different. For example, in some embodiments, one
hydrogel is different in volume from the other hydrogels (e.g., a
second hydrogel, a third hydrogel, or a fourth hydrogel). In some
embodiments, every hydrogel is different in volume from every other
hydrogel.
[0892] In some embodiments, members of the plurality of features
are cross-linked to a hydrogel (e.g., a first hydrogel, a second
hydrogel, a third hydrogel, or a fourth hydrogel).
[0893] In one embodiment, features of an array can be copied into a
hydrogel, and the volume of the hydrogel is reduced by removing
water. These steps can be performed multiple times. For example, a
method for preparing a high-density spatially-barcoded flexible
array can include copying a plurality of spatially-barcoded
features from an array into a first hydrogel, wherein the first
hydrogel is in contact with the array; reducing the volume of the
first hydrogel including the copied features by removing water,
forming a first shrunken hydrogel including the copied features;
copying the features in the first shrunken hydrogel into a second
hydrogel, where the second hydrogel is in contact with the first
hydrogel; and reducing the volume of the second hydrogel including
the copied features by removing water, forming a second shrunken
hydrogel including the copied features, thus generating a
high-density spatially-barcoded array. The process of copying
spatially-barcoded features from an array to a first hydrogel,
removing water from the first hydrogel to form a first shrunken
hydrogel, and copying spatially-barcoded features from the first
shrunken hydrogel to one or more subsequent hydrogels can be
performed multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10
times). The result is a high-density flexible array including
spatially-barcoded features.
[0894] In some embodiments, copying members of the plurality of
features from an array includes copy by PCR. In some embodiments,
the hydrogel (e.g., a first hydrogel, a second hydrogel, a third
hydrogel, and/or a fourth hydrogel) comprises PCR reagents as
described herein. In some embodiments, members of the plurality of
features are copied using replica plating techniques (see, e.g.,
Mitra and Church, Nucleic Acids Res. 1999 Dec. 15; 27(24):e34,
which is incorporated by reference herein in its entirety). In some
embodiments, after copying a plurality of features from an array
into a first hydrogel, the features of the array are amplified in
the first hydrogel (e.g., clonal amplification). In some
embodiments, members of the plurality of features are copied into
the first hydrogel such that the pattern of the plurality of
features of the first hydrogel is the same or substantially similar
(e.g., at least 80%) to the pattern of the plurality of features of
the array.
[0895] In some embodiments, one or more pluralities of features of
the array are partitioned. For example, each partition can comprise
a plurality of features different from the plurality of features of
other partitions. For example, the members of the plurality of
features are partitioned similar to the partitions of the plurality
of features of the array. In some embodiments, the features of the
array are copied into the first hydrogel, such that the volume or
diameter of the pre-shrunk first hydrogel features are similar to
the volume or diameter of the array features.
[0896] In some embodiments, the volume of a hydrogel comprising
copied features is reduced, thus increasing the density of the
copied features. In some embodiments, the copied features within a
hydrogel further increases in density with each subsequent hydrogel
copy and shrinking. For example, the density of the copied features
of a second shrunken hydrogel is higher than the density of the
copied features of a first shrunken hydrogel. Similarly, the
density of the copied features of a third shrunken hydrogel is
higher than the density of the copied features of a second shrunken
hydrogel. Similarly, the density of the copied features of a fourth
shrunken hydrogel is higher than the density of the copied features
of a third shrunken hydrogel. In some embodiments, the volume of a
partition of members of the plurality of features in a hydrogel is
reduced when the volume of the hydrogel is reduced.
[0897] In some embodiments of the methods described herein, an
array comprises shrunken gel features (e.g., beads). In some
embodiments, the methods described herein generate shrunken gel
bead arrays. In some embodiments, the shrunken gel beads of the
array are shrunken hydrogel beads.
[0898] A "shrunken array" includes a plurality of
spatially-barcoded features attached to, or embedded in, a
substrate that have been reduced in volume (e.g., reduction in
diameter or volume). A biological sample can be contacted with a
shrunken array and further contacted with a solution capable of
rehydrating the shrunken array. In some embodiments, analyte
transfer and capture is driven by molecular diffusion. The process
of rehydrating the shrunken array by providing a permeabilization
solution or tissue stain to the sample can promote analytes (e.g.,
transcripts) present in the biological sample towards the
spatially-barcoded features, thereby improving capture efficiency
of the analytes. See, e.g., J. Vlassakis, A. E. Herr. "Effect of
Polymer Hydration State on In-Gel Immunoassays." Anal. Chem. 2015,
87(21):11030-8, herein incorporated by reference in its
entirety.
[0899] A shrunken array can be generated with features (e.g.,
beads) containing spatial barcodes from an existing array. For
example, an array (e.g., hydrogel bead array) described and
prepared by any method herein can be contacted with reagents
capable of dehydrating (e.g., removing water) the features (e.g.,
beads) to generate a shrunken array (e.g., a shrunken bead array).
Methods of dehydrating features (e.g., beads) are known in the art.
Any suitable method of dehydration (e.g., removing water) can be
used. For example, in a non-limiting way, features (e.g., beads)
can be dehydrated by a ketone, such as methyl ethyl ketone (MEK),
isopropanol (IPA), acetone, 1-butanol, methanol (MeOH), dimethyl
sulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol,
ethanol, (k) 1,4-dioxane, propylene carbonate, furfuryl alcohol,
N,N-dimethylformamide (DMF), acetonitrile, aldehyde, such as
formaldehyde or glutaraldehyde, or any combinations thereof.
Additional dehydration agents include various salts, including
inorganic salts (See, e.g., Ahmed, E. M., Hydrogel: Preparation,
characterization, and applications: A review, Journal of Advanced
Research, 6 (2) 105-121 (2015), which is incorporated herein by
reference).
[0900] In some embodiments, the dehydrated features (e.g., beads)
can create a shrunken array (e.g., shrunken bead array or shrunken
hydrogel array) where the average diameter of the dehydrated
features (e.g., beads) can be smaller than the average diameter of
the features prior to dehydration. In some embodiments, the
dehydrated features (e.g., beads) can have an average diameter at
least two-fold smaller than the average diameter of the features
prior to dehydration. In some embodiments, the dehydrated features
(e.g., beads) can have an average diameter at least three-fold
smaller than the average diameter of the features prior to
dehydration. In some embodiments, the dehydrated features (e.g.,
beads) can have an average diameter at least four-fold or smaller
than the average diameter of the features (e.g., beads) prior to
dehydration.
[0901] After generating a shrunken array, a biological sample
(e.g., tissue sample) can be contacted with the shrunken array
(e.g., shrunken bead array). A rehydrating solution can be provided
to the biological sample and the shrunken array by any suitable
method (e.g., by pipetting). The rehydrating solution can contain
reagents to rehydrate (e.g., water or buffers) the features (e.g.,
beads) of the shrunken array. In some embodiments, the rehydrating
solution can be applied to the entire biological sample. In some
embodiments, the rehydrating solution can be selectively applied
(e.g., to a region of interest). In some embodiments, absorbing
water from the rehydrating solution can increase the diameter of at
least one feature (e.g., bead) in the shrunken array. In some
embodiments, the rehydrating solution can increase the diameter of
at least one feature (e.g., bead) by at least two-fold. In some
embodiments, the rehydrating solution can increase the diameter of
at least one feature (e.g., bead) by at least three-fold. In some
embodiments, the rehydrating solution can increase the diameter of
at least one feature (e.g., bead) by at least four-fold. In some
embodiments, the rehydrating solution can increase the diameter of
at least one feature (e.g., bead) by at least five-fold or
more.
[0902] In some embodiments, the rehydrating solution can contain
permeabilization reagents. The biological sample can be
permeabilized using permeabilization reagents and techniques known
in the art or otherwise described herein. Biological samples from
different sources (e.g., brain, liver, ovaries, kidney, breast,
colon, etc.) can require different permeabilization treatments. For
example, permeabilizing the biological sample (e.g., using a
protease) can facilitate the migration of analytes to the substrate
surface (e.g., spatially-barcoded features). In some embodiments,
the permeabilization reagents can be a detergent (e.g., saponin,
Triton X-100.TM., Tween-20.TM.). In some embodiments, an organic
solvent (e.g., methanol, acetone) can permeabilize cells of the
biological sample. In some embodiments, an enzyme (e.g., trypsin)
can permeabilize the biological sample. In another embodiment, an
enzyme (e.g., collagenase) can permeabilize the biological
sample.
[0903] In some embodiments the solution can permeabilize the
biological sample and rehydrate the features (e.g., beads) of the
shrunken array (e.g., shrunken hydrogel). In some embodiments, the
rehydrating solution can stain the biological sample and rehydrate
the features of the shrunken array (e.g., beads).
[0904] In some embodiments, the rehydrating solution (e.g.,
permeabilization or stain solution) can diffuse through the
biological sample. In some embodiments, the rehydrating solution
can reduce diffusion of analytes away from the substrate. In some
embodiments, while diffusing through the biological sample, the
rehydrating solution can migrate analytes toward the substrate
surface and improve the efficiency of analyte capture.
[0905] (viii) Microcapillary Arrays
[0906] A "microcapillary array" is an arrayed series of features
that are partitioned by microcapillaries. A "microcapillary
channel" is an individual partition created by the
microcapillaries. For example, microcapillary channels can be
fluidically isolated from other microcapillary channels, such that
fluid or other contents in one microcapillary channel in the array
are separated from fluid or other contents in a neighboring
microcapillary channel in the array. The density and order of the
microcapillary channels can be any suitable density or order of
discrete sites.
[0907] In some embodiments, microcapillary arrays are treated to
generate conditions that facilitate loading. An example is the use
of a corona wand (BD-20AC, Electro Technic Products) to generate a
hydrophilic surface. In some embodiments, a feature (e.g., a bead
with capture probes attached) is loaded onto a microcapillary array
such that the exact position of the feature within the array is
known. For example, a capture probe containing a spatial barcode
can be placed into a microcapillary channel so that the spatial
barcode can enable identification of the location from which the
barcoded nucleic acid molecule was derived.
[0908] In some embodiments, when random distribution is used to
distribute features, empirical testing can be performed to generate
loading/distribution conditions that facilitate a single feature
per microcapillary. In some embodiments, it can be desirable to
achieve distribution conditions that facilitate only a single
feature (e.g., bead) per microcapillary channel. In some
embodiments, it can be desirable to achieve distribution conditions
that facilitate more than one feature (e.g., bead) per
microcapillary channel, by flowing the features through the
microcapillary channel.
[0909] In some embodiments, the microcapillary array is placed in
contact with a sample (e.g., on top or below) so that
microcapillaries containing a feature (e.g., a bead, which can
include a capture probe) are in contact with the biological sample.
In some embodiments, a biological sample is placed onto an exposed
side of a microcapillary array and mechanical compression is
applied, moving the biological sample into the microcapillary
channel to create a fluidically isolated reaction chamber
containing the biological sample.
[0910] In some embodiments, a biological sample is partitioned by
contacting a microcapillary array to the biological sample, thereby
creating microcapillary channels including a bead and a portion of
the biological sample. In some embodiments, a portion of a
biological sample contained in a microcapillary channel is one or
more cells. In some embodiments, a feature is introduced into a
microcapillary array by flow after one or more cells are added to a
microcapillary channel.
[0911] In some embodiments, reagents are added to the
microcapillary array. The reagents can include enzymatic reagents
or reagent mixtures for performing amplification of a nucleic acid.
In some embodiments, the reagents include a reverse transcriptase,
a ligase, one or more nucleotides, or any combinations thereof. One
or more microcapillary channels can be sealed after reagents are
added to the microcapillary channels, e.g., by using silicone oil,
mineral oil, a non-porous material, or lid.
[0912] In some embodiments, a reagent solution is removed from each
microcapillary channel following an incubation for an amount of
time and at a certain temperature or range of temperatures, e.g.,
following a hybridization or an amplification reaction. Reagent
solutions can be processed individually for sequencing, or pooled
for sequencing analysis.
[0913] (ix) Hydrogel/Well Arrays
[0914] In some embodiments are methods for generating patterned
hydrogel arrays using wells (e.g., a nanowell or microwell array).
In some embodiments, the well is a 3-dimensional structure. In some
embodiments, the top view of a well is any suitable 2-dimensional
shape, which when extended along the z-axis, produces a
3-dimensional structure capable of containing one or more features
(e.g., beads) and/or reagents. Non-limiting examples of wells which
may form an array include a triangular prism, a square or
rectangular prism, a pentagonal prism, a hexagonal prism, a
heptagonal prism, an octagonal prism, an n-sided prism, or a
cylindrical array (e.g., "microcapillary array"). In some
embodiments, a well of the well array shares at least one well wall
(or a portion of the well wall, if a microcapillary array) with an
adjacent well. In some embodiments, a well does not share any walls
or portion of a wall in common with another well of the array. In
some embodiments, the well array is attached to a substrate, such
that the wells of the well array are fluidically isolated from each
other. In some embodiments, one end of the well array is open
(e.g., exposed), wherein the open end can be used to distribute
features or reagents into the well.
[0915] In some embodiments, the method includes providing shrunken
(e.g., dehydrated) hydrogel features (e.g., beads) to a well array.
The hydrogel features can be dehydrated (e.g., removing water) by
any of the variety of methods described herein. The features,
described elsewhere herein, can be provided such that the number of
features is less than the number of wells of the array, the
features can be provided such that the number of features equals
the number of wells of the array, or the features can be provided
in excess of the number of wells of the array. In some embodiments,
the well array is manipulated such that one or more shrunken
hydrogel features move from the top surface of the array down into
a well. For example, a well array can be placed on a shaker for a
length of time necessary for the features to distribute into the
wells. Other, non-limiting examples of manipulations that can cause
a shrunken hydrogel feature to enter a well are physically shaking,
tilting, or rolling the well array, or a combination thereof; using
forced air to blow features into a well, using a magnet to pull
down hydrogel features comprising magnetic particles, using
microfluidic systems to distribute features into wells, using a
printer to deposit a feature into a well, or any other method to
distribute features into wells. In some embodiments, once a well
contains a feature, the well cannot accept or retain another
feature. In other embodiments, a well can contain more than one
feature.
[0916] In some embodiments, the method includes rehydrating (e.g.,
adding water) the shrunken hydrogel features, wherein the shrunken
hydrogel features are located in the wells. Rehydrating shrunken
hydrogel features can be accomplished by any method described
herein. Rehydrating a shrunken hydrogel feature in the well can
cause the shrunken hydrogel feature to expand. In some embodiments,
the shrunken hydrogel feature expands to fill the well. In some
embodiments, the shrunken hydrogel feature expands in a z
direction, such that the feature expands out of the unenclosed
(i.e. open) end of the well. The exposed area of the rehydrated
feature can create a patterned hydrogel array (e.g., a well array).
A rehydrated feature contained within a well can be stable. In some
embodiments, a rehydrated feature (e.g., a rehydrated shrunken
hydrogel feature) is immobilized within a well, such that typical
array usage does not dissociate the rehydrated feature from the
well. The patterned hydrogel array can be used for analyte capture
according to the methods described herein.
[0917] (x) Bead Tethering
[0918] "Bead tethering" can refer to an arrangement of beads,
wherein the arrangement may or may not form an array. The tethered
beads can be contacted with a sample and processed according to
methods described herein. Further, contacting a biological sample
with a single bead or beads tethered together in various
arrangements can allow for more precise spatial detection of
analytes, e.g., a region of interest. Methods for tethering beads
together are known in the art. Some suitable, non-limiting, methods
of tethering beads together can be, e.g., chemical linkers,
proximity ligation, or any other method described herein. In some
embodiments, beads can be tethered together independent of a
substrate. In some embodiments, beads can be tethered in various
arrangements on an existing substrate. In some embodiments, a
substrate (e.g., a hydrogel) can be formed around existing tethered
beads. In some embodiments, the beads or bead arrangement can
contact a portion of the biological sample. In some embodiments,
the bead or bead arrangement can contact a region of interest. In
some embodiments, the beads or bead arrangement can contact the
entire biological sample. In some embodiments, the beads or bead
arrangements are contacted to random positions on the biological
sample. In some embodiments, the beads are contacted to according
to a specific pattern on the biological sample.
[0919] Beads can be tethered together in various arrangements. In
some embodiments, a single (e.g., one) bead can be contacted with a
biological sample. In some embodiments, two or more beads can be
tethered (e.g., connected to each other), in various arrangements.
For example, in a non-limiting way, beads can be tethered together
to form a cluster, a row, or arranged on a mesh (e.g., a net). In
some embodiments, at least three beads can be tethered together in
a two-dimensional (2D) array (e.g., a cluster). In some
embodiments, at least two beads can be tethered together in a
one-dimensional (1D) array (e.g., a row). In such embodiments, the
beads are arranged in such fashion that the beads can contact each
other directly. In some embodiments, at least two beads can be
tethered together in a string arrangement. In such embodiments, the
beads are arranged in such fashion that the beads can contact each
other indirectly (e.g., beads are connected via linker). In some
embodiments, at least two beads can be tethered together in a mesh
arrangement (e.g., net). In some embodiments, beads tethered
together in a 2D array, a 1D array, the beads on a string
arrangement, and the beads on the mesh arrangement can be used in
any combination with each other on the biological sample.
[0920] In some embodiments, at least about 2 to about 10 beads can
be tethered together in various arrangements. In some embodiments
at least about 3, about 4, about 5, about 6, about 7, about 8,
about 9, or more beads can be tethered together. In some
embodiments, about 10 to about 100 beads can be tethered together
in various arrangements. In some embodiments, about 10, about 20,
about 30, about 40, about 50, about 60, about 70, about 80, about
90, or more beads can be tethered together in various arrangements.
In some embodiments, about 100 to about 1,000 beads can be tethered
together in various arrangements. In some embodiments, about 100,
about 200, about 300, about 400, about 500, about 600, about 700,
about 800, or about 900 or more beads can be tethered together in
various arrangements. In some embodiments, about 1000 to about
10000 beads can be tethered together in various arrangements. In
some embodiments, about 1000, about 2000, about 3000, about 4000,
about 5000, about 6000, about 7000, about 8000, about 9000, about
10000 or more beads can be tethered together.
[0921] In some embodiments, the tethered beads can have capture
probes comprising spatial barcodes, functional domains, unique
molecular identifiers, cleavage domains, and capture domains, or
combinations thereof. In some embodiments, each bead can be
associated with a unique spatial barcode. In some embodiments, the
spatial barcode is known prior to contacting the bead or bead
arrangement to the biological sample. In some embodiments, the
spatial barcode is not known prior to contacting the bead or bead
arrangement to the biological sample. The identity of each bead
(e.g., spatial barcode) in the array can be deconvolved, for
example, by direct optical sequencing, as discussed herein.
[0922] (xi) Printing Arrays in Liquid
[0923] In some embodiments, an array can be printed in liquid. The
resolution of conventionally-printed arrays can be limited, due to
the diffusion of printed solutions. Printing the array in a highly
viscous liquid can increase resolution by preventing the diffusion
of the printed solution. Thus, disclosed herein are various methods
and materials for attaching and/or introducing a capture probe
(e.g., a nucleic acid capture probe) having a barcode (e.g., a
spatial barcode) to a substrate (e.g., a slide), wherein the
attaching (e.g., printing) is performed in liquid.
[0924] In some aspects, capture probes are printed on a substrate
(e.g., a slide or bead). In some aspects, the substrate is a slide.
In some aspects, the substrate is a 96-well microtiter plate. In
some aspects, methods provided herein can also be applied to other
substrates commonly used for nucleic acid analyses, including but
not limited to beads, particles, membranes, filters, dipsticks,
slides, plates, and microchips. In some aspects, such substrates
may be composed of a number of materials known to be compatible
with nucleic acid analysis, including but not limited to agarose,
styrene, nylon, glass, and silicon.
[0925] (1) First Solution
[0926] In some embodiments, provided herein are methods of printing
arrays on substrates using one or more liquid solution(s) (e.g.,
two or more solutions that include distinct capture probes). In
some aspects, methods of printing arrays on substrates using one or
more solution(s) can improve the resolution of the printed array.
In some aspects, methods provided herein include dispensing a first
solution (e.g., bulk solution) onto a substrate. In some aspects,
the first solution (e.g., bulk solution) has a lower Reynolds
Number relative to a second solution (e.g., a second solution that
includes capture probes to be attached to the substrate). The
Reynolds Number represents an inverse relationship between the
density and velocity of a fluid and its viscosity in a channel of
given length. More viscous, less dense, and/or slower moving fluids
will have a lower Reynolds Number, and are easier to divert, stop,
start, or reverse without turbulence. In some embodiments, the
first solution and the second solution are immiscible.
[0927] In some aspects, the first (e.g., bulk) solution is
hydrophobic. In some aspects, after dispensing the first (e.g.,
bulk) solution onto the slide, the first (e.g., bulk) solution
remains on the slide in discrete spatial areas on the slide. In
some aspects, the first (e.g., bulk) solution is made of a solution
that does not denature one or more probes and/or does not inhibit
probe-to-substrate binding. In some embodiments, the bulk solution
can include an aqueous solution, a high viscosity solution, or a
low nucleic acid diffusivity solution. In some aspects, the first
(e.g., bulk) solution is a gel. In some aspects, the first (e.g.,
bulk) solution is a hydrogel. In some aspects, the first (e.g.,
bulk) solution includes natural polymers, including for example,
glycerol, collagen, gelatin, sugars such as starch, alginate, and
agarose, or any combinations thereof. In some aspects, the first
(e.g., bulk) solution includes a synthetic polymer. In some
aspects, the synthetic polymer is prepared by any method known in
the art, including for example, chemical polymerization methods. In
some aspects, the gel or polymer is hydrophobic. In some aspects,
the gel or polymer is hydrophilic. In some aspects, the gel or
polymer is aqueous. In some aspects, the gel or polymer shrinks at
room temperature. In some aspects, the gel or polymer shrinks when
heated. In some aspects, the polymer is a film that shrinks when
heated.
[0928] In some aspects, the first (e.g., bulk) solution includes
glycerol. In some aspects, glycerol is present in the first (e.g.,
bulk) solution at a concentration of 5-100%. In some aspects,
glycerol is present in the solution at a concentration of 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.
[0929] In some aspects, the first (e.g., bulk) solution includes
sugar. In some aspects, the sugar is a monosaccharide, a
disaccharide, a polysaccharide, or combinations thereof. In some
aspects, the sugar is glucose, fructose, mannose, galactose,
ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose,
starch cellulose, or combinations thereof. In some aspects, sugar
is sourced from complex compounds such as molasses or other
by-products from sugar refinement. In some aspects, sugar is
present in the first (e.g., bulk) solution at a concentration of
5-100%. In some aspects, sugar is present in the solution at a
concentration of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, or 70%.
[0930] In some aspects, the first (e.g., bulk) solution has a
viscosity that is about 0.1.times.-fold, 0.2.times.-fold,
0.3.times.-fold, 0.4.times.-fold, 0.5.times.-fold, 0.6.times.-fold,
0.7.times.-fold, 0.8.times.-fold, 0.9.times.-fold, 1.0.times.-fold,
1.1.times.-fold, 1.2.times.-fold, 1.3.times.-fold, 1.4-fold,
1.5.times.-fold, 1.6.times.-fold, 1.7.times.-fold, 1.8.times.-fold,
1.9.times.-fold, 2.0.times.-fold, 2.5.times.-fold, 3.0.times.-fold,
4.0.times.-fold, 5.0.times. fold, 6.0.times.-fold, 7.0.times.-fold,
8.0.times. fold, 9.0.times.-fold, or 10.times.-fold greater than
the viscosity of the second solution.
[0931] (2) Second Solution
[0932] In some embodiments, printing arrays on substrates using two
or more solutions includes using a second solution. In some
embodiments, the second solution can include one or more capture
probes. In some embodiments, the second solution is dispensed in
the form of a droplet. Some embodiments include a plurality of
second solutions, wherein each member of the plurality includes one
or more capture probes comprising a spatial barcode unique to that
particular droplet. In some embodiments, the second solution is
dispensed onto a substrate covered or partially covered with the
first (e.g., bulk) solution. In some embodiments, the first (e.g.,
bulk) solution reduces or prevents the diffusion of one or more
capture probes from the second solution. In some aspects, the one
or more capture probes remain entirely within the second solution
when printed onto a substrate covered or partially covered with the
first (e.g., bulk) solution.
[0933] In some aspects, the second solution includes one or more
capture probes (e.g., any of the capture probes disclosed herein).
In some aspects, the second solution includes one
spatially-barcoded capture probe. In some aspects the second
solution includes at least 5, at least 10, at least 20, at least 30
at least 40, at least 50, at least 75, at least 100, at least 200,
at least 300, at least 400, at least 500, at least 600, at least
700, at least 800, at least 900, at least 1,000, at least 1,500, at
least 2,000, at least 3,000, at least 4,000, or at least 5,000
spatially-barcoded capture probes. In some aspects, the second
solution includes compounds that facilitate binding of the one or
more capture probes to the substrate. In some aspects, the second
solution does not inhibit the one or more capture probes from
binding the substrate and/or does not denature the one or more
capture probes. In some aspects, the second solution is less
viscous than the first (e.g., bulk) solution. In some aspects, the
second solution and the first (e.g., bulk) solution are wholly or
substantially immiscible (e.g., they do not mix). In some aspects,
the second solution is an aqueous solution. In some aspects, the
second solution is a hydrophilic solution.
[0934] (3) Dispensing
[0935] In some embodiments, spot printing a high-density pattern of
features can include dispensing the oligonucleotides and/or
features, in the form of a liquid droplet, onto the surface of the
substrate in the presence of a bulk solution. In some embodiments,
the second solution droplet(s) (e.g., oligonucleotides and/or
feature droplet(s)) and the first (e.g., bulk) solution does not
substantially mix with each other. In some aspects, the printing
methods disclosed herein that include dispensing a second solution
including one or more spatially-barcoded capture probes onto a
substrate in the presence (e.g., through) a first (e.g., bulk)
solution result in a spot size of the second solution (e.g., a
cross-sectional spot size of the second solution on the plane of
the substrate) that is smaller compared to the spot size that would
be obtained by dispensing the same second solution onto the
substrate in the absence of the first (e.g., bulk) solution. In
some aspects, the spot size (e.g., the cross-sectional spot size of
the second solution on the plane of the substrate) of the second
solution does not increase after printing the second solution onto
the substrate in the presence of the first (e.g., bulk) solution.
In some aspects, the area of the spot of the second solution does
not change after printing the second onto the substrate in the
presence of the first (e.g., bulk) solution. In some aspects,
printing of the second solution onto the substrate in the presence
of the first (e.g., bulk) solution results in a desired pattern on
the substrate surface. For example, a plurality of second solutions
can be printed onto the substrate in the presence of the first
solution such that the locations where the plurality of second
solutions are printed results in a desired pattern on the
substrate. In some embodiments, two or more members of a plurality
of second solutions printed onto a substrate include distinct
populations of capture probes, which capture probes attach to the
substrate, such that an array of capture probes is generated.
[0936] (4) Density
[0937] In some embodiments, the cross-sectional area of the
oligonucleotide and/or feature droplet(s) on the substrate is
smaller than a corresponding cross-sectional area of the
oligonucleotide and/or feature droplet(s) that would be generated
by dispensing the oligonucleotides and/or features onto the surface
of the substrate in the absence of the bulk solution. In some
embodiments, the cross-sectional area of the oligonucleotide and/or
feature droplet(s) on the substrate is about two-fold smaller than
the corresponding cross-sectional area of the oligonucleotide
and/or feature droplet(s) that would be generated by dispensing the
oligonucleotide and/or feature droplet(s) onto the surface of the
substrate in the absence of the bulk solution.
[0938] In some aspects, the cross-sectional area of the second
solution on the substrate generated by dispensing the second
solution onto the surface of the substrate in the presence of the
first solution is smaller than a corresponding cross-sectional area
of the second solution that would be generated by dispensing the
second solution onto the surface of the substrate in the absence of
the first solution. In some aspects, the cross-sectional area of
the second solution on the substrate generated by dispensing the
second solution onto the surface of the substrate in the presence
of the first solution is about two-fold, about three-fold, about
four-fold, or about five-fold, about 10-fold, about 20-fold, about
30-fold, about 40-fold, about 50-fold, about 60-fold, about
70-fold, about 80-fold, about 90-fold, or about 100-fold smaller
than the corresponding cross-sectional area of the second solution
that would be generated by dispensing the second solution onto the
surface of the substrate in the absence of the first solution.
[0939] (5) Curing
[0940] Any suitable technique or condition can be used to cure the
first solution, the second solution, and/or the spot formed by the
second solution after the second solution (including the capture
probes) is dispensed onto the substrate. As used herein, the term
"curing" and related terms can refer to treating a solution (e.g.,
a first solution, a second solution, or both) with an agent and/or
condition that transforms a solution from a liquid state to a solid
state (e.g., transformed into a matrix), wherein the solution
retains a three dimensional shape after the curing process.
Suitable examples of curing techniques and/or conditions include
ultraviolet (UV) radiation, infrared (IR) radiation, thermal
radiation, microwave radiation, visible radiation,
narrow-wavelength radiation, laser light, natural light, humidity,
or combinations thereof. Suitable examples of a curing source
include, for example, a UV light, a heating device, a radiation
device, a microwave device, a plasma device, or combinations
thereof.
[0941] In some embodiments, the first solution and/or the second
solution is chemically cured. In some embodiments, the
oligonucleotide and/or feature droplet(s) are chemically cured. In
some embodiments, the bulk solution is chemically cured. In some
aspects, the spot formed by the second solution after the second
solution (including the capture probes) is dispensed onto the
substrate is chemically cured. In some embodiments, the
oligonucleotides and/or features and the bulk solution are attached
to the substrate (e.g., by curing), thus generating a feature and
bulk solution-matrix. In some aspects, the matrix (e.g., the first
and second solution-matrix) is chemically cured. Chemically curing
a solution can be accomplished by any means known in the art. For
example, a solution can include one or more hydrogel subunits that
can be chemically polymerized (e.g., cross-linked) to form a
three-dimensional (3D) hydrogel network. Features dispensed, in the
form of a liquid droplet, onto the surface of a substrate in the
presence of a bulk solution can be polymerized. In some
embodiments, the features are co-polymerized with the bulk solution
to create a gel feature in hydrogel flexible array, whereas in
other embodiments the gel pad or feature is polymerized and the
bulk solution is removed leaving a spot or bead array. Non-limiting
examples of hydrogel subunits include acrylamide, bis-acrylamide,
polyacrylamide and derivatives thereof, poly(ethylene glycol) and
derivatives thereof (e.g., PEG-acrylate (PEG-DA), PEG-RGD),
gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA),
polyaliphatic polyurethanes, polyether polyurethanes, polyester
polyurethanes, polyethylene copolymers, polyamides, polyvinyl
alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl
pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and
poly(hydroxyethyl methacrylate), collagen, hyaluronic acid,
chitosan, dextran, agarose, gelatin, alginate, protein polymers,
methylcellulose, and the like, or combinations thereof. Other
materials and techniques useful in forming and/or cross-linking
hydrogels are described in more detail herein.
[0942] In some aspects, the first solution and/or second solution
is photo-reactively cured. In some embodiments, the oligonucleotide
and/or feature droplet(s) are photo-reactively cured. In some
embodiments, the bulk solution is photo-reactively cured. In some
aspects, the spot formed by the second solution after the second
solution including the capture probe is dispensed onto the
substrate is photo-reactively cured. In some aspects, the matrix
(e.g., the first and second solution-matrix) is photo-reactively
cured. Photo-reactive curing can be accomplished by any means known
in the art.
[0943] In some aspects, methods disclosed herein include curing the
first (e.g., bulk) and/or second solution. In some aspects, methods
disclosed herein include curing the spot formed by the second
solution after the second solution (e.g., a second solution
including the capture probe) is dispensed onto the substrate. In
some aspects, methods disclosed herein include curing the first
solution and the second solution after both are dispensed onto the
slide. In some embodiments, curing the first and second solutions
results in a first-and-second solution matrix. In some aspects,
methods disclosed herein include curing the second solution
followed by removal of the uncured first solution from the
substrate. In some aspects, the methods disclosed herein include
curing the first and second solutions on the substrate, thus
generating a matrix (e.g., a first and second solution-matrix).
[0944] (6) Expanding Matrices
[0945] In some embodiments, prior to dispensing the second solution
droplet(s) (e.g., oligonucleotide and/or feature droplet(s)) onto
the surface of a substrate in the presence of a first (e.g., bulk)
solution, the first (e.g., bulk) solution is cured to form a bulk
solution-matrix, and the bulk solution matrix is reversibly
expanded along one or more axes (e.g., one or more axes of a matrix
or a substrate surface). In some embodiments, second solution
droplet(s) can be dispensed in the presence of expanded first
(e.g., bulk) solution-matrix.
[0946] In some embodiments, dispensing a plurality of second
solution droplet(s) (e.g., including different capture probes
having different spatial barcodes) onto a substrate covered with a
first (e.g., bulk) solution, wherein the first solution is
initially stretched along one or more axes. In some embodiments,
the stretched first solution is cured. In some embodiments, the
stretched first solution is partially cured. In some embodiments,
the stretched first solution is uncured and is dispensed on a
surface that itself is stretched. In some aspects, the volume
(e.g., cross-sectional area) of the second solution droplet(s) is
decreased after being dispensed into a first solution that is
initially stretched. In some aspects, disclosed herein are methods
of preparing an array including (i) providing a gel or polymer
(e.g., a cured or partially cured solution) onto a substrate, (2)
stretching the gel or polymer, (3) dispensing a droplet of the
second solution onto the substrate while the gel or polymer is
stretched, and (4) allowing the gel or polymer to relax, thereby
decreasing the overall area (e.g., cross-sectional area) of the
second solution droplet(s) on the substrate. In some aspects, the
stretching step includes reversibly expanding the gel or polymer
along one or more axes coplanar with the surface of the
substrate.
[0947] (7) Removing Solution
[0948] In some embodiments, the first (e.g., bulk) solution can be
removed from the substrate after the oligonucleotide and/or feature
droplet(s) are attached to the substrate. In some aspects, the
first (e.g., bulk) solution is removed after the second solution
including the one or more capture probes (e.g., plurality of second
solutions that include different capture probes) is dispensed onto
the substrate. Methods of removing the first (e.g., bulk) solution
are known in the art. In some aspects, removal of the first (e.g.,
bulk) solution results in complete removal of the first solution,
leaving only the second solution including the one or more capture
probes (e.g., plurality of second solutions that include different
capture probes) on the substrate. In some aspects, removal of the
first (e.g., bulk) solution does not change the surface area of the
second solution (e.g., plurality of second solutions that include
different capture probes) in contact with the substrate. In some
aspects, removal of the first (e.g., bulk) solution does not change
the shape of the droplet of the second solution (e.g., plurality of
second solutions that include different capture probes) in contact
with the substrate. In some aspects, prior to removal of the first
(e.g., bulk) solution, the second solution including the one or
more probes (e.g., plurality of second solutions that include
different capture probes) is cured by methods disclosed herein but
that the first (e.g., bulk) solution is not cured. For example, the
first and second solutions can be subjected to agents and/or
conditions under which the second solution (e.g., plurality of
second solutions that include different capture probes) is cured,
while the first (e.g., bulk) solution is not cured. In some
embodiments, the second solution (e.g., plurality of second
solutions that include different capture probes) includes one or
more hydrogel subunits that can be polymerized (e.g., cross-linked)
to form a three-dimensional (3D) hydrogel network, while the first
(e.g., bulk) solution does not include such one or more hydrogel
subunits. Upon subjecting the first and second solutions to curing
conditions, only the second solution(s) will be cured, allowing the
first solution to be removed.
[0949] In some embodiments, the first (e.g., bulk) solution is not
removed from the substrate after the oligonucleotides and/or
features are attached to the substrate.
[0950] (8) Shrinking Droplet/Feature Arrays
[0951] In some embodiments, an expanded first (e.g., bulk)
solution-matrix containing a second solution (e.g., oligonucleotide
and/or feature droplet(s)) can be shrunk along one or more axes
(e.g., of the matrix or of the substrate surface) such that the
cross-sectional area of the second solution droplet(s) (e.g.,
oligonucleotide and/or feature droplet(s)) is smaller than a
corresponding cross-sectional area of a second solution droplet(s)
e.g., oligonucleotide and/or feature droplet(s)) that would be
generated if the first (e.g., bulk) solution-matrix containing the
second solution droplet(s) (e.g., oligonucleotide and/or feature
droplet(s)) were not shrunk along the one or more axes. In some
embodiments, shrinking the matrix (e.g., the first-and-second
solution matrix) generates a shrunken matrix (e.g., a shrunken
first-and-second solution matrix), wherein the volume of a second
solution droplet (e.g., a plurality of second solution droplets) of
the matrix is reduced as compared to the volume of the second
solution droplet in a non-shrunken matrix. In some aspects,
shrinking two or more second solutions (e.g., droplets of second
solutions) results in a higher density of the two or more second
solutions. In some embodiments, the second solution droplet(s)
(e.g., oligonucleotide and/or feature droplet(s)) and first (e.g.,
bulk) solution-matrix can be shrunk by any method disclosed herein.
In some embodiments, the shrinking can include removing water. In
some aspects, the resolution of the array is increased after the
droplet is shrunk. In some aspects, shrinking the second solution
droplet results in a decrease in the cross-sectional area of the
droplet (e.g., the cross-sectional area in the plane of the
substrate onto which the second solution droplet is printed or
dispensed). In some aspects, after shrinking the droplet, the
concentration of probes on the substrate will be increased, thereby
improving sensitivity.
[0952] The spatial array is contacted to the biological sample,
wherein the biological sample can be any described herein (e.g., a
FFPE tissue section). Once the spatial array has been placed on the
biological sample, a cellular and/or nuclear permeabilization
reaction can occur, such that the biological analytes (e.g., DNA,
RNA, proteins, metabolites, small molecules, lipids, and the like)
are released and captured onto the spatial array, preserving their
spatial information. The spatial array is removed, and the
molecular information therein is determined (e.g., by performing
library construction for next generation sequencing). Sequencing
can be followed by correlation of the expression value (e.g., gene
expression of the analyte) with the feature.
[0953] In some aspects, the cross-sectional area of a second
solution droplet is decreased upon shrinking by about 10%, by about
20%, by about 30%, by about 40%, by about 50%, by about 60%, by
about 70%, by about 80%, or by about 90% compared to the
cross-sectional area of a second solution droplet that is printed
or dispensed onto the substrate. In some aspects, the
cross-sectional area of a second solution droplet is decreased by
about 1.0-fold, by about 1.1-fold, by about 1.2-fold, by about
1.3-fold, by about 1.4 fold, by about 1.5-fold, by about 2-fold, by
about 3-fold, by about 4-fold, or by about 5-fold compared to the
cross-sectional area of a second solution droplet that is printed
or dispensed onto the substrate.
[0954] In some aspects, disclosed herein are methods of preparing
an array including dispensing a plurality of second solution
droplets of (e.g., including different capture probes having
different spatial barcodes) onto the substrate covered with a first
(e.g., bulk) solution, curing the first and second solutions to
generate a matrix (e.g., a first and second solution-matrix),
wherein the volume and shape of the plurality of second solution
droplets in the matrix are substantially the same as when the
plurality of second solution droplets was dispensed, and shrinking
the matrix, thereby decreasing the overall volume (e.g.,
cross-sectional area) of the plurality of second solution droplets.
In some aspects, shrinking can be accomplished using any of the
variety of shrinking agents or conditions described herein. In some
aspects, shrinking can be accomplished by dehydrating the
matrix.
[0955] In some aspects, after the second solution is dispensed, the
area of the second solution is shrunk along the one or more axes of
coplanar with the surface of the substrate such that the
cross-sectional area of the second solution on the substrate (e.g.,
the slide) is smaller than a corresponding cross-sectional area of
the second solution that would be generated if the first solution
matrix including the second solution were not shrunk along the one
or more axes of the surface of the substrate.
[0956] With respect to orientation, shrinkage need not be equal in
any two orthogonal directions on the substrate. However, in some
aspects, the shrinkage of a second solution droplet is
substantially uniform in shrinkage. In some aspects, a second
solution droplet shrinks in substantially the same amount in each
direction, regardless of position on the substrate plane.
[0957] In some aspects, after preparing the substrate with the one
or more capture probes (e.g., a spatial array that includes the
capture probes), disclosed herein are methods for spatially
profiling an analyte (e.g., a plurality of analytes) in a
biological sample, including (a) generating a spatial array
including a plurality of capture probes bound to a substrate;
wherein (i) at least a portion of the substrate is coated with a
first solution; and (ii) a plurality of second solutions, in the
form of a liquid droplets, are dispensed onto the surface of the
substrate in the presence of the first solution, wherein the first
solution and the plurality of second solutions do not substantially
mix with each other, wherein at least two members of the plurality
of the second solutions include one or more different capture
probes having different spatial barcodes, and wherein at least one
of the one or more capture probes of at least two members of the
plurality of second solutions is bound to the substrate; (b)
contacting the biological sample with the spatial array such that
the analyte(s) present in the biological sample are captured by one
or more of the capture probes; and (c) determining the spatial
profile of the captured analyte(s) in the biological sample.
[0958] (xii) Array/Feature Preservation
[0959] In some embodiments, the biological sample can be preserved
after completion of an assay with a feature or arrangement of
features for additional rounds of spatial detection of analytes. In
some embodiments, the biological sample, features, array, or any
combination thereof can be preserved after the spatial profiling.
In some embodiments, the biological sample, features, array, or
combinations thereof can be protected from dehydration (e.g.,
drying, desiccation). In some embodiments, the biological sample,
features, array, or combinations thereof, can be protected from
evaporation. Methods of preserving and/or protecting biological
samples, features, or arrays are known in the art. For example, in
a non-limiting way, the biological sample, features, array, or
combinations thereof can be covered by a reversible sealing agent.
Any suitable reversible sealing agent can be used. Methods of
reversible sealing are known in the art (See, e.g., WO 2019/104337,
which is incorporated herein by reference). In a non-limiting way,
suitable reversible sealing agents can include non-porous
materials, membranes, lids, or oils (e.g., silicone oil, mineral
oil). In further non-limiting examples, the biological sample,
features, array, or combinations thereof can be preserved in an
environmental chamber (e.g., hermetically sealed) and removed for
additional rounds of spatial analysis at a later time.
[0960] (e) Analyte Capture
[0961] In this section, general aspects of methods and systems for
capturing analytes are described. Individual method steps and
system features can be present in combination in many different
embodiments; the specific combinations described herein do not in
any way limit other combinations of steps or features.
[0962] (i) Conditions for capture Generally, analytes can be
captured when contacting a biological sample with a substrate
including capture probes (e.g., substrate with capture probes
embedded, spotted, printed on the substrate or a substrate with
features (e.g., beads, wells) comprising capture probes).
[0963] As used herein, "contact," "contacted," and/or "contacting,"
a biological sample with a substrate refers to any contact (e.g.,
direct or indirect) such that capture probes can interact (e.g.,
capture) with analytes from the biological sample. For example, a
substrate may be near or adjacent to the biological sample without
direct physical contact, yet capable of capturing analytes from the
biological sample. In some embodiments a biological sample is in
direct physical contact with a substrate. In some embodiments, a
biological sample is in indirect physical contact with a substrate.
For example, a liquid layer may be between the biological sample
and the substrate. In some embodiments, analytes diffuse through a
liquid layer. In some embodiments capture probes diffuse through a
liquid layer. In some embodiments reagents may be delivered via a
liquid layer between a biological sample and a substrate. In some
embodiments, indirect physical contact may include a second
substrate (e.g., a hydrogel, a film, a porous membrane) between the
biological sample and the first substrate comprising capture
probes. In some embodiments, reagents may be delivered by a second
substrate to a biological sample.
[0964] In some embodiments, a cell immobilization agent can be used
to contact a biological sample with a substrate (e.g., by
immobilizing non-aggregated or disaggregated sample on a
spatially-barcoded array prior to analyte capture). A "cell
immobilization agent" as used herein can refer to an agent (e.g.,
an antibody), attached to a substrate, which can bind to a cell
surface marker. Non-limiting examples of a cell surface marker
include CD45, CD3, CD4, CD8, CD56, CD19, CD20, CD11c, CD14, CD33,
CD66b, CD34, CD41, CD61, CD235a, CD146, and epithelial cellular
adhesion molecule (EpCAM). A cell immobilization agent can include
any probe or component that can bind to (e.g., immobilize) a cell
or tissue when on a substrate. A cell immobilization agent attached
to the surface of a substrate can be used to bind a cell that has a
cell surface maker. The cell surface marker can be a ubiquitous
cell surface marker, wherein the purpose of the cell immobilization
agent is to capture a high percentage of cells within the sample.
The cell surface marker can be a specific, or more rarely
expressed, cell surface marker, wherein the purpose of the cell
immobilization agent is to capture a specific cell population
expressing the target cell surface marker. Accordingly, a cell
immobilization agent can be used to selectively capture a cell
expressing the target cell surface marker from a population of
cells that do not have the same cell surface marker.
[0965] Capture probes on a substrate (or on a feature on the
substrate) may interact with released analytes through a capture
domain, described elsewhere, to capture analytes. In some
embodiments, certain steps are performed to enhance the transfer or
capture of analytes to the capture probes of the array. Examples of
such modifications include, but are not limited to, adjusting
conditions for contacting the substrate with a biological sample
(e.g., time, temperature, orientation, pH levels, pre-treating of
biological samples, etc.), using force to transport analytes (e.g.,
electrophoretic, centrifugal, mechanical, etc.), performing
amplification reactions to increase the amount of biological
analytes (e.g., PCR amplification, in situ amplification, clonal
amplification), and/or using labeled probes for detecting of
amplicons and barcodes.
[0966] In some embodiments, an array is adapted in order to
facilitate biological analyte migration. Non-limiting examples of
adapting an array to facilitate biological analyte migration
include arrays with substrates containing nanopores, nanowells,
and/or microfluidic channels; arrays with porous membranes; and
arrays with substrates that are made of hydrogel. In some cases,
the array substrate is liquid permeable. In some cases, the array
is a coverslip or slide that includes nanowells or patterning,
(e.g., via fabrication). In some cases where the substrate includes
nanopores, nanowells, and/or microfluidic channels, these
structures can facilitate exposure of the biological sample to
reagents (e.g., reagents for permeabilization, biological analyte
capture, and/or a nucleic acid extension reaction), thereby
increasing analyte capture efficiency as compared to a substrate
lacking such characteristics.
[0967] In some embodiments, analyte capture is facilitated by
treating a biological sample with permeabilization reagents. If a
biological sample is not permeabilized sufficiently, the amount of
analyte captured on a substrate can be too low to enable adequate
analysis. Conversely, if a biological sample is too permeable, an
analyte can diffuse away from its origin in the biological sample,
such that the relative spatial relationship of the analytes within
the biological sample is lost. Hence, a balance between
permeabilizing the biological sample enough to obtain good signal
intensity while still maintaining the spatial resolution of the
analyte distribution in the biological sample is desired. Methods
of preparing biological samples to facilitate analyte capture are
known in the art and can be modified depending on the biological
sample and how the biological sample is prepared (e.g., fresh
frozen, FFPE, etc.).
[0968] (ii) Substrate Holder
[0969] Described herein are methods in which an array with capture
probes located on a substrate and a biological sample located on a
different substrate, are contacted such that the array is in
contact with the biological sample (e.g., the substrates are
sandwiched together). In some embodiments, the array and the
biological sample can be contacted (e.g., sandwiched), without the
aid of a substrate holder. In some embodiments, the array and
biological sample substrates can be placed in a substrate holder
(e.g., an array alignment device) designed to align the biological
sample and the array. For example, the substrate holder can have
placeholders for two substrates. In some embodiments, an array
including capture probes can be positioned on one side of the
substrate holder (e.g., in a first substrate placeholder). In some
embodiments, a biological sample can be placed on the adjacent side
of the substrate holder in a second placeholder. In some
embodiments, a hinge can be located between the two substrate
placeholders that allows the substrate holder to close, e.g., make
a sandwich between the two substrate placeholders. In some
embodiments, when the substrate holder is closed the biological
sample and the array with capture probes are contacted with one
another under conditions sufficient to allow analytes present in
the biological sample to interact with the capture probes of the
array. For example, dried permeabilization reagents can be placed
on the biological sample and rehydrated. A permeabilization
solution can be flowed through the substrate holder to permeabilize
the biological sample and allow analytes in the biological sample
to interact with the capture probes. Additionally, the temperature
of the substrates or permeabilization solution can be used to
initiate or control the rate of permeabilization. For example, the
substrate including the array, the substrate including the
biological sample, or both substrates can be held at a low
temperature to slow diffusion and permeabilization efficiency. Once
sandwiched, in some embodiments, the substrates can be heated to
initiate permeabilization and/or increase diffusion efficiency.
Transcripts that are released from the permeabilized tissue can
diffuse to the array and be captured by the capture probes. The
sandwich can be opened, and cDNA synthesis can be performed on the
array.
[0970] Any of the variety of combinations described herein where a
sandwich including an array with capture probes and a biological
sample on two different substrates can be placed in a substrate
holder designed to align the biological sample and the array. For
example, the substrate holder can have placeholders for two
substrates. In some embodiments, an array including capture probes
can be positioned on one side of the substrate holder (e.g., in a
first substrate placeholder). In some embodiments, a biological
sample can be placed on the adjacent side of the substrate holder
in a second placeholder. In some embodiments, in between the two
substrate placeholders can be a hinge that allows the substrate
holder to close, e.g., make a sandwich between the two substrate
placeholders. In some embodiments, when the substrate holder is
closed the biological sample and the array with capture probes can
be contacted with one another under conditions sufficient to allow
analytes present in the biological sample to interact with the
capture probes of the array for spatial analysis by any method
described herein. For example, dried permeabilization reagents can
be placed on the biological sample and rehydrated. Additionally, a
permeabilization solution can be flowed through the substrate
holder to permeabilize the biological sample and allow analytes in
the biological sample to interact with the capture probes.
[0971] In some embodiments, a flexible array described herein can
be placed in the substrate holder, and sandwiched with a biological
sample. In some embodiments, the flexible array can include
spatially-barcoded cross-linked features. In some embodiments, the
flexible array can be presoaked in permeabilization reagents before
being placed into the substrate holder. In some embodiments, the
flexible array can be soaked in permeabilization reagents after
being placed in the substrate holder. In some embodiments, the
substrate holder including a biological sample in one placeholder
and a flexible array can be closed (e.g., form a sandwich) such
that the permeabilization reagents allow analytes present in the
biological sample to interact with capture probes of the flexible
array (e.g., capture probes on the spatially-barcoded
features).
[0972] In some embodiments, the substrate holder can be heated or
cooled to regulate permeabilization and/or diffusion
efficiency.
[0973] (iii) Passive Capture Methods
[0974] In some embodiments, analytes can be migrated from a sample
to a substrate. Methods for facilitating migration can be passive
(e.g., diffusion) and/or active (e.g., electrophoretic migration of
nucleic acids). Non-limiting examples of passive migration can
include simple diffusion and osmotic pressure created by the
rehydration of dehydrated objects.
[0975] Passive migration by diffusion uses concentration gradients.
Diffusion is movement of untethered objects toward equilibrium.
Therefore, when there is a region of high object concentration and
a region of low object concentration, the object (e.g., a capture
probe, an analyte, etc.) moves to an area of lower concentration.
In some embodiments, untethered analytes move down a concentration
gradient.
[0976] In some embodiments, different reagents may be added to the
biological sample, such that the biological sample is rehydrated
while improving capture of analytes. In some embodiments, the
biological sample can be contacted with a shrunken array as
described herein. In some embodiments, the biological sample and/or
the shrunken array can be rehydrated with permeabilization
reagents. In some embodiments, the biological sample and/or the
shrunken array can be rehydrated with a staining solution (e.g.,
hematoxylin and eosin stain).
[0977] (iv) Diffusion-Resistant Media/Lids
[0978] To increase efficiency by encouraging analyte diffusion
toward the spatially-barcoded capture probes, a diffusion-resistant
medium can be used. In general, molecular diffusion of biological
analytes occurs in all directions, including toward the capture
probes (i.e., toward the spatially-barcoded array), and away from
the capture probes (i.e., into the bulk solution). Increasing
diffusion toward the spatially-barcoded array reduces analyte
diffusion away from the spatially-barcoded array and increases the
capturing efficiency of the capture probes.
[0979] In some embodiments, a biological sample is placed on the
top of a spatially-barcoded substrate and a diffusion-resistant
medium is placed on top of the biological sample. For example, the
diffusion-resistant medium can be placed onto an array that has
been placed in contact with a biological sample. In some
embodiments, the diffusion-resistant medium and spatially-barcoded
array are the same component. For example, the diffusion-resistant
medium can contain spatially-barcoded capture probes within or on
the diffusion-resistant medium (e.g., coverslip, slide, hydrogel,
or membrane). In some embodiments, a sample is placed on a
substrate and a diffusion-resistant medium is placed on top of the
biological sample. Additionally, a spatially-barcoded capture probe
array can be placed in close proximity over a diffusion-resistant
medium. For example, a diffusion-resistant medium may be sandwiched
between a spatially-barcoded array and a sample on a substrate. In
some embodiments, a diffusion-resistant medium is disposed or
spotted onto a sample. In other embodiments, a diffusion-resistant
medium is placed in close proximity to a sample.
[0980] In general, a diffusion-resistant medium can be any material
known to limit diffusivity of biological analytes. For example, a
diffusion-resistant medium can be a solid lid (e.g., coverslip or
glass slide). In some embodiments, a diffusion-resistant medium may
be made of glass, silicon, paper, hydrogel polymer monoliths, or
other material. In some embodiments, the glass side can be an
acrylated glass slide. In some embodiments, the diffusion-resistant
medium is a porous membrane. In some embodiments, the material may
be naturally porous. In some embodiments, the material may have
pores or wells etched into solid material. In some embodiments, the
pore volume can be manipulated to minimize loss of target analytes.
In some embodiments, the membrane chemistry can be manipulated to
minimize loss of target analytes. In some embodiments, the
diffusion-resistant medium (e.g., hydrogel) is covalently attached
to a substrate (e.g., glass slide). In some embodiments, a
diffusion-resistant medium can be any material known to limit
diffusivity of poly(A) transcripts. In some embodiments, a
diffusion-resistant medium can be any material known to limit the
diffusivity of proteins. In some embodiments, a diffusion-resistant
medium can be any material know to limit the diffusivity of
macromolecular constituents.
[0981] In some embodiments, a diffusion-resistant medium includes
one or more diffusion-resistant media. For example, one or more
diffusion-resistant media can be combined in a variety of ways
prior to placing the media in contact with a biological sample
including, without limitation, coating, layering, or spotting. As
another example, a hydrogel can be placed onto a biological sample
followed by placement of a lid (e.g., glass slide) on top of the
hydrogel.
[0982] In some embodiments, a force (e.g., hydrodynamic pressure,
ultrasonic vibration, solute contrasts, microwave radiation,
vascular circulation, or other electrical, mechanical, magnetic,
centrifugal, and/or thermal forces) is applied to control diffusion
and enhance analyte capture. In some embodiments, one or more
forces and one or more diffusion-resistant media are used to
control diffusion and enhance capture. For example, a centrifugal
force and a glass slide can used contemporaneously. Any of a
variety of combinations of a force and a diffusion-resistant medium
can be used to control or mitigate diffusion and enhance analyte
capture.
[0983] In some embodiments, a diffusion-resistant medium, along
with the spatially-barcoded array and sample, is submerged in a
bulk solution. In some embodiments, a bulk solution includes
permeabilization reagents. In some embodiments, a
diffusion-resistant medium includes at least one permeabilization
reagent. In some embodiments, a diffusion-resistant medium (i.e.
hydrogel) is soaked in permeabilization reagents before contacting
the diffusion-resistant medium to the sample. In some embodiments,
a diffusion-resistant medium can include wells (e.g., micro-,
nano-, or picowells) containing a permeabilization buffer or
reagents. In some embodiments, a diffusion-resistant medium can
include permeabilization reagents. In some embodiments, a
diffusion-resistant medium can contain dried reagents or monomers
to deliver permeabilization reagents when the diffusion-resistant
medium is applied to a biological sample. In some embodiments, a
diffusion-resistant medium is added to the spatially-barcoded array
and sample assembly before the assembly is submerged in a bulk
solution. In some embodiments, a diffusion-resistant medium is
added to the spatially-barcoded array and sample assembly after the
sample has been exposed to permeabilization reagents. In some
embodiments, permeabilization reagents are flowed through a
microfluidic chamber or channel over the diffusion-resistant
medium. In some embodiments, the flow controls the sample's access
to the permeabilization reagents. In some embodiments, target
analytes diffuse out of the sample and toward a bulk solution and
get embedded in a spatially-barcoded capture probe-embedded
diffusion-resistant medium. In some embodiments, a free solution is
sandwiched between the biological sample and a diffusion-resistant
medium.
[0984] FIG. 13 is an illustration of an exemplary use of a
diffusion-resistant medium. A diffusion-resistant medium 1302 can
be contacted with a sample 1303. In FIG. 13, a glass slide 1304 is
populated with spatially-barcoded capture probes 1306, and the
sample 1303, 1305 is contacted with the array 1304, 1306. A
diffusion-resistant medium 1302 can be applied to the sample 1303,
wherein the sample 1303 is sandwiched between a diffusion-resistant
medium 1302 and a capture probe coated slide 1304. When a
permeabilization solution 1301 is applied to the sample, using the
diffusion-resistant medium/lid 1302 directs migration of the
analytes 1305 toward the capture probes 1306 by reducing diffusion
of the analytes out into the medium. Alternatively, the lid may
contain permeabilization reagents.
[0985] (v) Active Capture Methods
[0986] In some of the methods described herein, an analyte in a
biological sample (e.g., in a cell or tissue section) can be
transported (e.g., passively or actively) to a capture probe (e.g.,
a capture probe affixed to a substrate (e.g., a substrate or
bead)).
[0987] For example, analytes can be transported to a capture probe
(e.g., an immobilized capture probe) using an electric field (e.g.,
using electrophoresis), pressure, fluid flow, gravity, temperature,
and/or a magnetic field. For example, analytes can be transported
through, e.g., a gel (e.g., hydrogel), a fluid, or a permeabilized
cell, to a capture probe (e.g., an immobilized capture probe) using
a pressure gradient, a chemical concentration gradient, a
temperature gradient, and/or a pH gradient. For example, analytes
can be transported through a gel (e.g., hydrogel), a fluid, or a
permeabilized cell, to a capture probe (e.g., an immobilized
capture probe).
[0988] In some examples, an electrophoretic field can be applied to
analytes to facilitate migration of analytes towards a capture
probe. In some examples, a sample containing analytes contacts a
substrate having capture probes fixed on the substrate (e.g., a
slide, cover slip, or bead), and an electric current is applied to
promote the directional migration of charged analytes towards
capture probes on a substrate. An electrophoresis assembly (e.g.,
an electrophoretic chamber), where a biological sample is in
contact with a cathode and capture probes (e.g., capture probes
fixed on a substrate), and where the capture probes are in contact
with the biological sample and an anode, can be used to apply the
current.
[0989] In some embodiments, methods utilizing an active capture
method can employ a conductive substrate (e.g., any of the
conductive substrates described herein). In some embodiments, a
conductive substrate includes paper, a hydrogel film, or a glass
slide having a conductive coating. In some embodiments, a
conductive substrate (e.g., any of the conductive substrates
described herein) includes one or more capture probes.
[0990] In some embodiments, electrophoretic transfer of analytes
can be performed while retaining the relative spatial locations of
analytes in a biological sample while minimizing passive diffusion
of an analyte away from its location in a biological sample. In
some embodiments, an analyte captured by a capture probe (e.g.,
capture probes on a substrate) retains the spatial location of the
analyte present in the biological sample from which it was obtained
(e.g., the spatial location of the analyte that is captured by a
capture probe on a substrate when the analyte is actively migrated
to the capture probe by electrophoretic transfer can be more
precise or representative of the spatial location of the analyte in
the biological sample than when the analyte is not actively
migrated to the capture probe). In some embodiments,
electrophoretic transport and binding process is described by the
Damkohler number (Da), which is a ratio of reaction and mass
transport rates. The fraction of analytes bound and the shape of
the biological sample will depend on the parameters in the Da.
There parameters include electromigration velocity U.sub.e
(depending on analyte electrophoretic mobility .mu..sub.e and
electric field strength E), density of capture probes (e.g.,
barcoded oligonucleotides) p.sub.0, the binding rate between probes
(e.g., barcoded oligonucleotides) and analytes k.sub.on, and
capture area thickness L.
Da .about. k on .times. p 0 .times. L .mu. e .times. E
##EQU00001##
Fast migration (e.g., electromigration) can reduce assay time and
can minimize molecular diffusion of analytes.
[0991] In some embodiments, electrophoretic transfer of analytes
can be performed while retaining the relative spatial alignment of
the analytes in the sample. As such, an analyte captured by the
capture probes (e.g., capture probes on a substrate) retains the
spatial information of the cell or the biological sample from which
it was obtained. Applying an electrophoretic field to analytes can
also result in an increase in temperature (e.g., heat). In some
embodiments, the increased temperature (e.g., heat) can facilitate
the migration of the analytes towards a capture probe.
[0992] In some examples, a spatially-addressable microelectrode
array is used for spatially-constrained capture of at least one
charged analyte of interest by a capture probe. For example, a
spatially-addressable microelectrode array can allow for discrete
(e.g., localized) application of an electric field rather than a
uniform electric field. The spatially-addressable microelectrode
array can be independently addressable. In some embodiments, the
electric field can be applied to one or more regions of interest in
a biological sample. The electrodes may be adjacent to each other
or distant from each other. The microelectrode array can be
configured to include a high density of discrete sites having a
small area for applying an electric field to promote the migration
of charged analyte(s) of interest. For example, electrophoretic
capture can be performed on a region of interest using a
spatially-addressable microelectrode array.
[0993] A high density of discrete sites on a microelectrode array
can be used. The surface can include any suitable density of
discrete sites (e.g., a density suitable for processing the sample
on the conductive substrate in a given amount of time). In one
embodiment, the surface has a density of discrete sites greater
than or equal to about 500 sites per 1 mm.sup.2. In some
embodiments, the surface has a density of discrete sites of about
100, about 200, about 300, about 400, about 500, about 600, about
700, about 800, about 900, about 1,000, about 2,000, about 3,000,
about 4,000, about 5,000, about 6,000, about 7,000, about 8,000,
about 9,000, about 10,000, about 20,000, about 40,000, about
60,000, about 80,000, about 100,000, or about 500,000 sites per 1
mm.sup.2. In some embodiments, the surface has a density of
discrete sites of at least about 200, at least about 300, at least
about 400, at least about 500, at least about 600, at least about
700, at least about 800, at least about 900, at least about 1,000,
at least about 2,000, at least about 3,000, at least about 4,000,
at least about 5,000, at least about 6,000, at least about 7,000,
at least about 8,000, at least about 9,000, at least about 10,000,
at least about 20,000, at least about 40,000, at least about
60,000, at least about 80,000, at least about 100,000, or at least
about 500,000 sites per 1 mm.sup.2.
[0994] Schematics illustrating an electrophoretic transfer system
configured to direct nucleic acid analytes (e.g., mRNA transcripts)
toward a spatially-barcoded capture probe array are shown in FIG.
14A and FIG. 14B. In this exemplary configuration of an
electrophoretic system, a sample 1402 is sandwiched between the
cathode 1401 and the spatially-barcoded capture probe array 1404,
1405, and the spatially-barcoded capture probe array 1404, 1405 is
sandwiched between the sample 1402 and the anode 1403, such that
the sample 1402, 1406 is in contact with the spatially-barcoded
capture probes 1407. When an electric field is applied to the
electrophoretic transfer system, negatively charged nucleic acid
analytes 1406 will be pulled toward the positively charged anode
1403 and into the spatially-barcoded array 1404, 1405 containing
the spatially-barcoded capture probes 1407. The spatially-barcoded
capture probes 1407 interact with the nucleic acid analytes (e.g.,
mRNA transcripts hybridize to spatially-barcoded nucleic acid
capture probes forming DNA/RNA hybrids) 1406, making the analyte
capture more efficient. The electrophoretic system set-up may
change depending on the target analyte. For example, proteins may
be positive, negative, neutral, or polar depending on the protein
as well as other factors (e.g., isoelectric point, solubility,
etc.). The skilled practitioner has the knowledge and experience to
arrange the electrophoretic transfer system to facilitate capture
of a particular target analyte.
[0995] FIG. 15 is an illustration showing an exemplary workflow
protocol utilizing an electrophoretic transfer system. In the
example, Panel A depicts a flexible spatially-barcoded feature
array being contacted with a sample. The sample can be a flexible
array, wherein the array is immobilized on a hydrogel, membrane, or
other flexible substrate. Panel B depicts contact of the array with
the sample and imaging of the array-sample assembly. The image of
the sample/array assembly can be used to verify sample placement,
choose a region of interest, or any other reason for imaging a
sample on an array as described herein. Panel C depicts application
of an electric field using an electrophoretic transfer system to
aid in efficient capture of a target analyte. Here, negatively
charged mRNA target analytes migrate toward the positively charged
anode. Panel D depicts application of reverse transcription
reagents and first strand cDNA synthesis of the captured target
analytes. Panel E depicts array removal and preparation for library
construction (Panel F) and next-generation sequencing (Panel
G).
[0996] (vi) Targeted Analysis
[0997] In some aspects, arrays (e.g., glass slides) include a
plurality of capture probes that bind to one or more specific
biological targets in a sample. The capture probes can be directly
or indirectly attached to a substrate. The capture probe can be or
include, for example, DNA or RNA. In some aspects, the capture
probes on an array can be immobilized, e.g., attached or bound, to
the array via their 5' or 3' ends, depending on the chemical matrix
of the array. In some aspects, the probes are attached via a 3'
linkage, thereby leaving a free 5' end. In some aspects, the probes
are attached via a 5' linkage, thereby leaving a free 3' end. In
some aspects, the probes are immobilized indirectly. For example, a
probe can be attached to a bead, which bead can be deposited on a
substrate. A capture probe as disclosed in this section can include
any of the various components of a capture probe as provided
throughout this disclosure (e.g., spatial barcodes, UMIs,
functional domains, cleavage domains, etc.).
[0998] In some aspects, a capture probe or plurality of capture
probes interact with an analyte specific for a particular species
or organism (e.g., host or pathogen). In some aspects, the probe or
plurality of probes can be used to detect a viral, bacterial, or
plant protein or nucleic acid. In some aspects, the capture probe
or plurality of capture probes can be used to detect the presence
of a pathogen (e.g., bacteria or virus) in the biological sample.
In some aspects, the capture probe or plurality of capture probes
can be used to detect the expression of a particular nucleic acid
associated with a pathogen (e.g., presence of 16S ribosomal RNA or
Human Immunodeficiency Virus (HIV) RNA in a human sample).
[0999] In some aspects, the capture domain in the capture probe can
interact with one or more specific analytes (e.g., an analyte or a
subset of analytes out of the pool of total analytes). The specific
analyte(s) to be detected can be any of a variety of biological
molecules including but not limited to proteins, nucleic acids,
lipids, carbohydrates, ions, small molecules, subcellular targets,
or multicomponent complexes containing any of the above. In some
embodiments, the analyte(s) can be localized to subcellular
location(s), including, for example, organelles, e.g.,
mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts,
endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In
some embodiments, analyte(s) can be peptides or proteins, including
without limitation antibodies and enzymes.
[1000] In some aspects, analytes from a biological sample interact
with one or more capture probes (e.g., one or more capture probes
immobilized directly or indirectly on a substrate), and the capture
probes interact with specific analytes in the biological sample. In
some aspects, the capture probes are allowed to interact with
(e.g., hybridize to) specific analytes, e.g., under appropriate
conditions where oligonucleotide capture probes can hybridize to
the target nucleic acids. In some aspects, analytes that did not
hybridize to capture probes are removed (e.g., analytes that do not
interact with capture domains of the capture probes). In some
embodiments, removal of analytes that did not interact with a
capture probe can be accomplished by, e.g., washing the sample to
remove such analytes.
[1001] In some aspects, a capture probe or plurality of capture
probes includes a capture domain that interacts with an analyte or
analytes present in a biological sample. In some aspects, the
capture probe or plurality of capture probes includes a capture
domain that detects the presence or level amount (e.g., expression
level) of a particular analyte or analytes of interest. The capture
domain of a capture probe (immobilized directly or indirectly on a
substrate) can be capable of binding selectively to a desired
subtype or subset of nucleic acid. In some aspects, for example,
the capture domain binds to a subset of nucleic acids in a genome
or a subset of nucleic acids in a transcriptome. In some aspects,
the analyte(s) can include one or more nucleic acids. In some
aspects, the capture probe or plurality of capture probes can be
used to detect the expression of a particular transcript (e.g., a
particular mRNA). In some aspects, a capture probe or plurality of
capture probes can be specific for (e.g., binds to) an individual
change in a nucleic acid or protein (e.g., a mutation or single
nucleotide polymorphism (SNP)).
[1002] In some aspects, the biological sample includes an analyte
that is or includes a nucleic acid. The nucleic acid can be RNA or
DNA. In some aspects, the capture probe or plurality of capture
probes detects DNA copy number of a particular set of nucleic acid
analyte or analytes. For example, capture probe or plurality of
capture probes provided herein can be used to detect DNA copy
number of nucleic acids that share homology to each other.
[1003] In some aspects, the capture probe or plurality of capture
probes includes a capture domain that detects the presence or level
amount (e.g., expression level) of one or more RNA transcripts
(e.g., specific RNA transcripts). In some aspects, the capture
probe or plurality of capture probes includes a capture domain that
detects the presence or amount (e.g., expression level) of one or
more non-coding RNAs (e.g., microRNA, transfer RNA (tRNA),
ribosomal RNA (rRNA), small interfering RNA (siRNA) and small
nucleolar RNA (snoRNA). In some aspects, the probe or plurality of
probes includes a capture domain that detects the presence or level
amount (e.g., expression level) of one or more proteins (e.g.,
proteins expressed of a nucleic acid of interest).
[1004] In some aspects, the capture probe or plurality of capture
probes can be specific for a particular protein. In some aspects,
the capture probe or plurality of capture probes can be used to
detect the presence of a particular protein of interest. In some
aspects, the capture probe or plurality of capture probes can be
used to detect translation of a particular protein. In some
aspects, the capture probe or plurality of capture probes can
specifically interact with an active region of an enzyme, a binding
domain of an immunoglobulin, defined domains of proteins, whole
proteins, synthetic peptides, peptides with introduced mutations,
aptamer, or any combination thereof. In some aspects, the
analyte(s) can include one or more proteins. In some aspects, the
analyte(s) can include one or more nucleic acids and one or more
proteins.
[1005] In some aspects, the capture probe or plurality of capture
probes can be used to detect particular post-translational
modifications of a particular protein. In such embodiments, analyte
capture agents can be specific for cell surface analytes having a
given state of posttranslational modification (e.g.,
phosphorylation, glycosylation, ubiquitination, nitrosylation,
methylation, acetylation or lipidation), such that a cell surface
analyte profile can include posttranslational modification
information of one or more analytes.
[1006] In some aspects, the capture probe or plurality of capture
probes can be specific for a particular set of nucleic acids (e.g.,
nucleic acids that are associated with a specific cellular pathway
or pathways). In some aspects, the set of nucleic acids is DNA. In
some aspects, the set of nucleic acids is RNA. In some aspects, the
set of nucleic acids has similar and/or homologous sequences. In
some aspects, the set of nucleic acids encodes for analytes that
function in a similar cellular pathway. In some aspects, the set of
nucleic acids encodes for analytes that are expressed in a certain
pathological state (e.g., cancer, Alzheimer's, or Parkinson's
disease). In some aspects, the set of nucleic acids encodes for
analytes that are over-expressed in a certain pathological state.
In some aspects, the set of nucleic acids encodes for analytes that
are under-expressed in a certain pathological state.
[1007] In some aspects, the capture probe or plurality of capture
probes can be specific for a particular nucleic acid, or detection
or expression of a particular set of proteins (e.g., in a similar
cellular pathway). In some aspects, the set of proteins has similar
functional domains. In some aspects, the set of proteins functions
in a similar cellular pathway. In some aspects, the set of proteins
is expressed in a certain pathological state (e.g., cancer,
Alzheimer's or Parkinson's disease). In some aspects, the set of
proteins is over-expressed in a certain pathological state. In some
aspects, the set of proteins is under-expressed in a certain
pathological state.
[1008] In some embodiments, a capture probe includes a capture
domain that is capable of binding to more than one analyte. In some
embodiments, a capture domain can bind to one or more analytes that
are about 80% identical, about 85% identical, about 90% identical,
about 95% identical, about 96% identical, about 97% identical,
about 98% identical, about 99% identical, 100% identical to the
target analyte. In some aspects, the capture probe can bind to an
analyte that is about 80% identical, about 85% identical, about 90%
identical, about 95% identical, about 96% identical, about 97%
identical, about 98% identical, or about 99% identical to each
other. In some embodiments, a capture domain can bind to a
conserved region of one or more analytes, in which the conserved
regions are about 80% identical, about 85% identical, about 90%
identical, about 95% identical, about 96% identical, about 97%
identical, about 98% identical, about 99% identical, 100% identical
to the target analyte.
[1009] In some aspects, a capture probe or plurality of capture
probes interacts with two or more analytes (e.g., nucleic acids or
proteins) that are not similar in sequence and/or do not share a
conserved domain. In some embodiments, a capture probe includes two
or more capture domains, each of which interacts with a different
analyte. In such embodiments, members of the two or more capture
domains can be adjacent to each other in the capture probe and/or
members of the two or more capture domains can be separated from
each other in the capture probe by one or more domains (e.g.,
nucleic acid domains). For example, in some aspects, the sets of
analytes that are detected include mutational changes in the
targeted nucleic acids or proteins. In some aspects, the capture
probe or plurality of capture probes detects sets of nucleic acids
or proteins (e.g., non-homologous nucleic acids or proteins) that
are individually mutated during a pathogenic state. In some
aspects, the pathogenic state is cancer.
[1010] In some aspects, a capture probe or plurality of capture
probes include capture domains that can be used to detect analytes
that are typically detected using diagnostic panels. In some
aspects, the capture probe or plurality of capture probes are used
to detect changes in one or more analytes. In some aspects, the
analyte changes include one or more of increased analyte
expression, decreased analyte expression, mutated nucleic acid
sequences, or any combination thereof. In some aspects, the changes
in the analytes are associated with and/or lead to manifestation of
a pathogenic state in a subject. In some aspects, the detected
changes are compared to a reference analyte or analytes.
[1011] (vii) Polypeptide Capture
[1012] Provided herein are methods and materials for identifying
the location of a polypeptide in a biological sample. In some
embodiments, an analyte (e.g., a polypeptide analyte) can be
directly captured on a substrate. For example, polypeptide analytes
can be captured by amine groups on a functionalized substrate. In
other examples, an analyte (e.g., a polypeptide analyte) can be
captured via an analyte binding moiety directly attached to a
substrate. In some embodiments, the substrate may be populated with
analyte minding moieties directly attached to the substrate as well
as spatially-barcoded capture probes directly attached to the
substrate. In other embodiments, an analyte (e.g., a polypeptide
analyte) can be captured via an analyte binding moiety indirectly
attached to a substrate. In an example, the substrate may be
populated with capture probes that are bound to an analyte capture
agent, wherein the analyte capture domain of the analyte capture
agent binds to the capture domain of the capture probe and the
analyte binding moiety binds the polypeptide analyte.
[1013] In some embodiments, an analyte (e.g., a polypeptide
analyte) can be directly captured or immobilized on a substrate.
Direct immobilization may be achieved by covalently coupling the
polypeptide analyte to the substrate via amide bonds between the
carboxylic acid of the C-terminal amino acid residue and a
functionalized substrate surface. For example, a substrate (e.g., a
glass coverslip or slide) can be functionalized through
amino-silanization with aminopropyltriethoxysilane. The substrate
surfaces are further passivated by overnight incubation with
polyethylene glycol (PEG)-NHS solution, and functionalized slides
can be stored in a vacuum desiccator until use. The
t-butyloxycarbonyl protecting groups can be removed by incubating
the substrate with 90% TFA (v/v in water) for 5 hours before use,
thus exposing free amine groups for peptide immobilization. The
resulting functionalized substrate is stable to multiple cycles of
Edman degradation and washing steps.
[1014] In some embodiments, methods for capturing polypeptides in a
biological sample include providing a substrate where an analyte
binding moiety is directly immobilized on the substrate. In some
embodiments, direct immobilization is achieved through chemical
modification of the substrate and/or chemical modification of the
analyte binding moiety. For example, a substrate can be prepared
with free amines on the surface. When exposed to an analyte binding
moiety with a free carboxylic acid on the C-terminal residue, the
free amines can form amide bonds with the carboxylic acid thereby
covalently coupling the analyte binding moiety to the substrate.
Substrates and/or analyte binding moieties can be modified in any
manner that facilitates covalent bonding of the analyte binding
moiety to the substrate. Non-limiting examples of chemical
modification that can be used to covalently bind the analyte
binding moiety to the substrate include are described herein.
[1015] In some embodiments, methods for capturing analyte
polypeptides include providing a substrate (e.g., an array) where
the analyte binding moiety is indirectly attached to the substrate.
For example, an analyte binding moiety can be indirectly attached
to a substrate via an oligonucleotide (e.g., a capture agent
barcode domain or capture agent barcode domain hybridized to a
capture probe) or other domain capable of binding to both the
substrate and the analyte binding domain. The capture agent barcode
domain is described elsewhere herein. The capture agent barcode
domain can be modified to include a cleavage domain, which can
attach to a substrate using any of the chemistries described
herein. In some embodiments, the capture agent barcode domain can
include an analyte capture sequence as described herein, wherein
the analyte capture sequence can hybridize to the capture domain of
a capture probe. In some embodiments, a substrate (e.g., an array)
containing capture probes can be modified to capture polypeptide
analytes by hybridizing the analyte capture sequence of the analyte
capture agent to the capture domain of a capture probe.
[1016] In some embodiments, methods for capturing analyte
polypeptides include providing a substrate (e.g., an array) and
providing an analyte capture agent to the biological sample. For
example, after drying and fixing sectioned tissue samples, the
tissue samples can be positioned on a substrate (e.g., a spatial
array), rehydrated, blocked, and permeabilized (e.g., 3.times.SSC,
2% BSA, 0.1% Triton X, 1 U/.mu.l RNAse inhibitor for 10 min at
4.degree. C.) before being stained with fluorescent primary
antibodies (1:100) and a pool of analyte capture agents (in
3.times.SSC, 2% BSA, 0.1% Triton X, 1 U/.mu.l RNAse inhibitor for
30 min at 4.degree. C.). The biological sample can be washed,
coverslipped (in glycerol+1 U/.mu.l RNAse inhibitor), imaged for
detected analytes (e.g., using a confocal microscope or other
apparatus capable of fluorescent detection), and washed again. The
analyte-bound analyte capture agents can be released from the
biological sample (e.g., the biological sample can be treated with
proteinase, e.g., proteinase K) and migrated to the spatial array.
An analyte capture sequence of the analyte-bound analyte capture
agent can be captured by a capture probe capture domain, and the
capture agent barcode domain can be extended to produce a
spatially-tagged analyte capture agent. The spatially-tagged
analyte capture agents can be processed according to spatial
workflows described herein.
[1017] In some embodiments, methods for capturing analyte
polypeptides include providing blocking probes to analyte capture
agents before introducing the analyte capture agents to a
biological sample. In some embodiments, the blocking probes can be
alternatively or additionally provided in any of the rehydrating or
blocking buffers provided herein. In some embodiments, the analyte
capture agent analyte capture sequence can be blocked prior to
binding to the capture probe capture domain using a blocking probe
sequence complementary to the analyte capture sequence. Blocking
the capture agent barcode domain, particularly the free 3' end of
the capture agent barcode domain (e.g., analyte capture sequence),
prior to contacting the analyte capture agents with the biological
sample and/or substrate, can prevent binding of the analyte capture
sequence of the capture agent barcode domains, e.g., prevents the
binding of a poly(A) tail to the capture probe capture domain. In
some embodiments, blocking the analyte capture agent analyte
capture domain reduces non-specific background staining. In some
embodiments, the blocking probes are reversible, such that the
blocking probes can be removed from the analyte capture sequence
during or after the time that analyte capture agents are in contact
with the biological sample. In some embodiments, the blocking probe
can be removing with RNAse treatment (e.g., RNAse H treatment).
[1018] In some embodiments, methods for capturing polypeptides in a
biological sample include active transfer (e.g., electrophoresis).
For example, the biological sample is placed on a conductive
substrate and contacted with a spatial array including one or more
analyte binding moieties. An electric filed can be applied to the
conductive substrate to promote migration of the polypeptides
towards the analyte binding moieties, as described herein.
[1019] In some embodiments, methods for identifying the spatial
location of a polypeptide in a biological sample include
determining the sequence of a captured polypeptide. In some
embodiments, the sequence of the captured polypeptide is determined
through detection of amino acid residues labeled with a detectable
label (e.g., radiolabel of a fluorophore). Non-limiting examples of
detectable labels that can be used for labelling the captured
polypeptide include fluorophores and radiolabels. In some
embodiments, the polypeptides are labeled at specific amino acid
residues only (e.g., not all amino acid residues are labeled). In
some embodiments, the polypeptide is labeled prior to contacting
the biological sample with the substrate. In some embodiments, a
captured polypeptide is labeled with fluorophores using standard
coupling schemes (see Hernandez et al., New J. Chem. 41:462-469
(2017)). For example, polypeptides may be labeled by reaction with
Atto647N-NHS, Atto647Niodoacetamide, TMR-NHS, or JF549-NHS, as
appropriate, to label lysines (via NHS) or cysteines (via
iodoacetamide). In addition, serine or threonine phosphorylation
sites may be selectively labeled via beta elimination followed by
conjugate addition via thiols to substitute thiol-linked
fluorophores in place of phosphates (see Stevens et al., Rapid
Commun. Mass Specrtom., 15: 2157-2162 (2005)). The number of
fluorophores incorporated into a polypeptide is any number that may
be spectrally resolved. In some instances, four or more
fluorophores are utilized.
[1020] In some embodiments, a captured polypeptide is radiolabeled.
In some embodiments, specific amino acids can be labeled with an
isotope. Non-limiting examples of isotopes used to label amino
acids include .sup.3H, .sup.14C, .sup.15N, .sup.32P, and .sup.125I.
In some embodiments, the isotope is incorporated into the selected
amino acid prior to incorporation into a polypeptide. In some
embodiments, the radiolabeled amino acid can be incorporated into
the polypeptide after polypeptide formation.
[1021] In some embodiments, the sequence of the captured
polypeptide is determined using Edman degradation (and in some
embodiments successive rounds of Edman degradation). In such cases,
a polypeptide is captured, and the polypeptide sequence can be
resolved by imaging the substrate following repeated rounds of
Edman degradation. For example, the substrate is imaged following
each Edman reaction in order to capture the detectable labels that
are produced due to the removal of amino acids that are a byproduct
of the reaction. The information obtained by the Edman degradation
can be complied to identify a polypeptide. In some embodiments, the
biological sample is visualized or imaged using light or
fluorescence microscopy.
[1022] (viii) Enrichment of Captured Analytes after Capture
[1023] In some aspects, spatial analysis of targeted analytes
includes an enrichment step or steps post-capture to enrich the
captured analytes for the targeted analyte. For example, the
capture domain can be selected or designed for the selective
capture of more analytes than the practitioner desires to analyze.
In some embodiments, capture probes that include random sequences
(e.g., random hexamers or similar sequences) that form all or part
of the capture domain can be used to capture nucleic acids from a
biological sample in an unbiased way. For example, capture probes
having capture domains that include random sequences can be used to
generically capture DNA, RNA, or both from a biological sample.
Alternatively, capture probes can include capture domains can that
capture mRNA generally. As is well known in the art, this can be on
the basis of hybridization to the poly-A tail of mRNAs. In some
embodiments, the capture domain includes a sequence that interacts
with (e.g., hybridizes to) the poly-A tail of mRNAs. Non-limiting
examples of such sequences include poly-T DNA sequences and poly-U
RNA sequences. In some embodiments, random sequences can be used in
conjunction with poly-T (or poly-T analogue etc.) sequences. Thus,
where a capture domain includes a poly-T (or a "poly-T-like")
oligonucleotide, it can also include a random oligonucleotide
sequence.
[1024] In some aspects, after capture of more analytes than the
practitioner desires to analyze, methods disclosed herein include
enrichment of particular captured analytes. In some aspects,
methods include enrichment of analytes that include mutations
(e.g., SNPs) of interest, nucleic acid(s) of interest, and/or
proteins(s) of interest.
[1025] In some embodiments, methods of spatial analysis provided
herein include selectively enriching one or more analytes of
interest (e.g., target analytes) after analyte capture. For
example, one or more analytes of interest can be enriched by
addition of one or more oligonucleotides to the pool of captured
analytes. In some embodiments, one or more analytes of interest can
be enriched by addition of one or more oligonucleotides to the pool
of captured analytes on the array. In some embodiments, one or more
analytes of interest can be enriched by addition of one or more
oligonucleotides to the pool of captured analytes where the pool of
captured analytes have been released (e.g., removed) from the
array. In some embodiments, when captured analytes have been
released from the array the one or more nucleotides can be
complementary to a portion of a TSO and R1 sequence, or portion
thereof. In some embodiments, the additional oligonucleotide(s)
include a sequence used for priming a reaction by a polymerase. For
example, one or more primer sequences with sequence complementarity
to one or more analytes of interest can be used to amplify the one
or more analyte(s) of interest, thereby selectively enriching these
analytes. In some embodiments, one or more primer sequences can be
complementary to other domains on the capture probe (e.g., R1
sequence, or portion thereof, as above), and not complementary to
the analyte. In some embodiments, enrichment by amplification
(e.g., PCR) occurs by using a first primer complementary to an
analyte or analytes of interest (or another domain in the capture
probe and the TSO), or complement thereof, and a second primer
complementary to a region of the capture probe, or complement
thereof. In some embodiments, the region of the capture probe, or
complement thereof, is distal to a spatial barcode from the capture
domain, such that enrichment by amplification amplifies both the
captured analyte or analytes and its or their associated spatial
barcodes, thus permitting spatial analysis of the enriched analyte
or analytes.
[1026] In some embodiments, two or more capture probes capture two
or more distinct analytes, which analytes are enriched (e.g.,
simultaneously or sequentially) from the pool of captured analytes.
In some embodiments, enrichment by PCR amplification includes
multiple rounds of amplification. For example, enrichment by PCR
amplification can include nested PCR reactions using different
primers that are specific for the analyte or analytes of interest.
In some embodiments, enrichment by amplification can be performed
using an amplification method that is not PCR. A non-limiting
example of a non-PCR amplification method is rolling circle
amplification. Other non-PCR amplification methods are known in the
art.
[1027] In some embodiments, an oligonucleotide with sequence
complementarity to a captured analyte or analytes of interest, or
complement thereof, can be used to enrich the captured analyte or
analytes of interest from the pool of captured analytes. In some
embodiments, an oligonucleotide with sequence complementarity to a
captured analyte or analytes of interest (or another domain the
capture probe), or complement thereof, can include one or more
functional moieties that are useful in the enrichment process. For
example, biotinylated oligonucleotides with sequence complementary
to one or more analytes interest, or complements thereof, can bind
to the analyte(s) of interest and can be selected using
biotinylation-strepavidin affinity using any of a variety of
methods known in the art (e.g., streptavidin beads). In some
embodiments, oligonucleotides with sequence complementary to one or
more analytes interest, or complements thereof, include a magnetic
moiety (e.g., a magnetic bead) that can be used in the enrichment
process.
[1028] Additionally or alternatively, one or more species of
analyte (e.g., mitochondrial DNA or RNA) can be down-selected
(e.g., removed) using any of a variety of methods. In some
embodiments, such down-selection of analytes that are not of
interest can result in improved capture of other types of analytes
that are of interest. For example, probes can be administered to a
sample that selectively hybridize to ribosomal RNA (rRNA), thereby
reducing the pool and concentration of rRNA in the sample. In some
embodiments, such down-selection can result in improved capture of
other types of RNA due to the reduction in non-specific RNA present
in the sample. Additionally or alternatively, duplex-specific
nuclease (DSN) treatment can remove rRNA (see, e.g., Archer, et al,
Selective and flexible depletion of problematic sequences from
RNA-seq libraries at the cDNA stage, BMC Genomics, 15 401, (2014),
the entire contents of which is incorporated herein by reference).
In some embodiments, hydroxyapatite chromatography can be used to
remove abundant species (e.g., rRNA).
[1029] (ix) RNA-Templated Ligation
[1030] In some embodiments of methods provided here, RNA-templated
ligation is used to interrogate spatial gene expression in a
biological sample (e.g., an FFPE tissue section). RNA-templated
ligation enables sensitive measurement of specific nucleic acid
analytes of interest that otherwise might be analyzed less
sensitively with a whole transcriptomic approach. It provides
advantages of compatibility with common histochemical stains and
suitability for analysis of decade-old materials (e.g., FFPE
samples) and exceedingly small microdissected tissue fragments.
[1031] In some aspects, the steps of RNA-templated ligation
include: (1) hybridization of pairs of probes (e.g., DNA probes) to
RNA (e.g., formalin fixed RNA) within a tissue section; (2)
ligation of adjacently annealed probe pairs in situ; (3) RNase H
treatment that (i) releases RNA-templated ligation products from
the tissue (e.g., into solution) for downstream analysis and (ii)
destroys unwanted DNA-templated ligation products; and optionally,
(4) amplification of RNA-templated ligation products (e.g., by
multiplex PCR).
[1032] In some aspects, disclosed herein are methods of direct
detection of RNA target-DNA probe duplexes without first converting
RNA to cDNA by reverse transcription. In some aspects,
RNA-templated ligation can include a DNA ligase. In some aspects,
RNA-templated ligation can include RNA ligase. In some aspects,
RNA-templated ligation can include T4 RNA ligase.
[1033] In some aspects, RNA-templated ligation is used for
detection of RNA, determination of RNA sequence identity, and/or
expression monitoring and transcript analysis. In some aspects,
RNA-templated ligation allows for detection of a particular change
in a nucleic acid (e.g., a mutation or single nucleotide
polymorphism (SNP)), detection or expression of a particular
nucleic acid, or detection or expression of a particular set of
nucleic acids (e.g., in a similar cellular pathway or expressed in
a particular pathology). In some embodiments, the methods that
include RNA-templated ligation are used to analyze nucleic acids,
e.g., by genotyping, quantitation of DNA copy number or RNA
transcripts, localization of particular transcripts within samples,
and the like. In some aspects, systems and methods provided herein
that include RNA-templated ligation identify single nucleotide
polymorphisms (SNPs). In some aspects, such systems and methods
identify mutations.
[1034] In some aspects, disclosed herein are methods of detecting
RNA expression that include bringing into contact a first probe, a
second probe, and ligase (e.g., T4 RNA ligase). In some
embodiments, the first probe and the second probe are designed to
hybridize to a target sequence such that the 5' end of the first
probe and the 3' end of the second probe are adjacent and can be
ligated, wherein at least the 5'-terminal nucleotide of the first
probe and at least the 3'-terminal nucleotide of the second probe
are deoxyribonucleotides (DNA), and wherein the target sequence
includes (e.g., is composed of) ribonucleotides (RNA). After
hybridization, a ligase (e.g., T4 RNA ligase) ligates the first
probe and the second probe if the target sequence is present in the
target sample, but does not ligate the first probe and the second
probe if the target sequence is not present in the target sample.
The presence or absence of the target sequence in the biological
sample can be determined by determining whether or not the first
and second probes were ligated in the presence of ligase. Any of a
variety of methods can be used to determine whether or not the
first and second probes were ligated in the presence of ligase,
including but not limited to, sequencing the ligated product,
hybridizing the ligated product with a detection probe that
hybridizes only when the first and second probes were ligated in
the presence of ligase, restriction enzyme analysis, and other
methods known in the art.
[1035] In some aspects, two or more RNA analytes are analyzed using
methods that include RNA-templated ligation. In some aspects, when
two or more analytes are analyzed, a first and second probe that is
specific for (e.g., specifically hybridizes to) each RNA analyte
are used.
[1036] In some aspects, three or more probes are used in
RNA-templated ligation methods provided herein. In some
embodiments, the three or more probes are designed to hybridize to
a target sequence such that the three or more probes hybridize
adjacent to each other such that the 5' and 3' ends of adjacent
probes can be ligated. In some embodiments, the presence or absence
of the target sequence in the biological sample can be determined
by determining whether or not the three or more probes were ligated
in the presence of ligase.
[1037] In some aspects, the first probe is a DNA probe. In some
aspects, the first probe is a chimeric DNA/RNA probe. In some
aspects, the second probe is a DNA probe. In some aspects, the
second probe is a chimeric DNA/RNA probe.
[1038] In some aspects, methods of RNA-templated ligation utilize
the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA-DNA
probe pairs hybridized in situ on fixed RNA target sequences.
Subsequent treatment with RNase H releases RNA-templated ligation
products (e.g., into solution) for downstream analysis.
[1039] (x) Region of Interest
[1040] A biological sample can have regions that show morphological
feature(s) that may indicate the presence of disease or the
development of a disease phenotype. For example, morphological
features at a specific site within a tumor biopsy sample can
indicate the aggressiveness, therapeutic resistance, metastatic
potential, migration, stage, diagnosis, and/or prognosis of cancer
in a subject. A change in the morphological features at a specific
site within a tumor biopsy sample often correlate with a change in
the level or expression of an analyte in a cell within the specific
site, which can, in turn, be used to provide information regarding
the aggressiveness, therapeutic resistance, metastatic potential,
migration, stage, diagnosis, and/or prognosis of cancer in a
subject. A region or area within a biological sample that is
selected for specific analysis (e.g., a region in a biological
sample that has morphological features of interest) is often
described as "a region of interest."
[1041] A region of interest in a biological sample can be used to
analyze a specific area of interest within a biological sample, and
thereby, focus experimentation and data gathering to a specific
region of a biological sample (rather than an entire biological
sample). This results in increased time efficiency of the analysis
of a biological sample.
[1042] A region of interest can be identified in a biological
sample using a variety of different techniques, e.g., expansion
microscopy, bright field microscopy, dark field microscopy, phase
contrast microscopy, electron microscopy, fluorescence microscopy,
reflection microscopy, interference microscopy, confocal
microscopy, and visual identification (e.g., by eye), and
combinations thereof. For example, the staining and imaging of a
biological sample can be performed to identify a region of
interest. In some examples, the region of interest can correspond
to a specific structure of cytoarchitecture. In some embodiments, a
biological sample can be stained prior to visualization to provide
contrast between the different regions of the biological sample.
The type of stain can be chosen depending on the type of biological
sample and the region of the cells to be stained. In some
embodiments, more than one stain can be used to visualize different
aspects of the biological sample, e.g., different regions of the
sample, specific cell structures (e.g., organelles), or different
cell types. In other embodiments, the biological sample can be
visualized or imaged without staining the biological sample.
[1043] In some embodiments, staining and imaging a biological
sample prior to contacting the biological sample with a spatial
array is performed to select samples for spatial analysis. In some
embodiments, the staining includes applying a fiducial marker as
described herein, including fluorescent, radioactive,
chemiluminescent, or colorimetric detectable markers. In some
embodiments, the staining and imaging of biological samples allows
the user to identify the specific sample (or region of interest)
the user wishes to assess.
[1044] In some examples, an array (e.g., any of the exemplary
arrays described herein) can be contacted with only a portion of a
biological sample (e.g., a cell, a tissue section, or a region of
interest). In some examples, a biological sample is contacted with
only a portion of an array (e.g., any of the exemplary arrays
described herein). In some embodiments, capture probes on an array
corresponding to regions of interest of a biological sample (e.g.,
proximal to the region of interest) can be selectively cleaved and
analyzed. For example, capture probes on an array may be
deactivated or eliminated outside of areas corresponding to regions
of interest of a biological sample. In some embodiments, capture
probes including a photocleavable bond and on the array in areas
corresponding to regions of interest of a biological sample can be
selectively cleaved by using light. A mirror, mirror array, a lens,
a moving stage, and/or a photomask can be used to direct the light
to regions of the array that correspond areas outside one or more
regions of interest in the biological sample. Some embodiments
include deactivating or eliminating capture probes, e.g., capture
probes comprising a photocleavable bond as described herein, using
light. In some embodiments, a laser, e.g., a scanning laser, can be
used to deactivate or eliminate capture probes. In some
embodiments, the eliminated member of the plurality of capture
probes can be washed away. In some embodiments, regions of interest
can be labeled with different heavy metals, and a laser can
sequentially ablate these regions of interest before mass
spectrometry identification. A laser can, for example, deactivate
or eliminate capture probes through UV light destruction of DNA,
heat, inducing a chemical reaction that prevents the capture probes
from moving to the next step, inducing photocleavage of a
photocleavable bond, or a combination thereof. In some examples, a
portion of the array can be deactivated such that it does not
interact with the analytes in the biological sample (e.g., optical
deactivation, chemical deactivation, heat deactivation, or blocking
of the capture probes in the array (e.g., using blocking probes)).
In some examples, a region of interest can be removed from a
biological sample and then the region of interest can be contacted
to the array (e.g., any of the arrays described herein). A region
of interest can be removed from a biological sample using
microsurgery, laser capture microdissection, chunking, a microtome,
dicing, trypsinization, labelling, and/or fluorescence-assisted
cell sorting, and the like.
[1045] In some examples, a region of interest can be permeabilized
or lysed while areas outside the region of interest are not
permeabilized or lysed (e.g., Kashyap et al. Sci Rep. 2016; 6:
29579, herein incorporated by reference in its entirety). For
example, in some embodiments, a region of interest can be contacted
with a hydrogel comprising a permeabilization or lysing reagent. In
some embodiments, the area(s) outside the region of interest are
not contacted with the hydrogel comprising the permeabilization or
lysing reagent.
[1046] (f) Partitioning
[1047] As discussed above, in some embodiments, the sample can
optionally be separated into single cells, cell groups, or other
fragments/pieces that are smaller than the original, unfragmented
sample. Each of these smaller portions of the sample can be
analyzed to obtain spatially-resolved analyte information for the
sample.
[1048] For samples that have been separated into smaller
fragments--and particularly, for samples that have been
disaggregated, dissociated, or otherwise separated into individual
cells--one method for analyzing the fragments involves separating
the fragments into individual partitions (e.g., fluid droplets),
and then analyzing the contents of the partitions. In general, each
partition maintains separation of its own contents from the
contents of other partitions. The partition can be a droplet in an
emulsion, for example.
[1049] The partitions can be flowable within fluid streams. The
partitions can include, for example, micro-vesicles that have an
outer barrier surrounding an inner fluid center or core. In some
cases, the partitions can include a porous matrix that is capable
of entraining and/or retaining materials within its matrix. The
partitions can be droplets of a first phase within a second phase,
wherein the first and second phases are immiscible. For example,
the partitions can be droplets of aqueous fluid within a
non-aqueous continuous phase (e.g., oil phase). In another example,
the partitions can be droplets of a non-aqueous fluid within an
aqueous phase. In some examples, the partitions can be provided in
a water-in-oil emulsion or oil-in-water emulsion. A variety of
different vessels are described in, for example, U.S. Patent
Application Publication No. 2014/0155295, the entire contents of
which are incorporated herein by reference. Emulsion systems for
creating stable droplets in non-aqueous or oil continuous phases
are described, for example, in U.S. Patent Application Publication
No. 2010/0105112, the entire contents of which are incorporated
herein by reference.
[1050] For droplets in an emulsion, allocating individual particles
to discrete partitions can be accomplished, for example, by
introducing a flowing stream of particles in an aqueous fluid into
a flowing stream of a non-aqueous fluid, such that droplets are
generated at the junction of the two streams. Fluid properties
(e.g., fluid flow rates, fluid viscosities, etc.), particle
properties (e.g., volume fraction, particle volume, particle
concentration, etc.), microfluidic architectures (e.g., channel
geometry, etc.), and other parameters can be adjusted to control
the occupancy of the resulting partitions (e.g., number of analytes
per partition, number of beads per partition, etc.). For example,
partition occupancy can be controlled by providing the aqueous
stream at a certain concentration and/or flow rate of analytes.
[1051] To generate single analyte partitions, the relative flow
rates of the immiscible fluids can be selected such that, on
average, the partitions can contain less than one analyte per
partition to ensure that those partitions that are occupied are
primarily singly occupied. In some cases, partitions among a
plurality of partitions can contain at most one analyte. In some
embodiments, the various parameters (e.g., fluid properties,
particle properties, microfluidic architectures, etc.) can be
selected or adjusted such that a majority of partitions are
occupied, for example, allowing for only a small percentage of
unoccupied partitions. The flows and channel architectures can be
controlled as to ensure a given number of singly occupied
partitions, less than a certain level of unoccupied partitions
and/or less than a certain level of multiply occupied
partitions.
[1052] The channel segments described herein can be coupled to any
of a variety of different fluid sources or receiving components,
including reservoirs, tubing, manifolds, or fluidic components of
other systems. As will be appreciated, the microfluidic channel
structure can have a variety of geometries. For example, a
microfluidic channel structure can have one or more than one
channel junction. As another example, a microfluidic channel
structure can have 2, 3, 4, or 5 channel segments each carrying
particles that meet at a channel junction. Fluid can be directed to
flow along one or more channels or reservoirs via one or more fluid
flow units. A fluid flow unit can include compressors (e.g.,
providing positive pressure), pumps (e.g., providing negative
pressure), actuators, and the like to control flow of the fluid.
Fluid can also or otherwise be controlled via applied pressure
differentials, centrifugal force, electrokinetic pumping, vacuum,
capillary, and/or gravity flow.
[1053] In addition to cells and/or analytes, a partition can
include additional components, and in particular, one or more
beads. A partition can include a single gel bead, a single cell
bead, or both a single cell bead and single gel bead. A variety of
different beads can be incorporated into partitions. In some
embodiments, for example, non-barcoded beads can be incorporated
into the partitions. For example, where the biological particle
(e.g., a cell) that is incorporated into the partitions carries one
or more barcodes (e.g., spatial barcode(s), UMI(s), and
combinations thereof), the bead can be a non-barcoded bead.
[1054] In some embodiments, a barcode carrying bead can be
incorporated into partitions. In general, an individual bead can be
coupled to any number of individual nucleic acid molecules, for
example, from one to tens to hundreds of thousands or even millions
of individual nucleic acid molecules. The respective barcodes for
the individual nucleic acid molecules can include both common
sequence segments or relatively common sequence segments and
variable or unique sequence segments between different individual
nucleic acid molecules coupled to the same bead. For example, a
nucleic acid molecule (e.g., an oligonucleotide), can be coupled to
a bead by a releasable linkage (e.g., a disulfide linker), wherein
the nucleic acid molecule can be or include a barcode. For example,
barcodes can be injected into droplets previous to, subsequent to,
or concurrently with droplet generation. The delivery of the
barcodes to a particular partition allows for the later attribution
of the characteristics of the individual biological particle to the
particular partition. Barcodes can be delivered, for example on a
nucleic acid molecule (e.g., an oligonucleotide), to a partition
via any suitable mechanism. Barcoded nucleic acid molecules can be
delivered to a partition via a microcapsule. A microcapsule, in
some instances, can include a bead. The same bead can be coupled
(e.g., via releasable linkage) to one or more other nucleic acid
molecules.
[1055] The nucleic acid molecule can include a functional domain
that can be used in subsequent processing. For example, the
functional domain can include one or more of a sequencer specific
flow cell attachment sequence (e.g., a P5 sequence for
Illumina.RTM. sequencing systems) and a sequencing primer sequence
(e.g., a R1 primer for Illumina.RTM. sequencing systems). The
nucleic acid molecule can include a barcode sequence for use in
barcoding the sample (e.g., DNA, RNA, protein, etc.). In some
cases, the barcode sequence can be bead-specific such that the
barcode sequence is common to all nucleic acid molecules coupled to
the same bead. Alternatively or in addition, the barcode sequence
can be partition-specific such that the barcode sequence is common
to all nucleic acid molecules coupled to one or more beads that are
partitioned into the same partition. The nucleic acid molecule can
include a specific priming sequence, such as an mRNA specific
priming sequence (e.g., poly(T) sequence), a targeted priming
sequence, and/or a random priming sequence. The nucleic acid
molecule can include an anchoring sequence to ensure that the
specific priming sequence hybridizes at the sequence end (e.g., of
the mRNA). For example, the anchoring sequence can include a random
short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or
longer sequence, which can ensure that a poly(T) segment is more
likely to hybridize at the sequence end of the poly(A) tail of the
mRNA.
[1056] The nucleic acid molecule can include a unique molecular
identifying sequence (e.g., unique molecular identifier (UMI)). In
some embodiments, the unique molecular identifying sequence can
include from about 5 to about 8 nucleotides. Alternatively, the
unique molecular identifying sequence can include less than about 5
or more than about 8 nucleotides. The unique molecular identifying
sequence can be a unique sequence that varies across individual
nucleic acid molecules coupled to a single bead. In some
embodiments, the unique molecular identifying sequence can be a
random sequence (e.g., such as a random N-mer sequence). For
example, the UMI can provide a unique identifier of the starting
mRNA molecule that was captured, in order to allow quantitation of
the number of original expressed RNA.
[1057] A partition can also include one or more reagents. Unique
identifiers, such as barcodes, can be injected into the droplets
previous to, subsequent to, or concurrently with droplet
generation, such as via a microcapsule (e.g., bead). Microfluidic
channel networks (e.g., on a chip) can be utilized to generate
partitions. Alternative mechanisms can also be employed in the
partitioning of individual biological particles, including porous
membranes through which aqueous mixtures of cells are extruded into
non-aqueous fluids.
[1058] In some embodiments, barcoded nucleic acid molecules can be
initially associated with a microcapsule and then released from the
microcapsule. Release of the barcoded nucleic acid molecules can be
passive (e.g., by diffusion out of the microcapsule). In addition
or alternatively, release from the microcapsule can be upon
application of a stimulus which allows the barcoded nucleic acid
nucleic acid molecules to dissociate or to be released from the
microcapsule. Such stimulus can disrupt the microcapsule, an
interaction that couples the barcoded nucleic acid molecules to or
within the microcapsule, or both. Such stimulus can include, for
example, a thermal stimulus, photo-stimulus, chemical stimulus
(e.g., change in pH or use of a reducing agent(s)), a mechanical
stimulus, a radiation stimulus; a biological stimulus (e.g.,
enzyme), or any combination thereof.
[1059] In some embodiments, one more barcodes (e.g., spatial
barcodes, UMIs, or a combination thereof) can be introduced into a
partition as part of the analyte. As described previously, barcodes
can be bound to the analyte directly, or can form part of a capture
probe or analyte capture agent that is hybridized to, conjugated
to, or otherwise associated with an analyte, such that when the
analyte is introduced into the partition, the barcode(s) are
introduced as well. As described above, FIG. 16 shows an example of
a microfluidical channel structure for partitioning individual
analytes (e.g., cells) into discrete partitions.
[1060] FIG. 16 shows an example of a microfluidic channel structure
for partitioning individual analytes (e.g., cells) into discrete
partitions. The channel structure can include channel segments
1601, 1602, 1603, and 1604 communicating at a channel junction
1605. In operation, a first aqueous fluid 1606 that includes
suspended biological particles (or cells) 1607 may be transported
along channel segment 1601 into junction 1605, while a second fluid
1608 that is immiscible with the aqueous fluid 1606 is delivered to
the junction 1605 from each of channel segments 1602 and 1603 to
create discrete droplets 1609, 1610 of the first aqueous fluid 1606
flowing into channel segment 1604, and flowing away from junction
1605. The channel segment 1604 may be fluidically coupled to an
outlet reservoir where the discrete droplets can be stored and/or
harvested. A discrete droplet generated may include an individual
biological particle 1607 (such as droplets 1609). A discrete
droplet generated may include more than one individual biological
particle 1607. A discrete droplet may contain no biological
particle 1607 (such as droplet 1610). Each discrete partition may
maintain separation of its own contents (e.g., individual
biological particle 1607) from the contents of other
partitions.
[1061] FIG. 17A shows another example of a microfluidic channel
structure 1700 for delivering beads to droplets. The channel
structure includes channel segments 1701, 1702, 1703, 1704 and 1705
communicating at a channel junction 1706. During operation, the
channel segment 1701 can transport an aqueous fluid 1707 that
includes a plurality of beads 1708 along the channel segment 1701
into junction 1706. The plurality of beads 1708 can be sourced from
a suspension of beads. For example, the channel segment 1701 can be
connected to a reservoir that includes an aqueous suspension of
beads 1708. The channel segment 1702 can transport the aqueous
fluid 1707 that includes a plurality of particles 1709 (e.g.,
cells) along the channel segment 1702 into junction 1706. In some
embodiments, the aqueous fluid 1707 in either the first channel
segment 1701 or the second channel segment 1702, or in both
segments, can include one or more reagents, as further described
below.
[1062] A second fluid 1710 that is immiscible with the aqueous
fluid 1707 (e.g., oil) can be delivered to the junction 1706 from
each of channel segments 1703 and 1704. Upon meeting of the aqueous
fluid 1707 from each of channel segments 1701 and 1702 and the
second fluid 1710 from each of channel segments 1703 and 1704 at
the channel junction 1706, the aqueous fluid 1707 can be
partitioned as discrete droplets 1711 in the second fluid 1710 and
flow away from the junction 1706 along channel segment 1705. The
channel segment 1705 can deliver the discrete droplets to an outlet
reservoir fluidly coupled to the channel segment 1705, where they
can be harvested.
[1063] As an alternative, the channel segments 1701 and 1702 can
meet at another junction upstream of the junction 1706. At such
junction, beads and biological particles can form a mixture that is
directed along another channel to the junction 1706 to yield
droplets 1711. The mixture can provide the beads and biological
particles in an alternating fashion, such that, for example, a
droplet includes a single bead and a single biological
particle.
[1064] The second fluid 1710 can include an oil, such as a
fluorinated oil, that includes a fluorosurfactant for stabilizing
the resulting droplets, for example, inhibiting subsequent
coalescence of the resulting droplets 1711.
[1065] The partitions described herein can include small volumes,
for example, less than about 10 microliters (.mu.L), 5 .mu.L, 1
.mu.L, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL,
300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters
(nL), 100 nL, 50 nL, or less.
[1066] In the foregoing discussion, droplets with beads were formed
at the junction of different fluid streams. In some embodiments,
droplets can be formed by gravity-based partitioning methods.
[1067] FIG. 17B shows a cross-section view of another example of a
microfluidic channel structure 1750 with a geometric feature for
controlled partitioning. A channel structure 1750 can include a
channel segment 1752 communicating at a channel junction 1758 (or
intersection) with a reservoir 1754. In some instances, the channel
structure 1750 and one or more of its components can correspond to
the channel structure 1700 and one or more of its components.
[1068] An aqueous fluid 1760 comprising a plurality of particles
1756 may be transported along the channel segment 1752 into the
junction 1758 to meet a second fluid 1762 (e.g., oil, etc.) that is
immiscible with the aqueous fluid 1760 in the reservoir 1754 to
create droplets 1764 of the aqueous fluid 1760 flowing into the
reservoir 1754. At the junction 1758 where the aqueous fluid 1760
and the second fluid 1762 meet, droplets can form based on factors
such as the hydrodynamic forces at the junction 1758, relative flow
rates of the two fluids 1760, 1762, fluid properties, and certain
geometric parameters (e.g., .DELTA.h, etc.) of the channel
structure 1750. A plurality of droplets can be collected in the
reservoir 1754 by continuously injecting the aqueous fluid 1760
from the channel segment 1752 at the junction 1758.
[1069] A discrete droplet generated may comprise one or more
particles of the plurality of particles 1756. As described
elsewhere herein, a particle may be any particle, such as a bead,
cell bead, gel bead, biological particle, macromolecular
constituents of biological particle, or other particles.
Alternatively, a discrete droplet generated may not include any
particles.
[1070] In some instances, the aqueous fluid 1760 can have a
substantially uniform concentration or frequency of particles 1756.
As described elsewhere herein, the particles 1756 (e.g., beads) can
be introduced into the channel segment 1752 from a separate channel
(not shown in FIG. 17). The frequency of particles 1756 in the
channel segment 1752 may be controlled by controlling the frequency
in which the particles 1756 are introduced into the channel segment
1752 and/or the relative flow rates of the fluids in the channel
segment 1752 and the separate channel. In some instances, the
particles 1756 can be introduced into the channel segment 1752 from
a plurality of different channels, and the frequency controlled
accordingly. In some instances, different particles may be
introduced via separate channels. For example, a first separate
channel can introduce beads and a second separate channel can
introduce biological particles into the channel segment 1752. The
first separate channel introducing the beads may be upstream or
downstream of the second separate channel introducing the
biological particles.
[1071] In some instances, the second fluid 1762 may not be
subjected to and/or directed to any flow in or out of the reservoir
1754. For example, the second fluid 1762 may be substantially
stationary in the reservoir 1754. In some instances, the second
fluid 1762 may be subjected to flow within the reservoir 1754, but
not in or out of the reservoir 1754, such as via application of
pressure to the reservoir 1754 and/or as affected by the incoming
flow of the aqueous fluid 1760 at the junction 1758. Alternatively,
the second fluid 1762 may be subjected and/or directed to flow in
or out of the reservoir 1754. For example, the reservoir 1754 can
be a channel directing the second fluid 1762 from upstream to
downstream, transporting the generated droplets.
[1072] The channel structure 1750 at or near the junction 1758 may
have certain geometric features that at least partly determine the
volumes and/or shapes of the droplets formed by the channel
structure 1750. The channel segment 1752 can have a first
cross-section height, h1, and the reservoir 1754 can have a second
cross-section height, h2. The first cross-section height, h1, and
the second cross-section height, h2, may be different, such that at
the junction 1758, there is a height difference of .DELTA.h. The
second cross-section height, h2, may be greater than the first
cross-section height, h1. In some instances, the reservoir may
thereafter gradually increase in cross-section height, for example,
the more distant it is from the junction 1758. In some instances,
the cross-section height of the reservoir may increase in
accordance with expansion angle, .beta., at or near the junction
1758. The height difference, .DELTA.h, and/or expansion angle,
.beta., can allow the tongue (portion of the aqueous fluid 1760
leaving channel segment 1752 at junction 1758 and entering the
reservoir 1754 before droplet formation) to increase in depth and
facilitate decrease in curvature of the intermediately formed
droplet. For example, droplet volume may decrease with increasing
height difference and/or increasing expansion angle.
[1073] The height difference, .DELTA.h, can be at least about 1
.mu.m. Alternatively, the height difference can be at least about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500
.mu.m or more. Alternatively, the height difference can be at most
about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2, 1 .mu.m or less. In some instances, the expansion angle, (3,
may be between a range of from about 0.5.degree. to about
4.degree., from about 0.1.degree. to about 10.degree., or from
about 0.degree. to about 90.degree.. For example, the expansion
angle can be at least about 0.01.degree., 0.1.degree., 0.2.degree.,
0.3.degree., 0.4.degree., 0.5.degree., 0.6.degree., 0.7.degree.,
0.8.degree., 0.9.degree., 1.degree., 2.degree., 3.degree.,
4.degree., 5.degree., 6.degree., 7.degree., 8.degree., 9.degree.,
10.degree., 15.degree., 20.degree., 25.degree., 30.degree.,
35.degree., 40.degree., 45.degree., 50.degree., 55.degree.,
60.degree., 65.degree., 70.degree., 75.degree., 80.degree.,
85.degree., or higher. In some instances, the expansion angle can
be at most about 89.degree., 88.degree., 87.degree., 86.degree.,
85.degree., 84.degree., 83.degree., 82.degree., 81.degree.,
80.degree., 75.degree., 70.degree., 65.degree., 60.degree.,
55.degree., 50.degree., 45.degree., 40.degree., 35.degree.,
30.degree., 25.degree., 20.degree., 15.degree., 10.degree.,
9.degree., 8.degree., 7.degree., 6.degree., 5.degree., 4.degree.,
3.degree., 2.degree., 1.degree., 0.1.degree., 0.01.degree., or
less.
[1074] In some instances, the flow rate of the aqueous fluid 1760
entering the junction 1758 can be between about 0.04 microliters
(.mu.L)/minute (min) and about 40 .mu.L/min. In some instances, the
flow rate of the aqueous fluid 1760 entering the junction 1758 can
be between about 0.01 microliters (.mu.L)/minute (min) and about
100 .mu.L/min. Alternatively, the flow rate of the aqueous fluid
1760 entering the junction 1758 can be less than about 0.01
.mu.L/min. alternatively, the flow rate of the aqueous fluid 1760
entering the junction 1758 can be greater than about 40 .mu.L/min,
such as 45 .mu.L/min, 50 .mu.L/min, 55 .mu.L/min, 60 .mu.L/min, 65
.mu.L/min, 70 .mu.L/min, 75 .mu.L/min, 80 .mu.L/min, 85 .mu.L/min,
90 .mu.L/min, 95 .mu.L/min, 100 .mu.L/min, 110 .mu.L/min, 120
.mu.L/min, 130 .mu.L/min, 140 .mu.L/min, 150 .mu.L/min, or greater.
At lower flow rates, such as flow rates of about less than or equal
to 10 microliters/minute, the droplet radius may not be dependent
on the flow rate of the aqueous fluid 1760 entering the junction
1758. The second fluid 1762 may be stationary, or substantially
stationary, in the reservoir 1754. Alternatively, the second fluid
1762 may be flowing, such as at the above flow rates described for
the aqueous fluid 1760.
[1075] While FIG. 17B illustrates the height difference, .DELTA.h,
being abrupt at the junction 1758 (e.g., a step increase), the
height difference may increase gradually (e.g., from about 0 .mu.m
to a maximum height difference). Alternatively, the height
difference may decrease gradually (e.g., taper) from a maximum
height difference. A gradual increase or decrease in height
difference, as used herein, may refer to a continuous incremental
increase or decrease in height difference, wherein an angle between
any one differential segment of a height profile and an immediately
adjacent differential segment of the height profile is greater than
90.degree.. For example, at the junction 1758, a bottom wall of the
channel and a bottom wall of the reservoir can meet at an angle
greater than 90.degree.. Alternatively or in addition, a top wall
(e.g., ceiling) of the channel and a top wall (e.g., ceiling) of
the reservoir can meet an angle greater than 90.degree.. A gradual
increase or decrease may be linear or non-linear (e.g.,
exponential, sinusoidal, etc.). Alternatively or in addition, the
height difference may variably increase and/or decrease linearly or
non-linearly. While FIG. 17B illustrates the expanding reservoir
cross-section height as linear (e.g., constant expansion angle,
(3), the cross-section height may expand non-linearly. For example,
the reservoir may be defined at least partially by a dome-like
(e.g., hemispherical) shape having variable expansion angles. The
cross-section height may expand in any shape.
[1076] FIG. 17C depicts a workflow wherein cells are partitioned
into droplets along with barcode-bearing beads 1770. See FIG. 17A.
The droplet forms an isolated reaction chamber wherein the cells
can be lysed 1771 and target analytes within the cells can then be
captured 1772 and amplified 1773, 1774 according to previously
described methods. After sequence library preparation clean-up
1775, the material is sequenced and/or quantified 1776 according to
methods described herein.
[1077] It should be noted that while the example workflow in FIG.
17C includes steps specifically for the analysis of mRNA, analogous
workflows can be implemented for a wide variety of other analytes,
including any of the analytes described previously.
[1078] By way of example, in the context of analyzing sample RNA as
shown in FIG. 17C, the poly(T) segment of one of the released
nucleic acid molecules (e.g., from the bead) can hybridize to the
poly(A) tail of a mRNA molecule. Reverse transcription can result
in a cDNA transcript of the mRNA, which transcript includes each of
the sequence segments of the nucleic acid molecule. If the nucleic
acid molecule includes an anchoring sequence, it will more likely
hybridize to and prime reverse transcription at the sequence end of
the poly(A) tail of the mRNA.
[1079] Within any given partition, all of the cDNA transcripts of
the individual mRNA molecules can include a common barcode sequence
segment. However, the transcripts made from the different mRNA
molecules within a given partition can vary at the unique molecular
identifying sequence segment (e.g., UMI segment). Beneficially,
even following any subsequent amplification of the contents of a
given partition, the number of different UMIs can be indicative of
the quantity of mRNA originating from a given partition. As noted
above, the transcripts can be amplified, cleaned up and sequenced
to identify the sequence of the cDNA transcript of the mRNA, as
well as to sequence the barcode segment and the UMI segment. While
a poly(T) primer sequence is described, other targeted or random
priming sequences can also be used in priming the reverse
transcription reaction. Likewise, although described as releasing
the barcoded oligonucleotides into the partition, in some cases,
the nucleic acid molecules bound to the bead can be used to
hybridize and capture the mRNA on the solid phase of the bead, for
example, in order to facilitate the separation of the RNA from
other cell contents.
[1080] In some embodiments, partitions include precursors that
include a functional group that is reactive or capable of being
activated such that it becomes reactive can be polymerized with
other precursors to generate gel beads that include the activated
or activatable functional group. The functional group can then be
used to attach additional species (e.g., disulfide linkers,
primers, other oligonucleotides, etc.) to the gel beads. For
example, some precursors featuring a carboxylic acid (COOH) group
can co-polymerize with other precursors to form a bead that also
includes a COOH functional group. In some cases, acrylic acid (a
species comprising free COOH groups), acrylamide, and
bis(acryloyl)cystamine can be co-polymerized together to generate a
bead with free COOH groups. The COOH groups of the bead can be
activated (e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) and N-Hydroxysuccinimide (NHS) or
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM)) such that they are reactive (e.g., reactive to amine
functional groups where EDC/NHS or DMTMM are used for activation).
The activated COOH groups can then react with an appropriate
species (e.g., a species comprising an amine functional group where
the carboxylic acid groups are activated to be reactive with an
amine functional group) comprising a moiety to be linked to the
bead.
[1081] In some embodiments, a bead can be formed from materials
that include degradable chemical cross-linkers, such as BAC or
cystamine. Degradation of such degradable cross-linkers can be
accomplished through a number of mechanisms. In some examples, a
bead can be contacted with a chemical degrading agent that can
induce oxidation, reduction or other chemical changes. For example,
a chemical degrading agent can be a reducing agent, such as
dithiothreitol (DTT). Additional examples of reducing agents can
include .beta.-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane
(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP),
or combinations thereof. A reducing agent can degrade the disulfide
bonds formed between gel precursors forming the bead, and thus,
degrade the bead.
[1082] A degradable bead can include one or more species with a
labile bond such that, when the bead/species is exposed to the
appropriate stimulus, the bond is broken and the bead degrades
within the partition. The labile bond can be a chemical bond (e.g.,
covalent bond, ionic bond) or can be another type of physical
interaction (e.g., van der Waals interactions, dipole-dipole
interactions, etc.). In some embodiments, a cross-linker used to
generate a bead can include a labile bond. Upon exposure to the
appropriate conditions, the labile bond can be broken and the bead
degraded. For example, a polyacrylamide bead featuring cystamine
and linked, via a disulfide bond, to a barcode sequence, can be
combined with a reducing agent within a droplet of a water-in-oil
emulsion. Within the droplet, the reducing agent can break the
various disulfide bonds, resulting in bead degradation and release
of the barcode sequence into the aqueous, inner environment of the
droplet. In another example, heating of a droplet with a bead-bound
barcode sequence in basic solution can also result in bead
degradation and release of the attached barcode sequence into the
aqueous, inner environment of the droplet. The free species (e.g.,
oligonucleotides, nucleic acid molecules) can interact with other
reagents contained in the partition.
[1083] A degradable bead can be useful in more quickly releasing an
attached species (e.g., a nucleic acid molecule, a barcode
sequence, a primer, etc.) from the bead when the appropriate
stimulus is applied to the bead as compared to a bead that does not
degrade. For example, for a species bound to an inner surface of a
porous bead or in the case of an encapsulated species, the species
can have greater mobility and accessibility to other species in
solution upon degradation of the bead. In some embodiments, a
species can also be attached to a degradable bead via a degradable
linker (e.g., disulfide linker). The degradable linker can respond
to the same stimuli as the degradable bead or the two degradable
species can respond to different stimuli. For example, a barcode
sequence can be attached, via a disulfide bond, to a polyacrylamide
bead comprising cystamine. Upon exposure of the barcoded-bead to a
reducing agent, the bead degrades and the barcode sequence is
released upon breakage of both the disulfide linkage between the
barcode sequence and the bead and the disulfide linkages of the
cystamine in the bead.
[1084] Any suitable number of species (e.g., primer, barcoded
oligonucleotide) can be associated with a bead such that, upon
release from the bead, the species (e.g., primer, e.g., barcoded
oligonucleotide) are present in the partition at a pre-defined
concentration. Such pre-defined concentration can be selected to
facilitate certain reactions for generating a sequencing library,
e.g., amplification, within the partition. In some cases, the
pre-defined concentration of the primer can be limited by the
process of producing nucleic acid molecule (e.g., oligonucleotide)
bearing beads.
[1085] As will be appreciated from the above description, while
referred to as degradation of a bead, in many embodiments,
degradation can refer to the disassociation of a bound or entrained
species from a bead, both with and without structurally degrading
the physical bead itself. For example, entrained species can be
released from beads through osmotic pressure differences due to,
for example, changing chemical environments. By way of example,
alteration of bead pore volumes due to osmotic pressure differences
can generally occur without structural degradation of the bead
itself. In some cases, an increase in pore volume due to osmotic
swelling of a bead can permit the release of entrained species
within the bead. In some embodiments, osmotic shrinking of a bead
can cause a bead to better retain an entrained species due to pore
volume contraction.
[1086] Numerous chemical triggers can be used to trigger the
degradation of beads within partitions. Examples of these chemical
changes can include, but are not limited to pH-mediated changes to
the integrity of a component within the bead, degradation of a
component of a bead via cleavage of cross-linked bonds, and
depolymerization of a component of a bead.
[1087] In certain embodiments, a change in pH of a solution, such
as an increase in pH, can trigger degradation of a bead. In other
embodiments, exposure to an aqueous solution, such as water, can
trigger hydrolytic degradation, and thus degradation of the bead.
In some cases, any combination of stimuli can trigger degradation
of a bead. For example, a change in pH can enable a chemical agent
(e.g., DTT) to become an effective reducing agent.
[1088] Beads can also be induced to release their contents upon the
application of a thermal stimulus. A change in temperature can
cause a variety of changes to a bead. For example, heat can cause a
solid bead to liquefy. A change in heat can cause melting of a bead
such that a portion of the bead degrades. In other cases, heat can
increase the internal pressure of the bead components such that the
bead ruptures or explodes. Heat can also act upon heat-sensitive
polymers used as materials to construct beads.
[1089] In addition to beads and analytes, partitions that are
formed can include a variety of different reagents and species. For
example, when lysis reagents are present within the partitions, the
lysis reagents can facilitate the release of analytes within the
partition. Examples of lysis agents include bioactive reagents,
such as lysis enzymes that are used for lysis of different cell
types, e.g., gram positive or negative bacteria, plants, yeast,
mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin,
labiase, kitalase, lyticase, and a variety of other lysis enzymes
available from, e.g., Sigma-Aldrich, Inc. (St. Louis, Mo.), as well
as other commercially available lysis enzymes. Other lysis agents
can additionally or alternatively be co-partitioned to cause the
release analytes into the partitions. For example, in some cases,
surfactant-based lysis solutions can be used to lyse cells,
although these can be less desirable for emulsion based systems
where the surfactants can interfere with stable emulsions. In some
embodiments, lysis solutions can include non-ionic surfactants such
as, for example, TritonX-100 and Tween 20. In some embodiments,
lysis solutions can include ionic surfactants such as, for example,
sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,
thermal, acoustic or mechanical cellular disruption can also be
used in certain embodiments, e.g., non-emulsion based partitioning
such as encapsulation of analytes that can be in addition to or in
place of droplet partitioning, where any pore size of the
encapsulate is sufficiently small to retain nucleic acid fragments
of a given volume, following cellular disruption.
[1090] Examples of other species that can be co-partitioned with
analytes in the partitions include, but are not limited to, DNase
and RNase inactivating agents or inhibitors, such as proteinase K,
chelating agents, such as EDTA, and other reagents employed in
removing or otherwise reducing negative activity or impact of
different cell lysate components on subsequent processing of
nucleic acids. Additional reagents can also be co-partitioned,
including endonucleases to fragment DNA, DNA polymerase enzymes and
dNTPs used to amplify nucleic acid fragments and to attach the
barcode molecular tags to the amplified fragments.
[1091] Additional reagents can also include reverse transcriptase
enzymes, including enzymes with terminal transferase activity,
primers and oligonucleotides, and switch oligonucleotides (also
referred to herein as "switch oligos" or "template switching
oligonucleotides") which can be used for template switching. In
some embodiments, template switching can be used to increase the
length of a cDNA. Template switching can be used to append a
predefined nucleic acid sequence to the cDNA. In an example of
template switching, cDNA can be generated from reverse
transcription of a template, e.g., cellular mRNA, where a reverse
transcriptase with terminal transferase activity can add additional
nucleotides, e.g., poly(C), to the cDNA in a template independent
manner. Switch oligos can include sequences complementary to the
additional nucleotides, e.g., poly(G). The additional nucleotides
(e.g., poly(C)) on the cDNA can hybridize to the additional
nucleotides (e.g., poly(G)) on the switch oligo, whereby the switch
oligo can be used by the reverse transcriptase as template to
further extend the cDNA.
[1092] Template switching oligonucleotides can include a
hybridization region and a template region. The hybridization
region can include any sequence capable of hybridizing to the
target. In some cases, the hybridization region includes a series
of G bases to complement the overhanging C bases at the 3' end of a
cDNA molecule. The series of G bases can include 1 G base, 2 G
bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The
template sequence can include any sequence to be incorporated into
the cDNA. In some cases, the template region includes at least 1
(e.g., at least 2, 3, 4, 5 or more) tag sequences and/or functional
sequences. Switch oligos can include deoxyribonucleic acids;
ribonucleic acids; bridged nucleic acids, modified nucleic acids
including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted
dT, 5-Methyl dC, 2'-deoxylnosine, Super T
(5-hydroxybutynl-2'-deoxyuridine), Super G
(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked
nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG,
Iso-dC, 2' Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and
Fluoro G), and combinations of the foregoing.
[1093] In some embodiments, beads that are partitioned with the
analyte can include different types of oligonucleotides bound to
the bead, where the different types of oligonucleotides bind to
different types of analytes. For example, a bead can include one or
more first oligonucleotides (which can be capture probes, for
example) that can bind or hybridize to a first type of analyte,
such as mRNA for example, and one or more second oligonucleotides
(which can be capture probes, for example) that can bind or
hybridize to a second type of analyte, such as gDNA for example.
Partitions can also include lysis agents that aid in releasing
nucleic acids from the co-partitioned cell, and can also include an
agent (e.g., a reducing agent) that can degrade the bead and/or
break covalent linkages between the oligonucleotides and the bead,
releasing the oligonucleotides into the partition. The released
barcoded oligonucleotides (which can also be barcoded) can
hybridize with mRNA released from the cell and also with gDNA
released from the cell.
[1094] Barcoded constructs thus formed from hybridization can
include a first type of construct that includes a sequence
corresponding to an original barcode sequence from the bead and a
sequence corresponding to a transcript from the cell, and a second
type of construct that includes a sequence corresponding to the
original barcode sequence from the bead and a sequence
corresponding to genomic DNA from the cell. The barcoded constructs
can then be released/removed from the partition and, in some
embodiments, further processed to add any additional sequences. The
resulting constructs can then be sequenced, the sequencing data
processed, and the results used to spatially characterize the mRNA
and the gDNA from the cell.
[1095] In another example, a partition includes a bead that
includes a first type of oligonucleotide (e.g., a first capture
probe) with a first barcode sequence, a poly(T) priming sequence
that can hybridize with the poly(A) tail of an mRNA transcript, and
a UMI barcode sequence that can uniquely identify a given
transcript. The bead also includes a second type of oligonucleotide
(e.g., a second capture probe) with a second barcode sequence, a
targeted priming sequence that is capable of specifically
hybridizing with a third barcoded oligonucleotide (e.g., an analyte
capture agent) coupled to an antibody that is bound to the surface
of the partitioned cell. The third barcoded oligonucleotide
includes a UMI barcode sequence that uniquely identifies the
antibody (and thus, the particular cell surface feature to which it
is bound).
[1096] In this example, the first and second barcoded
oligonucleotides include the same spatial barcode sequence (e.g.,
the first and second barcode sequences are the same), which permits
downstream association of barcoded nucleic acids with the
partition. In some embodiments, however, the first and second
barcode sequences are different.
[1097] The partition also includes lysis agents that aid in
releasing nucleic acids from the cell and can also include an agent
(e.g., a reducing agent) that can degrade the bead and/or break a
covalent linkage between the barcoded oligonucleotides and the
bead, releasing them into the partition. The first type of released
barcoded oligonucleotide can hybridize with mRNA released from the
cell and the second type of released barcoded oligonucleotide can
hybridize with the third type of barcoded oligonucleotide, forming
barcoded constructs.
[1098] The first type of barcoded construct includes a spatial
barcode sequence corresponding to the first barcode sequence from
the bead and a sequence corresponding to the UMI barcode sequence
from the first type of oligonucleotide, which identifies cell
transcripts. The second type of barcoded construct includes a
spatial barcode sequence corresponding to the second barcode
sequence from the second type of oligonucleotide, and a UMI barcode
sequence corresponding to the third type of oligonucleotide (e.g.,
the analyte capture agent) and used to identify the cell surface
feature. The barcoded constructs can then be released/removed from
the partition and, in some embodiments, further processed to add
any additional sequences. The resulting constructs are then
sequenced, sequencing data processed, and the results used to
characterize the mRNA and cell surface feature of the cell.
[1099] The foregoing discussion involves two specific examples of
beads with oligonucleotides for analyzing two different analytes
within a partition. More generally, beads that are partitioned can
have any of the structures described previously, and can include
any of the described combinations of oligonucleotides for analysis
of two or more (e.g., three or more, four or more, five or more,
six or more, eight or more, ten or more, 12 or more, 15 or more, 20
or more, 25 or more, 30 or more, 40 or more, 50 or more) different
types of analytes within a partition. Examples of beads with
combinations of different types of oligonucleotides (e.g., capture
probes) for concurrently analyzing different combinations of
analytes within partitions include, but are not limited to: (a)
genomic DNA and cell surface features (e.g., using the analyte
capture agents described herein); (b) mRNA and a lineage tracing
construct; (c) mRNA and cell methylation status; (d) mRNA and
accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq);
(e) mRNA and cell surface or intracellular proteins and/or
metabolites; (f) a barcoded analyte capture agent (e.g., the MHC
multimers described herein) and a V(D)J sequence of an immune cell
receptor (e.g., T-cell receptor); and (g) mRNA and a perturbation
agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease,
and/or antisense oligonucleotide as described herein). In some
embodiments, a perturbation agent can be a small molecule, an
antibody, a drug, an aptamer, a miRNA, a physical environmental
(e.g., temperature change), or any other known perturbation
agents.
[1100] Additionally, in some embodiments, the unaggregated cell or
disaggregated cells introduced and processed within partitions or
droplets as described herein, can be removed from the partition,
contacted with a spatial array, and spatially-barcoded according to
methods described herein. For example, single cells of an
unaggregated cell sample can be partitioned into partitions or
droplets as described herein. The partitions or droplets can
include reagents to permeabilize a cell, barcode targeted cellular
analyte(s) with a cellular barcode, and amplify the barcoded
analytes. The partitions or droplets can be contacted with any of
the spatial arrays described herein. In some embodiments, the
partition can be dissolved, such that the contents of the partition
are placed in contact with the capture probes of the spatial array.
The capture probes of the spatial array can then capture target
analytes from the ruptured partitions or the droplets, and
processed by the spatial workflows described herein.
[1101] (g) Analysis of Captured Analytes
[1102] (i) Sample Removal from an Array
[1103] In some embodiments, after contacting a biological sample
with a substrate that includes capture probes, a removal step can
optionally be performed to remove all or a portion of the
biological sample from the substrate. In some embodiments, the
removal step includes enzymatic and/or chemical degradation of
cells of the biological sample. For example, the removal step can
include treating the biological sample with an enzyme (e.g., a
proteinase, e.g., proteinase K) to remove at least a portion of the
biological sample from the substrate. In some embodiments, the
removal step can include ablation of the tissue (e.g., laser
ablation).
[1104] In some embodiments, provided herein are methods for
spatially detecting an analyte (e.g., detecting the location of an
analyte, e.g., a biological analyte) from a biological sample
(e.g., present in a biological sample), the method comprising: (a)
optionally staining and/or imaging a biological sample on a
substrate; (b) permeabilizing (e.g., providing a solution
comprising a permeabilization reagent to) the biological sample on
the substrate; (c) contacting the biological sample with an array
comprising a plurality of capture probes, wherein a capture probe
of the plurality captures the biological analyte; and (d) analyzing
the captured biological analyte, thereby spatially detecting the
biological analyte; wherein the biological sample is fully or
partially removed from the substrate.
[1105] In some embodiments, a biological sample is not removed from
the substrate. For example, the biological sample is not removed
from the substrate prior to releasing a capture probe (e.g., a
capture probe bound to an analyte) from the substrate. In some
embodiments, such releasing comprises cleavage of the capture probe
from the substrate (e.g., via a cleavage domain). In some
embodiments, such releasing does not comprise releasing the capture
probe from the substrate (e.g., a copy of the capture probe bound
to an analyte can be made and the copy can be released from the
substrate, e.g., via denaturation). In some embodiments, the
biological sample is not removed from the substrate prior to
analysis of an analyte bound to a capture probe after it is
released from the substrate. In some embodiments, the biological
sample remains on the substrate during removal of a capture probe
from the substrate and/or analysis of an analyte bound to the
capture probe after it is released from the substrate. In some
embodiments, analysis of an analyte bound to capture probe from the
substrate can be performed without subjecting the biological sample
to enzymatic and/or chemical degradation of the cells (e.g.,
permeabilized cells) or ablation of the tissue (e.g., laser
ablation).
[1106] In some embodiments, at least a portion of the biological
sample is not removed from the substrate. For example, a portion of
the biological sample can remain on the substrate prior to
releasing a capture probe (e.g., a capture prove bound to an
analyte) from the substrate and/or analyzing an analyte bound to a
capture probe released from the substrate. In some embodiments, at
least a portion of the biological sample is not subjected to
enzymatic and/or chemical degradation of the cells (e.g.,
permeabilized cells) or ablation of the tissue (e.g., laser
ablation) prior to analysis of an analyte bound to a capture probe
from the substrate.
[1107] In some embodiments, provided herein are methods for
spatially detecting an analyte (e.g., detecting the location of an
analyte, e.g., a biological analyte) from a biological sample
(e.g., present in a biological sample) that include: (a) optionally
staining and/or imaging a biological sample on a substrate; (b)
permeabilizing (e.g., providing a solution comprising a
permeabilization reagent to) the biological sample on the
substrate; (c) contacting the biological sample with an array
comprising a plurality of capture probes, wherein a capture probe
of the plurality captures the biological analyte; and (d) analyzing
the captured biological analyte, thereby spatially detecting the
biological analyte; where the biological sample is not removed from
the substrate.
[1108] In some embodiments, provided herein are methods for
spatially detecting a biological analyte of interest from a
biological sample that include: (a) staining and imaging a
biological sample on a substrate; (b) providing a solution
comprising a permeabilization reagent to the biological sample on
the substrate; (c) contacting the biological sample with an array
on a substrate, wherein the array comprises one or more capture
probe pluralities thereby allowing the one or more pluralities of
capture probes to capture the biological analyte of interest; and
(d) analyzing the captured biological analyte, thereby spatially
detecting the biological analyte of interest; where the biological
sample is not removed from the substrate.
[1109] In some embodiments, the method further includes selecting a
region of interest in the biological sample to subject to spatial
transcriptomic analysis. In some embodiments, one or more of the
one or more capture probes include a capture domain. In some
embodiments, one or more of the one or more capture probe
pluralities comprise a unique molecular identifier (UMI). In some
embodiments, one or more of the one or more capture probe
pluralities comprise a cleavage domain. In some embodiments, the
cleavage domain comprises a sequence recognized and cleaved by a
uracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease
(APE1), U uracil-specific excision reagent (USER), and/or an
endonuclease VIII. In some embodiments, one or more capture probes
do not comprise a cleavage domain and is not cleaved from the
array.
[1110] (ii) Extended Capture Probes
[1111] In some embodiments, a capture probe can be extended (an
"extended capture probe," e.g., as described herein (e.g., Section
II(b)(vii))). For example, extending a capture probe can include
generating cDNA from a captured (hybridized) RNA. This process
involves synthesis of a complementary strand of the hybridized
nucleic acid, e.g., generating cDNA based on the captured RNA
template (the RNA hybridized to the capture domain of the capture
probe). Thus, in an initial step of extending a capture probe,
e.g., the cDNA generation, the captured (hybridized) nucleic acid,
e.g., RNA, acts as a template for the extension, e.g., reverse
transcription, step.
[1112] In some embodiments, the capture probe is extended using
reverse transcription. For example, reverse transcription includes
synthesizing cDNA (complementary or copy DNA) from RNA, e.g.,
(messenger RNA), using a reverse transcriptase. In some
embodiments, reverse transcription is performed while the tissue is
still in place, generating an analyte library, where the analyte
library includes the spatial barcodes from the adjacent capture
probes. In some embodiments, the capture probe is extended using
one or more DNA polymerases.
[1113] In some embodiments, a capture domain of a capture probe
includes a primer for producing the complementary strand of a
nucleic acid hybridized to the capture probe, e.g., a primer for
DNA polymerase and/or reverse transcription. The nucleic acid,
e.g., DNA and/or cDNA, molecules generated by the extension
reaction incorporate the sequence of the capture probe. The
extension of the capture probe, e.g., a DNA polymerase and/or
reverse transcription reaction, can be performed using a variety of
suitable enzymes and protocols.
[1114] In some embodiments, a full-length DNA (e.g., cDNA) molecule
is generated. In some embodiments, a "full-length" DNA molecule
refers to the whole of the captured nucleic acid molecule. However,
if a nucleic acid (e.g., RNA) was partially degraded in the tissue
sample, then the captured nucleic acid molecules will not be the
same length as the initial RNA in the tissue sample. In some
embodiments, the 3' end of the extended probes, e.g., first strand
cDNA molecules, is modified. For example, a linker or adaptor can
be ligated to the 3' end of the extended probes. This can be
achieved using single stranded ligation enzymes such as T4 RNA
ligase or Circligase.TM. (available from Epicentre Biotechnologies,
Madison, Wis.). In some embodiments, template switching
oligonucleotides are used to extend cDNA in order to generate a
full-length cDNA (or as close to a full-length cDNA as possible).
In some embodiments, a second strand synthesis helper probe (a
partially double stranded DNA molecule capable of hybridizing to
the 3' end of the extended capture probe), can be ligated to the 3'
end of the extended probe, e.g., first strand cDNA, molecule using
a double stranded ligation enzyme such as T4 DNA ligase. Other
enzymes appropriate for the ligation step are known in the art and
include, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp.
(strain 9.degree.N) DNA ligase (9.degree.N.TM. DNA ligase, New
England Biolabs), Ampligase.TM. (available from Epicentre
Biotechnologies, Madison, Wis.), and SplintR (available from New
England Biolabs, Ipswich, Mass.). In some embodiments, a
polynucleotide tail, e.g., a poly(A) tail, is incorporated at the
3' end of the extended probe molecules. In some embodiments, the
polynucleotide tail is incorporated using a terminal transferase
active enzyme.
[1115] In some embodiments, double-stranded extended capture probes
are treated to remove any unextended capture probes prior to
amplification and/or analysis, e.g., sequence analysis. This can be
achieved by a variety of methods, e.g., using an enzyme to degrade
the unextended probes, such as an exonuclease enzyme, or
purification columns.
[1116] In some embodiments, extended capture probes are amplified
to yield quantities that are sufficient for analysis, e.g., via DNA
sequencing. In some embodiments, the first strand of the extended
capture probes (e.g., DNA and/or cDNA molecules) acts as a template
for the amplification reaction (e.g., a polymerase chain
reaction).
[1117] In some embodiments, the amplification reaction incorporates
an affinity group onto the extended capture probe (e.g., RNA-cDNA
hybrid) using a primer including the affinity group. In some
embodiments, the primer includes an affinity group and the extended
capture probes includes the affinity group. The affinity group can
correspond to any of the affinity groups described previously.
[1118] In some embodiments, the extended capture probes including
the affinity group can be coupled to a substrate specific for the
affinity group. In some embodiments, the substrate can include an
antibody or antibody fragment. In some embodiments, the substrate
includes avidin or streptavidin and the affinity group includes
biotin. In some embodiments, the substrate includes maltose and the
affinity group includes maltose-binding protein. In some
embodiments, the substrate includes maltose-binding protein and the
affinity group includes maltose. In some embodiments, amplifying
the extended capture probes can function to release the extended
probes from the surface of the substrate, insofar as copies of the
extended probes are not immobilized on the substrate.
[1119] In some embodiments, the extended capture probe or
complement or amplicon thereof is released. The step of releasing
the extended capture probe or complement or amplicon thereof from
the surface of the substrate can be achieved in a number of ways.
In some embodiments, an extended capture probe or a complement
thereof is released from the array by nucleic acid cleavage and/or
by denaturation (e.g., by heating to denature a double-stranded
molecule).
[1120] In some embodiments, the extended capture probe or
complement or amplicon thereof is released from the surface of the
substrate (e.g., array) by physical means. For example, where the
extended capture probe is indirectly immobilized on the array
substrate, e.g., via hybridization to a surface probe, it can be
sufficient to disrupt the interaction between the extended capture
probe and the surface probe. Methods for disrupting the interaction
between nucleic acid molecules include denaturing double stranded
nucleic acid molecules are known in the art. A straightforward
method for releasing the DNA molecules (i.e., of stripping the
array of extended probes) is to use a solution that interferes with
the hydrogen bonds of the double stranded molecules. In some
embodiments, the extended capture probe is released by a applying
heated solution, such as water or buffer, of at least 85.degree.
C., e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or
99.degree. C. In some embodiments, a solution including salts,
surfactants, etc. that can further destabilize the interaction
between the nucleic acid molecules is added to release the extended
capture probe from the substrate.
[1121] In some embodiments, where the extended capture probe
includes a cleavage domain, the extended capture probe is released
from the surface of the substrate by cleavage. For example, the
cleavage domain of the extended capture probe can be cleaved by any
of the methods described herein. In some embodiments, the extended
capture probe is released from the surface of the substrate, e.g.,
via cleavage of a cleavage domain in the extended capture probe,
prior to the step of amplifying the extended capture probe.
[1122] (iii) Cleavage Domain
[1123] Capture probes can optionally include a "cleavage domain,"
where one or more segments or regions of the capture probe (e.g.,
spatial barcodes and/or UMIs) can be releasably, cleavably, or
reversibly attached to a feature, or some other substrate, so that
spatial barcodes and/or UMIs can be released or be releasable
through cleavage of a linkage between the capture probe and the
feature, or released through degradation of the underlying
substrate or chemical substrate, allowing the spatial barcode(s)
and/or UMI(s) of the cleaved capture probe to be accessed or be
accessible by other reagents, or both. Non-limiting aspects of
cleavage domains are described herein (e.g., in Section
II(b)(ii)).
[1124] In some embodiments, the capture probe is linked, (e.g., via
a disulfide bond), to a feature. In some embodiments, the capture
probe is linked to a feature via a propylene group (e.g., Spacer
C3). A reducing agent can be added to break the various disulfide
bonds, resulting in release of the capture probe including the
spatial barcode sequence. In another example, heating can also
result in degradation and release of the attached capture probe. In
some embodiments, the heating is done by laser (e.g., laser
ablation) and features at specific locations can be degraded. In
addition to thermally cleavable bonds, disulfide bonds,
photo-sensitive bonds, and UV sensitive bonds, other non-limiting
examples of labile bonds that can be coupled to a capture probe
(e.g., spatial barcode) include an ester linkage (e.g., cleavable
with an acid, a base, or hydroxylamine), a vicinal diol linkage
(e.g., cleavable via sodium periodate), a Diels-Alder linkage
(e.g., cleavable via heat), a sulfone linkage (e.g., cleavable via
a base), a silyl ether linkage (e.g., cleavable via an acid), a
glycosidic linkage (e.g., cleavable via an amylase), a peptide
linkage (e.g., cleavable via a protease), or a phosphodiester
linkage (e.g., cleavable via a nuclease (e.g., DNAase)).
[1125] In some embodiments, the cleavage domain includes a poly(U)
sequence which can be cleaved by a mixture of Uracil DNA
glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII,
commercially known as the USER.TM. enzyme. In some embodiments, the
cleavage domain can be a single U. In some embodiments, the
cleavage domain can be an abasic site that can be cleaved with an
abasic site-specific endonuclease (e.g., Endonuclease IV or
Endonuclease VIII).
[1126] In some embodiments, the cleavage domain of the capture
probe is a nucleotide sequence within the capture probe that is
cleaved specifically, e.g., physically by light or heat, chemically
or enzymatically. The location of the cleavage domain within the
capture probe will depend on whether or not the capture probe is
immobilized on the substrate such that it has a free 3' end capable
of functioning as an extension primer (e.g., by its 5' or 3' end).
For example, if the capture probe is immobilized by its 5' end, the
cleavage domain will be located 5' to the spatial barcode and/or
UMI, and cleavage of said domain results in the release of part of
the capture probe including the spatial barcode and/or UMI and the
sequence 3' to the spatial barcode, and optionally part of the
cleavage domain, from a feature. Alternatively, if the capture
probe is immobilized by its 3' end, the cleavage domain will be
located 3' to the capture domain (and spatial barcode) and cleavage
of said domain results in the release of part of the capture probe
including the spatial barcode and the sequence 3' to the spatial
barcode from a feature. In some embodiments, cleavage results in
partial removal of the cleavage domain. In some embodiments,
cleavage results in complete removal of the cleavage domain,
particularly when the capture probes are immobilized via their 3'
end as the presence of a part of the cleavage domain can interfere
with the hybridization of the capture domain and the target nucleic
acid and/or its subsequent extension.
[1127] (iv) Sequencing
[1128] After analytes from the sample have hybridized or otherwise
been associated with capture probes, analyte capture agents, or
other barcoded oligonucleotide sequences according to any of the
methods described above in connection with the general spatial
cell-based analytical methodology, the barcoded constructs that
result from hybridization/association are analyzed via sequencing
to identify the analytes.
[1129] In some embodiments, where a sample is barcoded directly via
hybridization with capture probes or analyte capture agents
hybridized, bound, or associated with either the cell surface, or
introduced into the cell, as described above, sequencing can be
performed on the intact sample. Alternatively, if the barcoded
sample has been separated into fragments, cell groups, or
individual cells, as described above, sequencing can be performed
on individual fragments, cell groups, or cells. For analytes that
have been barcoded via partitioning with beads, as described above,
individual analytes (e.g., cells, or cellular contents following
lysis of cells) can be extracted from the partitions by breaking
the partitions, and then analyzed by sequencing to identify the
analytes.
[1130] A wide variety of different sequencing methods can be used
to analyze barcoded analyte constructs. In general, sequenced
polynucleotides can be, for example, nucleic acid molecules such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including
variants or derivatives thereof (e.g., single stranded DNA or
DNA/RNA hybrids, and nucleic acid molecules with a nucleotide
analog).
[1131] Sequencing of polynucleotides can be performed by various
commercial systems. More generally, sequencing can be performed
using nucleic acid amplification, polymerase chain reaction (PCR)
(e.g., digital PCR and droplet digital PCR (ddPCR), quantitative
PCR, real time PCR, multiplex PCR, PCR-based singleplex methods,
emulsion PCR), and/or isothermal amplification.
[1132] Other examples of methods for sequencing genetic material
include, but are not limited to, DNA hybridization methods (e.g.,
Southern blotting), restriction enzyme digestion methods, Sanger
sequencing methods, next-generation sequencing methods (e.g.,
single-molecule real-time sequencing, nanopore sequencing, and
Polony sequencing), ligation methods, and microarray methods.
Additional examples of sequencing methods that can be used include
targeted sequencing, single molecule real-time sequencing, exon
sequencing, electron microscopy-based sequencing, panel sequencing,
transistor-mediated sequencing, direct sequencing, random shotgun
sequencing, Sanger dideoxy termination sequencing, whole-genome
sequencing, sequencing by hybridization, pyrosequencing, capillary
electrophoresis, gel electrophoresis, duplex sequencing, cycle
sequencing, single-base extension sequencing, solid-phase
sequencing, high-throughput sequencing, massively parallel
signature sequencing, co-amplification at lower denaturation
temperature-PCR (COLD-PCR), sequencing by reversible dye
terminator, paired-end sequencing, near-term sequencing,
exonuclease sequencing, sequencing by ligation, short-read
sequencing, single-molecule sequencing, sequencing-by-synthesis,
real-time sequencing, reverse-terminator sequencing, nanopore
sequencing, 454 sequencing, Solexa Genome Analyzer sequencing,
SOLiD.TM. sequencing, MS-PET sequencing, and any combinations
thereof.
[1133] Sequence analysis of the nucleic acid molecules (including
barcoded nucleic acid molecules or derivatives thereof) can be
direct or indirect. Thus, the sequence analysis substrate (which
can be viewed as the molecule which is subjected to the sequence
analysis step or process) can be the barcoded nucleic acid molecule
or it can be a molecule which is derived therefrom (e.g., a
complement thereof). Thus, for example, in the sequence analysis
step of a sequencing reaction, the sequencing template can be the
barcoded nucleic acid molecule or it can be a molecule derived
therefrom. For example, a first and/or second strand DNA molecule
can be directly subjected to sequence analysis (e.g., sequencing),
i.e., can directly take part in the sequence analysis reaction or
process (e.g., the sequencing reaction or sequencing process, or be
the molecule which is sequenced or otherwise identified).
Alternatively, the barcoded nucleic acid molecule can be subjected
to a step of second strand synthesis or amplification before
sequence analysis (e.g., sequencing or identification by another
technique). The sequence analysis substrate (e.g., template) can
thus be an amplicon or a second strand of a barcoded nucleic acid
molecule.
[1134] In some embodiments, both strands of a double stranded
molecule can be subjected to sequence analysis (e.g., sequenced).
In some embodiments, single stranded molecules (e.g., barcoded
nucleic acid molecules) can be analyzed (e.g., sequenced). To
perform single molecule sequencing, the nucleic acid strand can be
modified at the 3' end.
[1135] Massively parallel pyrosequencing techniques can be used for
sequencing nucleic acids. In pyrosequencing, the nucleic acid is
amplified inside water droplets in an oil solution (emulsion PCR),
with each droplet containing a single nucleic acid template
attached to a single primer-coated bead that then forms a clonal
colony. The sequencing system contains many picolitre-volume wells
each containing a single bead and sequencing enzymes.
Pyrosequencing uses luciferase to generate light for detection of
the individual nucleotides added to the nascent nucleic acid and
the combined data are used to generate sequence reads.
[1136] As another example application of pyrosequencing, released
PPi can be detected by being immediately converted to adenosine
triphosphate (ATP) by ATP sulfurylase, and the level of ATP
generated can be detected via luciferase-produced photons, such as
described in Ronaghi, et al., Anal. Biochem. 242(1), 84-9 (1996);
Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281
(5375), 363 (1998); and U.S. Pat. Nos. 6,210,891, 6,258,568, and
6,274,320, the entire contents of each of which are incorporated
herein by reference.
[1137] In some embodiments, a massively parallel sequencing
technique can be based on reversible dye-terminators. As an
example, DNA molecules are first attached to primers on, e.g., a
glass or silicon substrate, and amplified so that local clonal
colonies are formed (bridge amplification). Four types of ddNTPs
are added, and non-incorporated nucleotides are washed away. Unlike
pyrosequencing, the DNA is only extended one nucleotide at a time
due to a blocking group (e.g., 3' blocking group present on the
sugar moiety of the ddNTP). A detector acquires images of the
fluorescently labelled nucleotides, and then the dye along with the
terminal 3' blocking group is chemically removed from the DNA, as a
precursor to a subsequent cycle. This process can be repeated until
the required sequence data is obtained.
[1138] In some embodiments, sequencing is performed by detection of
hydrogen ions that are released during the polymerization of DNA. A
microwell containing a template DNA strand to be sequenced can be
flooded with a single type of nucleotide. If the introduced
nucleotide is complementary to the leading template nucleotide, it
is incorporated into the growing complementary strand. This causes
the release of a hydrogen ion that triggers a hypersensitive ion
sensor, which indicates that a reaction has occurred. If
homopolymer repeats are present in the template sequence, multiple
nucleotides will be incorporated in a single cycle. This leads to a
corresponding number of released hydrogen ions and a proportionally
higher electronic signal.
[1139] In some embodiments, sequencing can be performed in situ. In
situ sequencing methods are particularly useful, for example, when
the biological sample remains intact after analytes on the sample
surface (e.g., cell surface analytes) or within the sample (e.g.,
intracellular analytes) have been barcoded. In situ sequencing
typically involves incorporation of a labeled nucleotide (e.g.,
fluorescently labeled mononucleotides or dinucleotides) in a
sequential, template-dependent manner or hybridization of a labeled
primer (e.g., a labeled random hexamer) to a nucleic acid template
such that the identities (i.e., nucleotide sequence) of the
incorporated nucleotides or labeled primer extension products can
be determined, and consequently, the nucleotide sequence of the
corresponding template nucleic acid. Aspects of in situ sequencing
are described, for example, in Mitra et al., (2003) Anal. Biochem.
320, 55-65, and Lee et al., (2014) Science, 343(6177), 1360-1363,
the entire contents of each of which are incorporated herein by
reference.
[1140] In addition, examples of methods and systems for performing
in situ sequencing are described in PCT Patent Application
Publication Nos. WO2014/163886, WO2018/045181, WO2018/045186, and
in U.S. Pat. Nos. 10,138,509 and 10,179,932, the entire contents of
each of which are incorporated herein by reference. Exemplary
techniques for in situ sequencing include, but are not limited to,
STARmap (described for example in Wang et al., (2018) Science,
361(6499) 5691), MERFISH (described for example in Moffitt, (2016)
Methods in Enzymology, 572, 1-49), and FISSEQ (described for
example in U.S. Patent Application Publication No. 2019/0032121).
The entire contents of each of the foregoing references are
incorporated herein by reference.
[1141] For analytes that have been barcoded via partitioning,
barcoded nucleic acid molecules or derivatives thereof (e.g.,
barcoded nucleic acid molecules to which one or more functional
sequences have been added, or from which one or more features have
been removed) can be pooled and processed together for subsequent
analysis such as sequencing on high throughput sequencers.
Processing with pooling can be implemented using barcode sequences.
For example, barcoded nucleic acid molecules of a given partition
can have the same barcode, which is different from barcodes of
other spatial partitions. Alternatively, barcoded nucleic acid
molecules of different partitions can be processed separately for
subsequent analysis (e.g., sequencing).
[1142] In some embodiments, where capture probes do not contain a
spatial barcode, the spatial barcode can be added after the capture
probe captures analytes from a biological sample and before
analysis of the analytes. When a spatial barcode is added after an
analyte is captured, the barcode can be added after amplification
of the analyte (e.g., reverse transcription and polymerase
amplification of RNA). In some embodiments, analyte analysis uses
direct sequencing of one or more captured analytes, such as direct
sequencing of hybridized RNA. In some embodiments, direct
sequencing is performed after reverse transcription of hybridized
RNA. In some embodiments direct sequencing is performed after
amplification of reverse transcription of hybridized RNA.
[1143] In some embodiments, direct sequencing of captured RNA is
performed by sequencing-by-synthesis (SBS). In some embodiments, a
sequencing primer is complementary to a sequence in one or more of
the domains of a capture probe (e.g., functional domain). In such
embodiments, sequencing-by-synthesis can include reverse
transcription and/or amplification in order to generate a template
sequence (e.g., functional domain) from which a primer sequence can
bind.
[1144] SBS can involve hybridizing an appropriate primer, sometimes
referred to as a sequencing primer, with the nucleic acid template
to be sequenced, extending the primer, and detecting the
nucleotides used to extend the primer. Preferably, the nucleic acid
used to extend the primer is detected before a further nucleotide
is added to the growing nucleic acid chain, thus allowing
base-by-base in situ nucleic acid sequencing. The detection of
incorporated nucleotides is facilitated by including one or more
labelled nucleotides in the primer extension reaction. To allow the
hybridization of an appropriate sequencing primer to the nucleic
acid template to be sequenced, the nucleic acid template should
normally be in a single stranded form. If the nucleic acid
templates making up the nucleic acid features are present in a
double stranded form these can be processed to provide single
stranded nucleic acid templates using methods well known in the
art, for example by denaturation, cleavage, etc. The sequencing
primers which are hybridized to the nucleic acid template and used
for primer extension are preferably short oligonucleotides, for
example, 15 to 25 nucleotides in length. The sequencing primers can
be provided in solution or in an immobilized form. Once the
sequencing primer has been annealed to the nucleic acid template to
be sequenced by subjecting the nucleic acid template and sequencing
primer to appropriate conditions, primer extension is carried out,
for example using a nucleic acid polymerase and a supply of
nucleotides, at least some of which are provided in a labelled
form, and conditions suitable for primer extension if a suitable
nucleotide is provided.
[1145] Preferably after each primer extension step, a washing step
is included in order to remove unincorporated nucleotides which can
interfere with subsequent steps. Once the primer extension step has
been carried out, the nucleic acid colony is monitored to determine
whether a labelled nucleotide has been incorporated into an
extended primer. The primer extension step can then be repeated to
determine the next and subsequent nucleotides incorporated into an
extended primer. If the sequence being determined is unknown, the
nucleotides applied to a given colony are usually applied in a
chosen order which is then repeated throughout the analysis, for
example dATP, dTTP, dCTP, dGTP.
[1146] SBS techniques which can be used are described for example,
but not limited to, those in U.S. Patent App. Pub. No.
2007/0166705, U.S. Patent App. Pub. No. 2006/0188901, U.S. Pat. No.
7,057,026, U.S. Patent App. Pub. No. 2006/0240439, U.S. Patent App.
Pub. No. 2006/0281109, PCT Patent App. Pub. No. WO 05/065814, U.S.
Patent App. Pub. No. 2005/0100900, PCT Patent App. Pub. No. WO
06/064199, PCT Patent App. Pub. No. WO07/010,251, U.S. Patent App.
Pub. No. 2012/0270305, U.S. Patent App. Pub. No. 2013/0260372, and
U.S. Patent App. Pub. No. 2013/0079232, the entire contents of each
of which are incorporated herein by reference.
[1147] In some embodiments, direct sequencing of captured RNA is
performed by sequential fluorescence hybridization (e.g.,
sequencing by hybridization). In some embodiments, a hybridization
reaction where RNA is hybridized to a capture probe is performed in
situ. In some embodiments, captured RNA is not amplified prior to
hybridization with a sequencing probe. In some embodiments, RNA is
amplified prior to hybridization with sequencing probes (e.g.,
reverse transcription to cDNA and amplification of cDNA). In some
embodiments, amplification is performed using single-molecule
hybridization chain reaction. In some embodiments, amplification is
performed using rolling chain amplification.
[1148] Sequential fluorescence hybridization can involve sequential
hybridization of probes including degenerate primer sequences and a
detectable label. A degenerate primer sequence is a short
oligonucleotide sequence which is capable of hybridizing to any
nucleic acid fragment independent of the sequence of said nucleic
acid fragment. For example, such a method could include the steps
of: (a) providing a mixture including four probes, each of which
includes either A, C, G, or T at the 5'-terminus, further including
degenerate nucleotide sequence of 5 to 11 nucleotides in length,
and further including a functional domain (e.g., fluorescent
molecule) that is distinct for probes with A, C, G, or T at the
5'-terminus; (b) associating the probes of step (a) to the target
polynucleotide sequences, whose sequence needs will be determined
by this method; (c) measuring the activities of the four functional
domains and recording the relative spatial location of the
activities; (d) removing the reagents from steps (a)-(b) from the
target polynucleotide sequences; and repeating steps (a)-(d) for n
cycles, until the nucleotide sequence of the spatial domain for
each bead is determined, with modification that the
oligonucleotides used in step (a) are complementary to part of the
target polynucleotide sequences and the positions 1 through n
flanking the part of the sequences. Because the barcode sequences
are different, in some embodiments, these additional flanking
sequences are degenerate sequences. The fluorescent signal from
each spot on the array for cycles 1 through n can be used to
determine the sequence of the target polynucleotide sequences.
[1149] In some embodiments, direct sequencing of captured RNA using
sequential fluorescence hybridization is performed in vitro. In
some embodiments, captured RNA is amplified prior to hybridization
with a sequencing probe (e.g., reverse transcription to cDNA and
amplification of cDNA). In some embodiments, a capture probe
containing captured RNA is exposed to the sequencing probe
targeting coding regions of RNA. In some embodiments, one or more
sequencing probes are targeted to each coding region. In some
embodiments, the sequencing probe is designed to hybridize with
sequencing reagents (e.g., a dye-labeled readout oligonucleotides).
A sequencing probe can then hybridize with sequencing reagents. In
some embodiments, output from the sequencing reaction is imaged. In
some embodiments, a specific sequence of cDNA is resolved from an
image of a sequencing reaction. In some embodiments, reverse
transcription of captured RNA is performed prior to hybridization
to the sequencing probe. In some embodiments, the sequencing probe
is designed to target complementary sequences of the coding regions
of RNA (e.g., targeting cDNA).
[1150] In some embodiments, a captured RNA is directly sequenced
using a nanopore-based method. In some embodiments, direct
sequencing is performed using nanopore direct RNA sequencing in
which captured RNA is translocated through a nanopore. A nanopore
current can be recorded and converted into a base sequence. In some
embodiments, captured RNA remains attached to a substrate during
nanopore sequencing. In some embodiments, captured RNA is released
from the substrate prior to nanopore sequencing. In some
embodiments, where the analyte of interest is a protein, direct
sequencing of the protein can be performed using nanopore-based
methods. Examples of nanopore-based sequencing methods that can be
used are described in Deamer et al., Trends Biotechnol. 18, 14
7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li
et al., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,
1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007);
Cockroft et al., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S.
Pat. No. 7,001,792. The entire contents of each of the foregoing
references are incorporated herein by reference.
[1151] In some embodiments, direct sequencing of captured RNA is
performed using single molecule sequencing by ligation. Such
techniques utilize DNA ligase to incorporate oligonucleotides and
identify the incorporation of such oligonucleotides. The
oligonucleotides typically have different labels that are
correlated with the identity of a particular nucleotide in a
sequence to which the oligonucleotides hybridize. Aspects and
features involved in sequencing by ligation are described, for
example, in Shendure et al. Science (2005), 309: 1728-1732, and in
U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488; 6,172,218; and
6,306,597, the entire contents of each of which are incorporated
herein by reference.
[1152] In some embodiments, nucleic acid hybridization can be used
for sequencing. These methods utilize labeled nucleic acid decoder
probes that are complementary to at least a portion of a barcode
sequence. Multiplex decoding can be performed with pools of many
different probes with distinguishable labels. Non-limiting examples
of nucleic acid hybridization sequencing are described for example
in U.S. Pat. No. 8,460,865, and in Gunderson et al., Genome
Research 14:870-877 (2004), the entire contents of each of which
are incorporated herein by reference.
[1153] In some embodiments, commercial high-throughput digital
sequencing techniques can be used to analyze barcode sequences, in
which DNA templates are prepared for sequencing not one at a time,
but in a bulk process, and where many sequences are read out
preferably in parallel, or alternatively using an ultra-high
throughput serial process that itself may be parallelized. Examples
of such techniques include Illumina.RTM. sequencing (e.g., flow
cell-based sequencing techniques), sequencing by synthesis using
modified nucleotides (such as commercialized in TruSeq.TM. and
HiSeq.TM. technology by Illumina, Inc., San Diego, Calif.),
HeliScope.TM. by Helicos Biosciences Corporation, Cambridge, Mass.,
and PacBio RS by Pacific Biosciences of California, Inc., Menlo
Park, Calif.), sequencing by ion detection technologies (Ion
Torrent, Inc., South San Francisco, Calif.), and sequencing of DNA
nanoballs (Complete Genomics, Inc., Mountain View, Calif.).
[1154] In some embodiments, detection of a proton released upon
incorporation of a nucleotide into an extension product can be used
in the methods described herein. For example, the sequencing
methods and systems described in U.S. Patent Application
Publication Nos. 2009/0026082, 2009/0127589, 2010/0137143, and
2010/0282617, can be used to directly sequence barcodes.
[1155] In some embodiments, real-time monitoring of DNA polymerase
activity can be used during sequencing. For example, nucleotide
incorporations can be detected through fluorescence resonance
energy transfer (FRET), as described for example in Levene et al.,
Science (2003), 299, 682-686, Lundquist et al., Opt. Lett. (2008),
33, 1026-1028, and Korlach et al., Proc. Natl. Acad. Sci. USA
(2008), 105, 1176-1181. The entire contents of each of the
foregoing references are incorporated herein by reference
herein.
[1156] (v) Temporal Analysis
[1157] In some embodiments, the methods described herein can be
used to assess analyte levels and/or expression in a cell or a
biological sample over time (e.g., before or after treatment with
an agent or different stages of differentiation). In some examples,
the methods described herein can be performed on multiple similar
biological samples or cells obtained from the subject at a
different time points (e.g., before or after treatment with an
agent, different stages of differentiation, different stages of
disease progression, different ages of the subject, before or after
physical perturbation, before or after treatment with a
perturbation agent as described herein, or before or after
development of resistance to an agent). As described herein, a
"perturbation agent" or "perturbation reagent" can be a small
molecule, an antibody, a drug, an aptamer, a nucleic acid (e.g.,
miRNA), a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease,
antisense oligonucleotide a physical environmental (e.g.,
temperature change), and/or any other known perturbation agents
where the agent alters equilibrium or homeostasis.
[1158] In some embodiments, the methods described herein can be
performed on multiple similar biological samples or cells obtained
from the subject at 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. For
example, the multiple similar biological samples can be repetitive
samples from the same subject, the same tissue, the same organoid,
the same cell suspension, or any other biological sample described
herein. In some embodiments, the methods described herein can be
performed on the same biological sample or cells obtained from the
subject at a different time points (e.g., before or after treatment
with a perturbation agent, different stages of differentiation,
different stages of disease progression, different ages of the
subject, or before or after development of resistance to an agent).
In some embodiments, a perturbation agent can be small-molecules,
antibodies, nucleic acids, peptides, and/or other external stimuli
(e.g., temperature change). In some embodiments, the biological
sample is contacted with a different array at each time point.
[1159] In some embodiments, a sample can be placed in a controlled
environment permissive for cellular growth and/or maintenance,
and/or to prevent hypoxia. In some embodiments, a controlled
environment allows a sample to be analyzed at different time
points. Barcoded arrays can be placed proximal to (e.g., on top of)
the sample and imaged using a microscope or other suitable
instrument to register the relative position of the biological
sample to the barcoded array, optionally using optically encoded
fiducial markers. An electric field can be applied for a period of
time, such that biological analytes (e.g., DNA, RNA, proteins,
metabolites, small molecules, lipids, and the like) are released
from the sample and captured by capture probes on the
spatially-barcoded array, preserving spatial information of the
sample. The barcoded array can be removed, and the spatial and
molecular information therein is determined (e.g., by performing
library construction for next generation sequencing or in situ
sequencing). Sequencing can be followed by computational analysis
to correlate the molecular information (e.g., gene expression
values with the spatial barcode). These steps can be repeated one
or more times to capture the spatial information of analytes at
different time-points.
[1160] In some embodiments, methods as described herein can be
combined with a cell migration assay. A cell migration assay can
comprise one or more microprinted lines, or suspended 3D
nanofibers, on which the cells migrate. Migration using these
assays can be measured by imaging cell migration and/or contacting
migrated cells with a spatially-barcoded array. An array used in a
cell migration assay can comprise one or more channels on the
substrate of the array, e.g., to confine cell migration to one
dimension along the substrate. Additionally, the channels can
direct the migration of a cell such that it does not contact
another cell on the array (e.g., the channels do not overlap with
each other), and in some embodiments, the channels are about the
same width as or wider than a cell (e.g., for a mammalian cell, a
channel can have a width of about 2 .mu.m to about 10 .mu.m).
Cellular location on the spatially-barcoded array can be identified
using any method described herein.
[1161] In some embodiments, cells can be disposed on an array as
described herein and allowed to migrate. Cell migration in cell
migration assays can be used to measure target phenotypes (e.g.,
phenotype for invasiveness). In some embodiments, the cell
migration distance can be measured and correlated to a biological
analyte. Reagents can be added to the array to facilitate cell
migration. For example, the array can be coated with one or more
extracellular matrix (ECM) components (e.g., basement membrane
extract (BME), laminin I, collagen I, collagen IV, fibronectin,
vitronectin, elastin), a cell culture medium, a chemoattractant, a
chemorepellant, or a combination thereof. In some embodiments, a
reagent such as a chemoattractant or chemorepellant can be disposed
on only a portion of the array, present as a gradient along the one
or more axis or channels of the array, or a combination
thereof.
[1162] (vi) Spatially Resolving Analyte Information
[1163] In some embodiments, a lookup table (LUT) can be used to
associate one property with another property of a feature. These
properties include, e.g., locations, barcodes (e.g., nucleic acid
barcode molecules), spatial barcodes, optical labels, molecular
tags, and other properties.
[1164] In some embodiments, a lookup table can associate a nucleic
acid barcode molecule with a feature. In some embodiments, an
optical label of a feature can permit associating the feature with
a biological particle (e.g., cell or nuclei). The association of a
feature with a biological particle can further permit associating a
nucleic acid sequence of a nucleic acid molecule of the biological
particle to one or more physical properties of the biological
particle (e.g., a type of a cell or a location of the cell). For
example, based on the relationship between the barcode and the
optical label, the optical label can be used to determine the
location of a feature, thus associating the location of the feature
with the barcode sequence of the feature. Subsequent analysis
(e.g., sequencing) can associate the barcode sequence and the
analyte from the sample. Accordingly, based on the relationship
between the location and the barcode sequence, the location of the
biological analyte can be determined (e.g., in a specific type of
cell or in a cell at a specific location of the biological
sample).
[1165] In some embodiments, a feature can have a plurality of
nucleic acid barcode molecules attached thereto. The plurality of
nucleic acid barcode molecules can include barcode sequences. The
plurality of nucleic acid molecules attached to a given feature can
have the same barcode sequences, or two or more different barcode
sequences. Different barcode sequences can be used to provide
improved spatial location accuracy.
[1166] As discussed above, analytes obtained from a sample, such as
RNA, DNA, peptides, lipids, and proteins, can be further processed.
In particular, the contents of individual cells from the sample can
be provided with unique spatial barcode sequences such that, upon
characterization of the analytes, the analytes can be attributed as
having been derived from the same cell. More generally, spatial
barcodes can be used to attribute analytes to corresponding spatial
locations in the sample. For example, hierarchical spatial
positioning of multiple pluralities of spatial barcodes can be used
to identify and characterize analytes over a particular spatial
region of the sample. In some embodiments, the spatial region
corresponds to a particular spatial region of interest previously
identified, e.g., a particular structure of cytoarchitecture
previously identified. In some embodiments, the spatial region
corresponds to a small structure or group of cells that cannot be
seen with the naked eye. In some embodiments, a unique molecular
identifier can be used to identify and characterize analytes at a
single cell level.
[1167] The analyte can include a nucleic acid molecule, which can
be barcoded with a barcode sequence of a nucleic acid barcode
molecule. In some embodiments, the barcoded analyte can be
sequenced to obtain a nucleic acid sequence. In some embodiments,
the nucleic acid sequence can include genetic information
associated with the sample. The nucleic acid sequence can include
the barcode sequence, or a complement thereof. The barcode
sequence, or a complement thereof, of the nucleic acid sequence can
be electronically associated with the property (e.g., color and/or
intensity) of the analyte using the LUT to identify the associated
feature in an array.
[1168] (vii) Proximity Capture
[1169] In some embodiments, two- or three-dimensional spatial
profiling of one or more analytes present in a biological sample
can be performed using a proximity capture reaction, which is a
reaction that detects two analytes that are spatially close to each
other and/or interacting with each other. For example, a proximity
capture reaction can be used to detect sequences of DNA that are
close in space to each other, e.g., the DNA sequences can be within
the same chromosome, but separated by about 700 bp or less. As
another example, a proximity capture reaction can be used to detect
protein associations, e.g., two proteins that interact with each
other. A proximity capture reaction can be performed in situ to
detect two analytes that are spatially close to each other and/or
interacting with each other inside a cell. Non-limiting examples of
proximity capture reactions include DNA nanoscopy, DNA microscopy,
and chromosome conformation capture methods. Chromosome
conformation capture (3C) and derivative experimental procedures
can be used to estimate the spatial proximity between different
genomic elements. Non-limiting examples of chromatin capture
methods include chromosome conformation capture (3-C), conformation
capture-on-chip (4-C), 5-C, ChIA-PET, Hi-C, targeted chromatin
capture (T2C). Examples of such methods are described, for example,
in Miele et al., Methods Mol Biol. (2009), 464, Simonis et al.,
Nat. Genet. (2006), 38(11): 1348-54, Raab et al., Embo. J. (2012),
31(2): 330-350, and Eagen et al., Trends Biochem. Sci. (2018)
43(6): 469-478, the entire contents of each of which is
incorporated herein by reference.
[1170] In some embodiments, the proximity capture reaction includes
proximity ligation. In some embodiments, proximity ligation can
include using antibodies with attached DNA strands that can
participate in ligation, replication, and sequence decoding
reactions. For example, a proximity ligation reaction can include
oligonucleotides attached to pairs of antibodies that can be joined
by ligation if the antibodies have been brought in proximity to
each oligonucleotide, e.g., by binding the same target protein
(complex), and the DNA ligation products that form are then used to
template PCR amplification, as described for example in Soderberg
et al., Methods. (2008), 45(3): 227-32, the entire contents of
which are incorporated herein by reference. In some embodiments,
proximity ligation can include chromosome conformation capture
methods.
[1171] In some embodiments, the proximity capture reaction is
performed on analytes within about 400 nm distance (e.g., about 300
nm, about 200 nm, about 150 nm, about 100 nm, about 50 nm, about 25
nm, about 10 nm, or about 5 nm) from each other. In general,
proximity capture reactions can be reversible or irreversible.
[1172] (viii) Feature Removal from an Array
[1173] A spatially-barcoded array can be contacted with a
biological sample to spatially detect analytes present in the
biological sample. In some embodiments, the features (e.g., gel
pads, beads) can be removed from the substrate surface for
additional analysis (e.g., imaging, sequencing, or quantification).
In some embodiments, the features (e.g., gel pads, beads) can be
removed mechanically (e.g., scraping), by an enzymatic reaction, or
by a chemical reaction. In some embodiments, the features (e.g.,
gel pads, beads) can be aspirated. In some embodiments, after the
features are removed (by any method), the features can be combined
with a uniquely barcoded bead. In some embodiments, the
oligonucleotides within a feature can be ligated or hybridized to
the barcode sequence on the barcoded bead. For example, the spatial
barcode oligonucleotide within a feature can be ligated to the
barcode sequence on the barcoded bead. Additionally, the capture
probes can be ligated to the barcode sequence on the barcoded bead.
In some embodiments, the features and the bead can be partitioned.
In some embodiments, the features (e.g., gels pads, beads) and the
uniquely barcoded bead can be partitioned into a vesicle. In some
embodiments, the vesicle can have a lipid bilayer. In some
embodiments, the features and the bead can be encapsulated. In some
embodiments, the features and the bead can be encapsulated in an
oil emulsion. In some embodiments, the features and the bead can be
encapsulated in a water-in-oil emulsion. Once partitioned, the
features (e.g., gel pads, beads) can be processed for further
analysis (e.g., sequencing) according to any method described
herein.
[1174] (ix) Other Applications
[1175] The spatial analysis methods described herein can be used to
detect and characterize the spatial distribution of one or more
haplotypes in a biological sample. As used in the present
disclosure, a haplotype is used to describe one or more mutations,
DNA variations, polymorphisms in a given segment of the genome,
which can be used to classify the genetic segment, or a collection
of alleles or genetic segments containing single nucleotide
polymorphisms (SNPs). Haplotype association studies are used to
inform a greater understanding of biological conditions. For
example, identifying and characterizing haplotype variants at or
associated with putative disease loci in humans can provide a
foundation for mapping genetic causes underlying disease
susceptibility. The term "locus" (plural "loci"), as used in the
art, can be a fixed location on a chromosome, including the
location of a gene or a genetic marker, which can contain a
plurality of haplotypes, including alleles and SNPs.
[1176] Variant haplotype detection is a technique used to identify
heterozygous cells in single cell studies. In combination with
spatial analysis, variant haplotype detection can further provide
novel information on the distribution of heterozygous cells in
biological samples (e.g., tissues) affected by or exhibiting a
variety of biological conditions. These data may reveal causal
relationships between variant haplotypes and disease outcomes, to
aid in identification of disease-associated variants, or to reveal
heterogeneity within a biological sample.
[1177] In some embodiments, variant haplotype detection is a
technique that can be used in combination with, in addition to, or
as a part of, the spatial analysis methods described herein.
Briefly, variant haplotype detection can include providing inputs
for executing an algorithm on a computer system, and performing an
analysis to identify and determine the spatial distribution of
haplotypes. One input can be a plurality of sequence reads obtained
from a two-dimensional spatial array in contact with a biological
sample and subsequently aligned to a genome. The sequence reads can
also contain spatial barcodes with positional information, such
that the sequence reads can be mapped to a location on the
biological sample. Other inputs can include electronic data files
of gene sequence variations, or haplotypes, and a reference genome.
For each locus, the corresponding sequence reads and variant
haplotypes are aligned to determine the haplotype identity of each
sequence read. The haplotype identity and the spatial barcode of
the sequence reads are then categorized to determine the spatial
distribution of haplotypes within the biological sample. As
described above, this spatial distribution can be used to
characterize a biological condition of the sample. In some
embodiments, sequence reads are obtained by in situ sequencing of
the two-dimensional array of positions on the substrate, while in
some embodiments, sequence reads are obtained by high-throughput
sequencing. In some embodiments, other methods for generating
sequence reads described herein are used, such as paired end
sequencings.
[1178] In some embodiments, a respective loci in the plurality of
loci is bi-allelic and the corresponding set of haplotypes for the
respective loci consists of a first allele and a second allele. In
some such embodiments, the respective loci includes a heterozygous
single nucleotide polymorphism (SNP), a heterozygous insert, or a
heterozygous deletion.
[1179] In some embodiments, analytes captured by any of the spatial
analysis methods described herein can be analyzed (e.g., sequenced)
via in situ sequencing methods. For example, a substrate including
a plurality of capture probes (e.g., an array), attached either
directly or indirectly (e.g., via a feature), that include a
spatial barcode and a capture domain. In some embodiments, the
capture domain can be configured to interact (e.g., hybridize) with
an analyte (e.g., mRNA). In some embodiments, a biological sample
can be contacted to the array such that the capture domain of the
capture probe interacts with (e.g., hybridizes) the analyte. In
some embodiments, the capture probe can function as a template for
a hybridization or ligation reaction with the captured analyte. For
example, a reverse transcription reaction can be performed to
extend the 3' end of a capture probe hybridized to the analyte
using any of the exemplary reverse transcriptases described herein,
thereby generating an extended capture probe (e.g., an extended
capture probe including the spatial barcode and a sequence that is
complementary to a sequence in the analyte). After the extended
capture probe is synthesized, a second strand that is complementary
to the extended capture probe can be synthesized. In some
embodiments, second strand synthesis can be performed using any of
the methods described herein. In some embodiments, amine-modified
nucleotides can be used when generating the extended capture probe
or the second strand, or both. For example, the amine-modified
nucleotides can be aminoallyl (aa)-dUTP, aa-dCTP, aa-dGTP, and/or
aa-dATP.
[1180] In some embodiments, after generation of the extended
capture probe, the second strand, or both the extended capture
probe and/or the second strand can be released from the surface of
the substrate. For example, the extended capture probe and/or the
second strand can be released by any of the methods described
herein (e.g., heat or cleavage via a cleavage domain). In some
embodiments, the amine-modified nucleotides incorporated into the
extended capture probe can be cross-linked to the surface of a
substrate or cross-linked to the biological sample using its
amine-modified nucleotides. In some embodiments, the surface of the
substrate can be coated in a hydrogel. In some embodiments, the
surface of the substrate can be coated in a protein matrix. In some
embodiments, the cross-linking can be irreversible. In some
embodiments, the cross-linked extended capture probe and/or second
strand can be circularized. For example, circular template ligation
can be performed by a DNA ligase (e.g., T4 DNA ligase) or circular
template-free ligation can be performed by a template independent
ligase (e.g., CircLigase). In some embodiments, the extended
capture probe is circularized with CircLigase. In some embodiments,
the circularized extended capture probe can be amplified. For
example, rolling circle amplification can be performed with a
suitable DNA polymerase (e.g., phi29). In some embodiments, the
capture probe includes a functional domain (e.g., sequencing
adapter). In some embodiments, rolling circle amplification can be
performed with a primer complementary to the functional domain
(e.g., sequencing adapter). In some embodiments, the rolling circle
amplification can be performed to generate two or more amplicons
(e.g., one or more amplicons including any of the amine-modified
nucleotides described herein). In some embodiments, the two or more
amplicons produced by the rolling circle amplification can be
cross-linked to the surface of the substrate and/or cross-linked to
the biological sample. In some embodiments, the two or more
amplicons can be sequenced in situ. The in situ sequencing can be
performed by any method described herein (See, Lee, J. H.,
Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression
profiling, Nat Protoc., 10(3): 442-458, doi:10.1038/nprot.2014.191
(2015), which is incorporated herein by reference). In some
embodiments, the two or more amplicons can be imaged.
[1181] In some embodiments, spatial analysis by any of the methods
described herein can be performed on ribosomal RNA (rRNA),
including, endogenous ribosomal RNA (e.g., native to the biological
sample), and/or exogenous RNA (e.g., microbial ribosomal RNA and/or
viral RNA also present in the biological sample). As used herein,
"metagenomics," can refer to the study of exogenous nucleic acids
(e.g., DNA, RNA, or other nucleic acids described herein) present
in a biological sample. As used herein, "spatial metagenomics," can
refer to the study of the spatial location of exogenous nucleic
acid present in a biological sample. Spatial metagenomics can also
refer to the identification of one or more species (e.g., viral or
microbial) present in the biological sample and/or the study of
identifying patterns of proximity (e.g., co-localization) amongst
species.
[1182] In some embodiments, microbial rRNA can be spatially
detected, quantified, and/or amplified from a biological sample. In
some embodiments, rRNA (e.g., 16S ribosomal RNA) can be associated
with a particular microbial species. For example, microbial
ribosomal RNA (e.g., 16S ribosomal RNA) can be used to identify one
or more species of microbe present in the biological sample (See
e.g., Kolbert, C. P., and Persing, D. H., Ribosomal DNA sequencing
as a tool for identification of bacterial pathogens, Current
Opinion in Microbiology. 2 (3): 299-305.
doi:10.1016/S1369-5274(99)80052-6. PMID 10383862 (1999), which is
incorporated herein by reference). In some embodiments,
identification of microbial species in proximity to one or more
other microbial species can be identified.
[1183] In some embodiments, a substrate can be covered (e.g.,
coated) with a photo-crosslinkable coating (e.g., conditionally
dissolvable polymer, e.g., DTT sensitive hydrogel). A biological
sample can be contacted with the photo-crosslinkable coated
substrate. In some embodiments, the biological sample and
photo-crosslinkable substrate are assembled into a flow-cell and
the photo-crosslinkable polymer can be incubated with the
biological sample. The biological sample can be cross-linked into
hydrogel-voxels of defined dimensions using a light source and a
photomask. In some embodiments, the flow-cell can be dismantled and
washed to remove unpolymerized hydrogel. The photo-crosslinkable
coating can be treated with DTT to yield single-cell partitions or
approximately single-cell partitions.
[1184] In some embodiments, the single-cell or approximately
single-cell partitions can be encapsulated in a vesicle. The
vesicle can contain a barcoded feature (e.g., a bead), and the
barcoded feature can contain a capture domain. In some embodiments,
the capture domain can capture microbial rRNA (e.g., microbial 16S
rRNA). In some embodiments, the captured microbial rRNA can be
amplified and analyzed (e.g., sequenced) by any of the methods
described herein. In some embodiments, the amplified and sequenced
microbial rRNA can identify microbial species and/or patterns of
proximity (e.g., co-localization) of one or more species.
[1185] Alternatively, spatial analysis can be performed on
exogenous rRNA (e.g., microbial or viral) with a plurality of
capture probes on a substrate (e.g., an array), wherein the capture
probes include a spatial barcode and a capture domain. In some
embodiments, the capture domain can be configured to interact
(e.g., hybridize) with microbial rRNA present in the biological
sample. The capture probe can be configured to interact with any
microbial rRNA. In some embodiments, the capture probe is
configured to interact with microbial 16S rRNA. The biological
sample can be treated (e.g., permeabilized) such that the capture
domain and the analyte (e.g., microbial rRNA) interact (e.g.,
hybridize). In some embodiments, the captured analyte (e.g.,
microbial rRNA) can be reverse transcribed generating a first
strand cDNA, followed by second strand cDNA synthesis as described
herein. The first stand cDNA and/or the second strand cDNA can
include a portion or all of a capture probe sequence, or a
complement thereof. The capture probe sequence, or complement
thereof, can include the spatial barcode, or complement thereof. In
some embodiments, the first strand cDNA, and optionally, the second
strand cDNA can be amplified by any method described herein. The
amplified capture probes and analytes can be analyzed (e.g.,
sequenced) by any method described herein. The spatial information
of the spatially-barcoded features can be used to determine the
spatial location of the captured analytes (e.g., microbial rRNA) in
the biological sample. In some embodiments, the captured analyte
can identify the microbial species present in the biological
sample. In some embodiments, the spatial information and identity
of microbial species present in the biological sample can be
correlated with one another, thus revealing whether certain
microbial species may be found in proximity (e.g., co-localize)
with one another.
[1186] Provided herein are methods for spatially profiling analytes
within a biological sample. Profiles of biological samples (e.g.,
individual cells, populations of cells, tissue sections, etc.) can
be compared to profiles of other cells, e.g., "normal," or
"healthy," biological samples. In some embodiments of any the
methods for spatially profiling analytes described herein, the
method can provide for diagnosis of a disease (e.g., cancer,
Alzheimer's disease, Parkinson's disease). In some embodiments of
any the methods for spatially profiling analytes described herein,
the methods can be used in drug screening. In some embodiments of
any the methods for spatially profiling analytes described herein,
the methods can be used to perform drug screening with an organoid.
In some embodiments of any the methods for spatially profiling
analytes described herein, the methods can be used to detect
changes in (e.g., altered) cellular signaling. In some embodiments
of any the methods for spatially profiling analytes described
herein, the methods can include the introduction of a pathogen to
the biological sample and evaluation of the response of the
biological sample to the pathogen. In some embodiments of any the
methods for spatially profiling analytes described herein, the
methods include exposing the biological sample to a perturbation
agent (e.g., any of the perturbation agents described herein) and
evaluating the response of the biological sample to the
perturbation agent. In some embodiments of any the methods for
spatially profiling analytes described herein, the methods include
monitoring cell differentiation in a biological sample (e.g., an
organoid). In some embodiments of any the methods for spatially
profiling analytes described herein, the methods include analyzing
tissue morphogenesis. In some embodiments of any the methods for
spatially profiling analytes described herein, the methods include
identifying spatial heterogeneity in a biological sample (e.g.,
identifying different cell types or populations in a biological
sample). In some embodiments of any the methods for spatially
profiling analytes described herein, the methods include analyzing
the spatiotemporal order (e.g., timing) of molecular events. For
example, the methods for spatially profiling analytes can include
monitoring expression levels over the course of a disease.
[1187] The methods provided herein can also be used to determine a
relative level of inflammation in a subject (e.g., determine an
inflammatory score) or a subject's response to treatment or the
development of resistance to treatment. The methods described
herein can also be used to identify candidate targets for potential
therapeutic intervention and/or to identify biomarkers associated
with different disease states in a subject.
[1188] (h) Quality Control
[1189] (i) Control Sample
[1190] As used herein, the term "control sample" typically refers
to a substrate that is insoluble in aqueous liquid and that allows
for an accurate and traceable positioning of test analytes on the
substrate. The control sample can be any suitable substrate known
to the person skilled in the art. Exemplary control samples
comprise a semi-porous material. Non-limiting examples of a
semi-porous material include a nitrocellulose membrane, a hydrogel,
and a nylon filter.
[1191] A control sample can be of any appropriate dimension or
volume (e.g., size or shape). In some embodiments, a control sample
is a regular shape (e.g., a square, circle, or a rectangle). In
some embodiments, a surface of a control sample has any appropriate
form or format. For example, the surface of a control sample can be
flat or curved (e.g., convexly or concavely curved towards the area
where the interaction between the substrate and the control sample
takes place). In some embodiments, a control sample has rounded
corners (e.g., for increased safety or robustness). In some
embodiments, a control sample has one or more cut-off corners
(e.g., for use with a slide clamp or cross-table).
[1192] A control sample can comprise a plurality of test analytes.
In some embodiments, the members of the plurality of test analytes
are disposed on the substrate in a known amount and in a known
location. For example, a plurality of test analytes are disposed at
a known amount on the control sample at one or more locations. In
some embodiments, the plurality of test analytes are disposed on
the substrate in a defined pattern (e.g., an x-y grid pattern). In
some embodiments, the defined pattern includes one or more
locations or spots.
[1193] In some embodiments, each location comprises a plurality of
the same species of test analyte. In some embodiments, each
location comprises a plurality of one or more different species of
test analytes. In some embodiments, each location on the control
sample represents a different region of a biological sample, e.g.,
a tissue sample. In some embodiments, an area on the control sample
that does not comprise a plurality of test analytes represents an
area where no biological sample is present.
[1194] In some embodiments, the plurality of test analytes
comprises one or more test analytes, e.g., a first test analyte, a
second test analyte, a third test analyte, a fourth test analyte,
etc. In some embodiments, the plurality of test analytes comprises
nucleic acids. In some embodiments, each location or feature
comprises a population of nucleic acid sequences. In some
embodiments, the nucleic acid sequence of a first test analyte
differs from the nucleic acid sequence of a second test analyte by
a single nucleic acid residue. In some embodiments, each location
or feature comprises a population of RNA transcripts and one or
more specific surface marker proteins or one or more CRISPR guide
RNAs. In some embodiments, the plurality of test analytes comprises
a bacterial artificial chromosomes (BAC). In some embodiments, each
location on the control sample comprises a unique blend of BACs. In
some embodiments, proteins are cross-linked to the BACs, for
example, to mimic histone binding on DNA.
[1195] In some embodiments, the concentration of a first test
analyte differs from the concentration of a second test analyte at
a different location or feature on the control sample. In some
embodiments, the first test analyte and the second test analyte
comprise an identical nucleotide sequence.
[1196] (ii) Spatial RNA Integrity Number (sRIN)
[1197] As used herein, the term "spatial RNA Integrity Number" or
"sRIN" refers to the in situ indication of RNA quality based on an
integrity score. Higher sRIN scores correlate with higher data
quality in the spatial profiling assays described herein. For
example, a first biological sample with a high sRIN score will have
higher data quality compared to a second biological sample with
sRIN score lower than the first biological sample. In some
embodiments, a sRIN is calculated for a tissue section, one or more
regions of a tissue section, or a single cell.
[1198] In some embodiments, one or more sRINs for a given
biological sample (e.g., tissue section, one or more regions of a
tissue, or a single cell) are calculated by: (a) providing (i) a
spatial array including a plurality of capture probes on a
substrate, where a capture probe comprises a capture domain and
(ii) a tissue stained with a histology stain (e.g., any of the
stains described herein); (b) contacting the spatial array with the
biological sample (e.g., tissue); (c) capturing a biological
analyte (e.g., an 18S rRNA molecule) from the biological sample
(e.g., tissue) with the capture domain; (d) generating a cDNA
molecule from the captured biological analyte (e.g., 18S rRNA); (e)
hybridizing one or more labeled oligonucleotide probes to the cDNA;
(f) imaging the labeled cDNA and the histology stain (e.g., any of
the stains described herein), and (g) generating a spatial RNA
integrity number for a location in the spatial array, wherein the
spatial RNA integrity number comprises an analysis of a labeled
cDNA image and a histology stain (e.g., any of the stains described
herein) image for the location.
[1199] In some embodiments, the biological sample (e.g., tissue) is
stained with a histology stain. As used herein, a "histology stain"
can be any stain described herein. For example, the biological
sample can be stained with IF/IHC stains described herein. For
example, the biological sample (e.g., tissue) can be stained with
Hematoxylin & Eosin ("H&E"). In some embodiments, the
biological sample (e.g., tissue) is stained with a histology stain
(e.g., any of the stains described herein) before,
contemporaneously with, of after labelling of the cDNA with labeled
oligonucleotide probes. In some embodiments, the stained biological
sample can be, optionally, destained (e.g., washed in HCl). For
example, Hematoxylin, from the H&E stain, can be optionally
removed from the biological sample by washing in dilute HCl (0.01M)
prior to further processing. In some embodiments, the stained
biological sample can be optionally destained after imaging and
prior to permeabilization.
[1200] In some embodiments, the spatial array includes a plurality
of capture probes immobilized on a substrate where the capture
probes include at least a capture domain. In some embodiments, the
capture domain includes a poly(T) sequence. For example, a capture
domain includes a poly(T) sequence that is capable of capturing an
18S rRNA transcript from a biological sample.
[1201] In some embodiments, calculating one or more spatial RNA
Integrity Numbers for a biological sample includes hybridizing at
least one (e.g., at least two, at least three, at least four, or at
least five) labeled oligonucleotide probes to the cDNA generated
from the 18s rRNA. In some embodiments, a labeled oligonucleotide
probe includes a sequence that is complementary to a portion of the
18S cDNA. In some embodiments, four labeled oligonucleotide probes
(P1-P4) are designed to hybridize at four different locations
spanning the entire gene body of the 18S rRNA. In some embodiments,
a labeled oligonucleotide probe can include any of the detectable
labels as described herein. For example, an oligonucleotide labeled
probe can include a fluorescent label (e.g., Cy3). In some
embodiments, one or more of the labeled oligonucleotide probes
designed with complementarity to different locations within the 18S
cDNA sequence include the same detectable label. For example, four
labeled oligonucleotide probes, (P1-P4) each designed to have
complementarity to a different location within the 18S cDNA
sequence can all have the same detectable label (e.g., Cy3). In
some embodiments, one or more of the labeled oligonucleotide probes
designed with complementarity to different locations within the 18S
cDNA sequence include a different detectable label. For example,
four labeled oligonucleotide probes, (P1-P4) each designed to have
complementarity to a different location within the 18S cDNA
sequence can include different detectable labels.
[1202] In some embodiments, determining a spatial RNA Integrity
Number for a biological sample (e.g., tissue section, one or more
regions of a tissue, or a single cell) includes analyzing the
images taken from a spatial array and a histology stain (e.g., any
of the stains described herein) for the same location. For example,
for the spatial array, all images are generated by scanning with a
laser (e.g., a 532 nm wavelength) after the fluorescently labeled
(e.g., Cy3) oligonucleotide probes have been hybridized to the 18S
cDNA. One image is generated per probe (P1-P4) and one image is
generated where no fluorescently labeled probes were hybridized
(P0). Normalization of Fluorescence Units (FU) data is performed by
subtraction of the auto-fluorescence recorded with P0 and division
with P1. After alignment, the five images (one image from each
probe, P1-P4, and one image from an area without bound probe) are
loaded into a script. The script generates two different plots, one
heat-map of spatial RIN values and one image alignment error plot,
which combines the histology stain (e.g., any of the stains
described herein) image. The image alignment error plot is used to
visualize which pixels and positions should be excluded from the
analysis due to alignment errors between the images from P0-P4.
III. General Spatial Cell-Based Analytical Methodology
[1203] (a) Barcoding a Biological Sample
[1204] In some embodiments, provided herein are methods and
materials for attaching and/or introducing a molecule (e.g., a
peptide, a lipid, or a nucleic acid molecule) having a barcode
(e.g., a spatial barcode) to a biological sample (e.g., to a cell
in a biological sample) for use in spatial analysis. In some
embodiments, a plurality of molecules (e.g., a plurality of nucleic
acid molecules) having a plurality of barcodes (e.g., a plurality
of spatial barcodes) are introduced to a biological sample (e.g.,
to a plurality of cells in a biological sample) for use in spatial
analysis.
[1205] FIG. 18 is a schematic diagram depicting cell tagging using
either covalent conjugation of an analyte binding moiety to a cell
surface or non-covalent interactions with cell membrane elements.
FIG. 18 lists non-exhaustive examples of a covalent analyte binding
moiety/cell surface interactions, including protein targeting,
amine conjugation using NHS chemistry, cyanuric chloride, thiol
conjugation via maleimide addition, as well as targeting
glycoproteins/glycolipids expressed on the cell surface via click
chemistry. Non-exhaustive examples of non-covalent interactions
with cell membrane elements include lipid modified oligos,
biocompatible anchor for cell membrane (BAM, e.g., oleyl-PEG-NHS),
lipid modified positive neutral polymer, and antibody to membrane
proteins. A cell tag can be used in combination with an analyte
capture agent and cleavable or non-cleavable spatially-barcoded
capture probes for spatial and multiplexing applications.
[1206] In some embodiments, a plurality of molecules (e.g., a
plurality of lipid or nucleic acid molecules) having a plurality of
barcodes (e.g., a plurality of spatial barcodes) are introduced to
a biological sample (e.g., to a plurality of cells in a biological
sample) for use in spatial analysis, wherein the plurality of
molecules are introduced to the biological sample in an arrayed
format. In some embodiments, a plurality of molecules (e.g., a
plurality of lipid or nucleic acid molecules) having a plurality of
barcodes are provided on a substrate (e.g., any of the variety of
substrates described herein) in any of the variety of arrayed
formats described herein, and the biological sample is contacted
with the molecules on the substrate such that the molecules are
introduced to the biological sample. In some embodiments, the
molecules that are introduced to the biological sample are
cleavably attached to the substrate, and are cleaved from the
substrate and released to the biological sample when contacted with
the biological sample. In some embodiments, the molecules
introduced to the biological sample are covalently attached to the
substrate prior to cleavage. In some embodiments, the molecules
that are introduced to the biological sample are non-covalently
attached to the substrate (e.g., via hybridization), and are
released from the substrate to the biological sample when contacted
with the biological sample.
[1207] In some embodiments, a plurality of molecules (e.g., a
plurality of lipid or nucleic acid molecules) having a plurality of
barcodes (e.g., a plurality of spatial barcodes) are migrated or
transferred from a substrate to cells of a biological sample. In
some embodiments, migrating a plurality of molecules from a
substrate to cells of a biological sample includes applying a force
(e.g., mechanical, centrifugal, or electrophoretic) to the
substrate and/or the biological sample to facilitate migration of
the plurality of molecules from the substrate to the biological
sample.
[1208] In some embodiments of any of the spatial analysis methods
described herein, physical force is used to facilitate attachment
to or introduction of a molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode) into a biological sample
(e.g., a cell present in a biological sample). As used herein,
"physical force" refers to the use of a physical force to
counteract the cell membrane barrier in facilitating intracellular
delivery of molecules. Examples of physical force instruments and
methods that can be used in accordance with materials and methods
described herein include the use of a needle, ballistic DNA,
electroporation, sonoporation, photoporation, magnetofection,
hydroporation, and combinations thereof.
[1209] (i) Introducing a Cell-Tagging Agent to the Surface of a
Cell
[1210] In some embodiments, biological samples (e.g., cells in a
biological sample) can be labelled using cell-tagging agents where
the cell-tagging agents facilitate the introduction of the
molecules (e.g., nucleic acid molecules) having barcodes (e.g.,
spatial barcodes) into the biological sample (e.g., into cells in a
biological sample). As used herein, the term "cell-tagging agent"
refers to a molecule having a moiety that is capable of attaching
to the surface of a cell (e.g., thus attaching the barcode to the
surface of the cell) and/or penetrating and passing through the
cell membrane (e.g., thus introducing the barcode to the interior
of the cell). In some embodiments, a cell-tagging agent includes a
barcode (e.g., a spatial barcode). The barcode of a barcoded
cell-tagging agent can be any of the variety of barcodes described
herein. In some embodiments, the barcode of a barcoded cell-tagging
agent is a spatial barcode. In some embodiments, a cell-tagging
agent comprises a nucleic acid molecule that includes the barcode
(e.g., the spatial barcode). In some embodiments, the barcode of a
barcoded cell-tagging agent identifies the associated molecule,
where each barcode is associated with a particular molecule. In
some embodiments, one or more molecules are applied to a sample. In
some embodiments, a nucleic acid molecule that includes the barcode
is covalently attached to the cell-tagging agent. In some
embodiments, a nucleic acid molecule that includes the barcode is
non-covalently attached to the cell-tagging agent. A non-limiting
example of non-covalent attachment includes hybridizing the nucleic
acid molecule that includes the barcode to a nucleic acid molecule
on the cell-tagging agent (which nucleic acid molecule on the
cell-tagging agent can be bound to the cell-tagging agent
covalently or non-covalently). In some embodiments, a nucleic acid
molecule attached to a cell-tagging agent that includes a barcode
(e.g., a spatial barcode) also includes one or more additional
domains. Such additional domains include, without limitation, a PCR
handle, a sequencing priming site, a domain for hybridizing to
another nucleic acid molecule, and combinations thereof.
[1211] In some embodiments, a cell-tagging agent attaches to the
surface of a cell. When the cell-tagging agent includes a barcode
(e.g., a nucleic acid that includes a spatial barcode), the barcode
is also attached to the surface of the cell. In some embodiments of
any of the spatial analysis methods described herein, a
cell-tagging agent attaches covalently to the cell surface to
facilitate introduction of the spatial analysis reagents. In some
embodiments of any of the spatial analysis methods described
herein, a cell-tagging agent attaches non-covalently to the cell
surface to facilitate introduction of the spatial analysis
reagents.
[1212] In some embodiments, once a cell or cells in a biological
sample is spatially tagged with a cell-tagging agent(s), spatial
analysis of analytes present in the biological sample is performed.
In some embodiments, such spatial analysis includes dissociating
the spatially-tagged cells of the biological sample (or a subset of
the spatially-tagged cells of the biological sample) and analyzing
analytes present in those cells on a cell-by-cell basis. Any of a
variety of methods for analyzing analytes present in cells on a
cell-by-cell basis can be used. Non-limiting examples include any
of the variety of methods described herein and methods described in
PCT Application Publication No. WO 2019/113533A1, the content of
which is incorporated herein by reference in its entirety. For
example, the spatially-tagged cells can be encapsulated with beads
comprising one or more nucleic acid molecules having a barcode
(e.g., a cellular barcode) (e.g., an emulsion). The nucleic acid
present on the bead can have a domain that hybridizes to a domain
on a nucleic acid present on the tagged cell (e.g., a domain on a
nucleic acid that is attached to a cell-tagging agent), thus
linking the spatial barcode of the cell to the cellular barcode of
the bead. Once the spatial barcode of the cell and the cellular
barcode of the bead are linked, analytes present in the cell can be
analyzed using capture probes (e.g., capture probes present on the
bead). This allows the nucleic acids produced (using these methods)
from specific cells to be amplified and sequenced separately (e.g.,
within separate partitions or droplets).
[1213] In some embodiments, once a cell or cells in a biological
sample is spatially tagged with a cell-tagging agent(s), spatial
analysis of analytes present in the biological sample is performed
in which the cells of the biological sample are not dissociated
into single cells. In such embodiments, various methods of spatial
analysis such as any of those provided herein can be employed. For
example, once a cell or cells in a biological sample is spatially
tagged with a cell-tagging agent(s), analytes in the cells can be
captured and assayed. In some embodiments, cell-tagging agents
include both a spatial barcode and a capture domain that can be
used to capture analytes present in a cell. For example,
cell-tagging agents that include both a spatial barcode and a
capture domain can be introduced to cells of the biological sample
in a way such that locations of the cell-tagging agents are known
(or can be determined after introducing them to the cells). One
non-limiting example of introducing cell-tagging agents to a
biological sample is to provide the cell-tagging agents in an
arrayed format (e.g., arrayed on a substrate such as any of the
variety of substrates and arrays provided herein), where the
positions of the cell-tagging agents on the array are known at the
time of introduction (or can be determined after introduction). The
cells can be permeabilized as necessary (e.g., using
permeabilization agents and methods described herein), reagents for
analyte analysis can be provided to the cells (e.g., a reverse
transcriptase, a polymerase, nucleotides, etc., in the case where
the analyte is a nucleic acid that binds to the capture probe), and
the analytes can be assayed. In some embodiments, the assayed
analytes (and/or copies thereof) can be released from the substrate
and analyzed. In some embodiments, the assayed analytes (and/or
copies thereof) are assayed in situ.
[1214] Non-limiting examples of cell-tagging agents and systems
that attach to the surface of a cell (e.g., thus introducing the
cell-tagging agent and any barcode attached thereto to the exterior
of the cell) that can be used in accordance with materials and
methods provided herein for spatially analyzing an analyte or
analytes in a biological sample include: lipid tagged
primers/lipophilic-tagged moieties, positive or neutral
oligo-conjugated polymers, antibody-tagged primers,
streptavidin-conjugated oligonucleotides, dye-tagged
oligonucleotides, click-chemistry, receptor-ligand systems,
covalent binding systems via amine or thiol functionalities, and
combinations thereof.
[1215] (ii) Introducing a Cell-Tagging Agent to the Interior of a
Cell
[1216] Non-limiting examples of cell-tagging agents and systems
that penetrate and/or pass through the cell membrane (e.g., thus
introducing the cell-tagging agent and any barcode attached thereto
to the interior of the cell) that can be used in accordance with
materials and methods provided herein for spatially profiling an
analyte or analytes in a biological sample include: a
cell-penetrating agent (e.g., a cell-penetrating peptide), a
nanoparticle, a liposome, a polymersome, a peptide-based chemical
vector, electroporation, sonoporation, lentiviral vectors,
retroviral vectors, and combinations thereof.
[1217] FIG. 19 is a schematic showing an exemplary cell tagging
method. Non-exhaustive examples of oligo delivery vehicles may
include a cell penetrating peptide or a nanoparticle.
Non-exhaustive examples of the delivery systems can include
lipid-based polymeric and metallic nanoparticles or oligos that can
be conjugated or encapsulated within the delivery system. The cell
tag can be used in combination with a capture agent barcode domain
and a cleavable or non-cleavable spatially-barcoded capture probes
for spatial and multiplexing applications.
[1218] 1. Cell-Penetrating Agent
[1219] In some embodiments of any of the spatial profiling methods
described herein, identification of a biological analyte by a
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) and a capture domain is facilitated by a
cell-penetrating agent. In some embodiments, a molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
and a capture domain is coupled to a cell-penetrating agent, and
the cell-penetrating agent allows the molecule to interact with an
analyte inside the cell. A "cell-penetrating agent" as used herein
can refer to an agent capable of facilitating the introduction of a
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) and a capture domain into a cell of a biological
sample (see, e.g., Lovatt et al. Nat Methods. 2014 February;
11(2):190-6, which is incorporated herein by reference in its
entirety). In some embodiments, a cell-penetrating agent is a
cell-penetrating peptide. A "cell-penetrating peptide" as used
herein refers to a peptide (e.g., a short peptide, e.g., a peptide
not usually exceeding 30 residues) that has the capacity to cross
cellular membranes. In some embodiments, cell-penetrating agents or
cell penetrating peptides may be covalently or non-covalently
coupled to a molecule (e.g., a barcoded nucleic acid molecule),
likely at the 5' end of the molecule. A cell-penetrating peptide
may direct the barcoded nucleic acid molecule to a specific
organelle.
[1220] In some embodiments of any of the spatial profiling methods
described herein, a cell-penetrating peptide coupled to a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain can cross a cellular membrane using
an energy dependent or an energy independent mechanism. For
example, a cell-penetrating peptide can cross a cellular membrane
through direct translocation through physical perturbation of the
plasma membrane, endocytosis (e.g., mediated via clathrin),
adaptive translocation, pore-formation, electroporation-like
permeabilization, and/or entry at microdomain boundaries.
Non-limiting examples of a cell-penetrating peptide include:
penetratin, tat peptide, pVEC, transportan, MPG, Pep-1, a
polyarginine peptide, MAP, R6W3, (D-Arg)9, Cys(Npys)-(D-Arg)9,
Anti-BetaGamma (MPS--Phosducin-like protein C terminus), Cys(Npys)
antennapedia, Cys(Npys)-(Arg)9, Cys(Npys)-TAT (47-57), HIV-1 Tat
(48-60), KALA, mastoparan, penetratin-Arg, pep-1-cysteamine,
TAT(47-57)GGG-Cys(Npys), Tat-NR2Bct, transdermal peptide, SynB1,
SynB3, PTD-4, PTD-5, FHV Coat-(35-49), BMV Gag-(7-25), HTLV-II
Rex-(4-16), R9-tat, SBP, FBP, MPG, MPG(.DELTA.NLS), Pep-2, MTS,
plsl, and a polylysine peptide (see, e.g., Bechara et al. FEBS
Lett. 2013 Jun. 19; 587(12):1693-702, which is incorporated by
reference herein in its entirety).
[1221] In some embodiments, there could be two orientations for
cell-penetrating peptide (CPP) conjugation. For example, one
orientation can be
(N-terminus)-CPP-Cys-(C-terminus)-linker-NH2C6-5'-oligo-3';
3'-oligo-5'-NH2C6-linker-(N-terminus)-Cys-CPP-(C-terminus). The
methods herein can be performed with other CPP conjugations and
orientations.
[1222] 2. Nanoparticles
[1223] In some embodiments of any of the spatial profiling methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by an inorganic
particle (e.g., a nanoparticle). In some embodiments, a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is coupled to an inorganic particle
(e.g., a nanoparticle), and the molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) and a capture
domain uses the nanoparticle to get access to analytes inside the
cell. Non-limiting examples of nanoparticles that can be used in
embodiments herein to deliver a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) and a capture
domain into a cell and/or cell bead include inorganic nanoparticles
prepared from metals, (e.g., iron, gold, and silver), inorganic
salts, and ceramics (e.g., phosphate or carbonate salts of calcium,
magnesium, or silicon). The surface of a nanoparticle can be coated
to facilitate binding of the molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) and a capture
domain, or the surface can be chemically modified to facilitate
attachment of the molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) and a capture domain. Magnetic
nanoparticles (e.g., supermagnetic iron oxide), fullerenes (e.g.,
soluble carbon molecules), carbon nanotubes (e.g., cylindrical
fullerenes), quantum dots, and supramolecular systems can also be
used.
[1224] 3. Liposomes
[1225] In some embodiments of any of the spatial analysis methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by a liposome. Various
types of lipids, including cationic lipids, can be used in liposome
delivery. In some cases, a molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode) and a capture domain is
delivered to a cell via a lipid nano-emulsion. A lipid emulsion
refers to a dispersion of one immiscible liquid in another
stabilized by emulsifying agent. Labeling cells can comprise use of
a solid lipid nanoparticle.
[1226] 4. Polymersomes
[1227] In some embodiments of any of the spatial analysis methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by a polymersome. In
some embodiments, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) and a capture domain is
contained in the polymersome, and the molecule (e.g., a nucleic
acid molecule) having a barcode (e.g., a spatial barcode) and a
capture domain uses the polymersome to get access to analytes
inside the cell. A "polymersome" as referred to herein is an
artificial vesicle. For example, a polymersome can be a vesicle
similar to a liposome, but the membrane comprises amphiphilic
synthetic block copolymers (see, e.g., Rideau et al. Chem. Soc.
Rev., 2018, 47, 8572-8610, which is incorporated by reference
herein in its entirety). In some embodiments, polymersomes comprise
di-(AB) or tri-block copolymers (e.g., ABA or ABC), where A and C
are a hydrophilic block and B is a hydrophobic block. In some
embodiments, a polymersome comprises
poly(butadiene)-b-poly(ethylene oxide), poly(ethyl
ethylene)-b-poly(ethylene oxide), polystyrene-b-poly(ethylene
oxide), poly(2-vinylpyridine)-b-poly(ethylene oxide),
polydimethylsiloxane-b-poly(ethylene oxide),
polydimethylsiloxane-g-poly(ethylene oxide),
polycaprolactone-b-poly(ethylene oxide),
polyisobutylene-b-poly(ethylene oxide), polystyrene-b-polyacrylic
acid, polydimethylsiloxane-b-poly-2-methyl-2-oxazoline, or a
combination thereof (wherein b=block and g=grafted).
[1228] 5. Peptide-Based Chemical Vectors
[1229] In some embodiments of any of the spatial analysis methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by a peptide-based
chemical vector, e.g., a cationic peptide-based chemical vector.
Cationic peptides can be rich in basic residues like lysine and/or
arginine. In some embodiments of any of the spatial analysis
methods described herein, capture of a biological analyte by a
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) and a capture domain is facilitated by a
polymer-based chemical vector. Cationic polymers, when mixed with a
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) and a capture domain, can form nanosized complexes
called polyplexes. Polymer based vectors can comprise natural
proteins, peptides and/or polysaccharides. Polymer based vectors
can comprise synthetic polymers. In some embodiments, a
polymer-based vector comprises polyethylenimine (PEI). PEI can
condense DNA into positively-charged particles, which bind to
anionic cell surface residues and are brought into the cell via
endocytosis. In some embodiments, a polymer-based chemical vector
comprises poly(L)-lysine (PLL), poly (DL-lactic acid) (PLA), poly
(DL-lactide-co-glycoside) (PLGA), polyornithine, polyarginine,
histones, protamines, or a combination thereof. Polymer-based
vectors can comprise a mixture of polymers, for example, PEG and
PLL. Other non-limiting examples of polymers include dendrimers,
chitosans, synthetic amino derivatives of dextran, and cationic
acrylic polymers.
[1230] 6. Electroporation
[1231] In some embodiments of any of the spatial analysis methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by electroporation.
With electroporation, a biological analyte by a molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
and a capture domain can enter a cell through one or more pores in
the cellular membrane formed by applied electricity. The pore of
the membrane can be reversible based on the applied field strength
and pulse duration.
[1232] 7. Sonoporation
[1233] In some embodiments of any of the spatial analysis methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by sonoporation. Cell
membranes can be temporarily permeabilized using sound waves,
allowing cellular uptake of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain.
[1234] 8. Lentiviral Vectors and Retroviral Vectors
[1235] In some embodiments of any of the spatial analysis methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by vectors. For
example, a vector as described herein can be an expression vector
where the expression vector includes a promoter sequence operably
linked to the sequence encoding the molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) and a capture
domain. Non-limiting examples of vectors include plasmids,
transposons, cosmids, and viral vectors (e.g., any adenoviral
vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV)
vectors, lentivirus vectors, and retroviral vectors), and any
Gateway.RTM. vectors. A vector can, for example, include sufficient
cis-acting elements for expression where other elements for
expression can be supplied by the host mammalian cell or in an in
vitro expression system. Skilled practitioners will be capable of
selecting suitable vectors and mammalian cells for introducing any
of spatial profiling reagents described herein.
[1236] 9. Other Methods and Cell-Tagging Agents for Intracellular
Introduction of a Molecule
[1237] In some embodiments of any of the spatial analysis methods
described herein, capture of a biological analyte by a molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) and a capture domain is facilitated by the use of a
needle, for example for injection (e.g., microinjection), particle
bombardment, photoporation, magnetofection, and/or hydroporation.
For example, with particle bombardment, a molecule (e.g., a nucleic
acid molecule) having a barcode (e.g., a spatial barcode) and a
capture domain can be coated with heavy metal particles and
delivered to a cell at a high speed. In photoporation, a transient
pore in a cell membrane can be generated using a laser pulse,
allowing cellular uptake of a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) and a capture
domain. In magnetofection, a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) and a capture
domain can be coupled to a magnetic particle (e.g., magnetic
nanoparticle, nanowires, etc.) and localized to a target cell via
an applied magnetic field. In hydroporation, a molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
and a capture domain can be delivered to a cell and/or cell bead
via hydrodynamic pressure.
[1238] (iii) Lipid Tagged Primers/Lipophilic-Tagged Moieties
[1239] In some embodiments of any of the spatial profiling methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) is coupled to a lipophilic
molecule. In some embodiments, the lipophilic molecule enables
delivery of the lipophilic molecule to the cell membrane or the
nuclear membrane. In some embodiments, a molecule (e.g., a nucleic
acid molecule) having a barcode (e.g., a spatial barcode) coupled
to a lipophilic molecule can associate with and/or insert into
lipid membranes such as cell membranes and nuclear membranes. In
some cases, the insertion can be reversible. In some cases, the
association between the lipophilic molecule and the cell may be
such that the cell retains the lipophilic molecule (e.g., and
associated components, such as nucleic acid barcode molecules)
during subsequent processing (e.g., partitioning, cell
permeabilization, amplification, pooling, etc.). In some
embodiments, a molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) coupled to a lipophilic molecule
may enter into the intracellular space and/or a cell nucleus.
[1240] Non-limiting examples of lipophilic molecules that can be
used in embodiments described herein include sterol lipids such as
cholesterol, tocopherol, steryl, palmitate, lignoceric acid, and
derivatives thereof. In some embodiments, the lipophilic molecules
are neutral lipids that are conjugated to hydrophobic moieties
(e.g., cholesterol, squalene, or fatty acids) (See Raouane et al.
Bioconjugate Chem., 23(6):1091-1104 (2012) which is herein
incorporated by reference in its entirety). In some embodiments, a
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) may be attached to the lipophilic moiety via a
linker, using covalent or direct attachment. In some embodiments,
the linker is a tetra-ethylene glycol (TEG) linker. Other exemplary
linkers include, but are not limited to, Amino Linker C6, Amino
Linker C12, Spacer C3, Spacer C6, Spacer C12, Spacer 9, and Spacer
18. In some embodiments, a molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode) is indirectly coupled
(e.g., via hybridization or ligand-ligand interactions, such as
biotin-streptavidin) to a lipophilic molecule. In some embodiments,
the lipophilic moiety can be attached to a capture probe, spatial
barcode, or other DNA sequence, at either the 5' or 3' end of the
specified DNA sequence. In some embodiments, the lipophilic moiety
can be coupled to a capture probe, spatial barcode, or other DNA
sequence in a lipid-dependent manner. Other lipophilic molecules
that may be used in accordance with methods provided herein include
amphiphilic molecules wherein the headgroup (e.g., charge,
aliphatic content, and/or aromatic content) and/or fatty acid chain
length (e.g., C12, C14, C16, or C18) can be varied. For instance,
fatty acid side chains (e.g., C12, C14, C16, or C18) can be coupled
to glycerol or glycerol derivatives (e.g.,
3-t-butyldiphenylsilylglycerol), which can also comprise, e.g., a
cationic head group. In some embodiments, a molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
disclosed herein can then be coupled (either directly or
indirectly) to these amphiphilic molecules. In some embodiments, a
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) coupled to an amphiphilic molecule may associate
with and/or insert into a membrane (e.g., a cell, cell bead, or
nuclear membrane). In some cases, an amphiphilic or lipophilic
moiety may cross a cell membrane and provide a molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
to an internal region of a cell and/or cell bead.
[1241] In some embodiments, additives can be added to supplement
lipid-based modifications. In some embodiments, the additive is low
density lipoprotein (LDL). In some embodiments, the additive is the
cholesterol trafficking inhibitor U-18666A. In some embodiments,
U-18666A inhibits cholesterol transport from late endosomes at
micromolar concentrations and/or lysosomes to the endoplasmic
reticulum (ER) at nanomolar concentrations. In some embodiments,
U-18666A can inhibit oxidosqualene cyclase, a key enzyme in the
cholesterol biosynthesis pathway, at sufficiently high
concentrations (e.g., at or about >0.5 mM).
[1242] In some embodiments, wherein the molecule (e.g., with a
nucleic acid sequence) has an amino group within the molecule, the
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) and an amino group can be coupled to an
amine-reactive lipophilic molecule. For example, a molecule (e.g.,
a nucleic acid molecule) having a barcode (e.g., a spatial barcode)
and an amino group can be conjugated to DSPE-PEG(2000)-cyanuric
chloride
(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene
glycol)-2000]).
[1243] In some embodiments, a cell-tagging agent can attach to a
surface of a cell through a combination of lipophilic and covalent
attachment. For example, a cell-tagging agent can include an
oligonucleotide attached to a lipid to target the oligonucleotide
to a cell membrane, and an amine group that can be covalently
linked to a cell surface protein(s) via any number of chemistries
described herein. In these embodiments, the lipid can increase the
surface concentration of the oligonucleotide and can promote the
covalent reaction.
[1244] As used herein, an "anchor oligonucleotide" and/or
"co-anchor oligonucleotide" can include a lipid-conjugated
oligonucleotide, wherein the lipid is capable of becoming embedded
within a cell membrane. In some embodiments, the lipid capable of
becoming embedded within a cell membrane includes but is not
limited to, sterol lipids such as cholesterol, tocopherol, steryl,
palmitate, lignoceric acid, and derivatives thereof. In some
embodiments, the sterol lipid of the anchor oligonucleotide and/or
co-anchor oligonucleotide can be attached to either the 5' or 3'
end of the oligonucleotide portion. In some embodiments, the anchor
oligonucleotide and/or the co-anchor oligonucleotide can integrate
into the cell membrane of a cell in a biological sample (e.g., the
sterol lipid of the anchor oligonucleotide and/or co-anchor
oligonucleotide).
[1245] In some embodiments, a sterol lipid (e.g., lignoceric acid)
anchor oligonucleotide is attached to the 5' end of the
oligonucleotide. In some embodiments, the anchor oligonucleotide
can have a constant sequence. In some embodiments the constant
sequence of the anchor oligonucleotide can be about 15 to about 30
nucleotides long. In some embodiments, the anchor oligonucleotide
can have an additional domain 3' to the constant sequence. In some
embodiments, the additional domain can be an adapter sequence
(e.g., sequencing adapter). In some embodiments, the adapter
sequence can be about 15 to about 35 nucleotides long.
[1246] In some embodiments, the lipid (e.g., sterol lipid) of the
co-anchor oligonucleotide (e.g., palmitic acid), is attached to the
3' end of the oligonucleotide. In some embodiments, the co-anchor
oligonucleotide can have a constant sequence. For example, the
constant sequence of the co-anchor oligonucleotide can be a reverse
complement of the constant sequence from the anchor
oligonucleotide. In some embodiments, the constant sequence of the
anchor oligonucleotide and the constant sequence of the co-anchor
oligonucleotide can bind (e.g., hybridize) to each other. In some
embodiments, the lipid (e.g., sterol lipid) of the anchor
oligonucleotide and the co-anchor oligonucleotide can integrate
into a cell membrane in the biological sample and the respective
constant sequences can hybridize to each other at the same time. In
some embodiments, a barcoded oligonucleotide, which can include
several domains, can be introduced to the integrated anchor
oligonucleotide and co-anchor oligonucleotide hybridized to each
other. The barcoded oligonucleotide can include, in a 5' to 3'
direction, a functional domain (e.g., a sequencing adapter domain),
a unique molecular identifier, a sample barcode, a second unique
molecular identifier, and the reverse complement of a constant
sequence. For example, after tagging a cell with any of the
cell-tagging agents described herein the cells can be partitioned
(e.g., encapsulated in a vesicle) with a barcoded feature (e.g., a
bead). In some embodiments, the reverse complement of the constant
sequence of the barcoded oligonucleotide can interact (e.g.,
hybridize) with the constant sequence (e.g., a portion of the
sequence) on the barcoded feature.
[1247] (iv) Intracellular Cleavage Domain
[1248] As used herein, capture probes can optionally include an
"intracellular cleavage domain," wherein one or more segments or
regions of the capture probe (e.g., capture domains, spatial
barcodes, and/or UMIs) can be releasably or cleavably attached to
one or more other segments or regions of the capture probe, such as
a cell-penetrating agent, such that the capture domain, spatial
barcode, and/or UMI can be released or be releasable through
cleavage of a linkage between the capture domain, spatial barcode,
and/or UMI and the cell-penetrating agent and/or cell penetration
tag. In some embodiments, the cleavage of the linkage between the
capture domain, spatial barcode, and/or UMI and the
cell-penetrating agent is induced in an intracellular environment
(e.g., the intracellular cleavage domain is cleaved after the
capture probes is introduced into the cell). For example, the
linkage between the capture domain, spatial barcode, and/or UMI and
the cell-penetrating agent can be a disulfide bond that is cleaved
by the reducing conditions in the cell, for example, when the
intracellular cleavage domain comprises a disulfide bond. Any other
suitable linker can be used to release or cleave the intracellular
cleavage domain of the capture probe.
[1249] (v) Positive or Neutral Oligo-Conjugated Polymers
[1250] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can be coupled to a glycol
chitosan derivative. In some embodiments, the glycol chitosan
derivative can be coupled with two or more molecules (e.g., nucleic
acid molecules) having a barcode (e.g., a spatial barcode). In some
embodiments, the glycol chitosan derivative can be coupled with
about 3, about 4, about 5, about 6, about 7, about 8, about 9,
about 10 or more molecules. The glycol chitosan derivative (e.g.,
glycol chitosan-cholesterol) can serve as a hydrophobic anchor (see
Wang et al. J. Mater. Chem. B., 30:6165 (2015), which is herein
incorporated by reference in its entirety). Non-limiting examples
of chitosan derivatives that can be coupled to a molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
can be found in Cheung et al., Marine Drugs, 13(8): 5156-5186
(2015), which is herein incorporated by reference in its
entirety.
[1251] (vi) Bifunctional NHS Linker Cell-Tagging
[1252] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can be coupled to a
bifunctional NHS linker. In some embodiments, the coupled
bifunctional NHS linker (e.g., bifunctional linker and the molecule
having a barcode) can facilitate the attachment of the spatial
barcode to the surface of the cell. In some embodiments, after
facilitating attachment to the surface of the cell, excess NHS
linker can be removed (e.g., washed away). In some embodiments, the
process of coupling the molecule having a barcode can be performed
under non-anhydrous conditions to maintain the activity of
unreacted bifunctional NHS. In some embodiments, the non-anhydrous
condition can be in the presence of DMSO. In some embodiments, the
non-anhydrous condition can be in the presence of DMF.
[1253] (vii) Antibody-Tagged Primers
[1254] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can be coupled to an antibody
or antigen binding fragment thereof in a manner that facilitates
attachment of the molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) to the surface of a cell. In some
embodiments, facilitating attachment to the cell surface
facilitates introduction of the molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) into the cell.
In some embodiments, the molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode) can be coupled to an
antibody that is directed to an antigen that is present on the
surface of a cell. In some embodiments, the molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
can be coupled to an antibody that is directed to an antigen that
is present on the surface of a plurality of cells (e.g., a
plurality of cells in a biological sample). In some embodiments,
the molecule (e.g., a nucleic acid molecule) having a barcode
(e.g., a spatial barcode) can be coupled to an antibody that is
directed to an antigen that is present on the surface of a cell, a
plurality of cells, or substantially all the cells present in a
biological sample. In some embodiments, the barcoded-antibody is
directed to an intracellular antigen. Any of the exemplary methods
described herein of attaching a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) to another
molecule (e.g., an antibody or antigen fragment thereof) can be
used.
[1255] (viii) Streptavidin-Conjugated Oligonucleotides
[1256] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can attach to the surface of a
cell using biotin-streptavidin. In some embodiments, primary amines
in the side chain of lysine residues of cell surface polypeptides
are labelled with NHS-activated biotin reagents. For example, the
N-terminus of a polypeptide can react with NHS-activated biotin
reagents to form stable amide bonds. In some embodiments,
cell-tagging agents include molecules (e.g., a nucleic acid
molecule) having barcodes (e.g., a spatial barcode) conjugated to
streptavidin. In some cases, streptavidin can be conjugated to the
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode) using click chemistry (e.g., maleimide
modification) as described herein. In some embodiments, a cell
containing NHS-activated biotin incorporated into lysine side
chains of a cell surface protein forms a non-covalent bond with the
streptavidin conjugated to the molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode). In some
embodiments, the molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) conjugated to streptavidin is
itself part of a cell-tagging agent.
[1257] (ix) Dye-Tagged Oligonucleotides
[1258] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) is directly linked to a
detectable label. In some embodiments, the detectable label is any
of the detectable labels described herein. In some embodiments the
detectable label is a fluorescent tag. In some embodiments, the
physical properties of the fluorescent tags (e.g., a fluorescent
tag having hydrophobic properties) can overcome the hydrophilic
nature of the molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode). For example, in some
embodiments, wherein the molecule is a nucleic acid molecule, a
fluorescent tag (e.g., BODIPY, Cy3, Atto 647N, and Rhodamine Red
C2) can be coupled to a 5' end of the nucleic acid molecule having
a barcode (e.g., a spatial barcode). In some embodiments, wherein
the molecule is a nucleic acid molecule, any fluorescent tag having
hydrophobic properties can be coupled to the nucleic acid molecule
having a barcode (e.g., a spatial barcode) in a manner that
overcomes the hydrophilic nature of the nucleic acid molecule.
Non-limiting examples of fluorescent tags with hydrophobic
properties include BODIPY, Cy3, Atto 647N, and Rhodamine Red
C2.
[1259] (x) Click-Chemistry
[1260] In some embodiments of any of the spatial analysis methods
described herein, molecules (e.g., a nucleic acid molecule) having
barcodes (e.g., a spatial barcode) are coupled to click-chemistry
moieties. As used herein, the term "click chemistry," generally
refers to reactions that are modular, wide in scope, give high
yields, generate byproducts, such as those that can be removed by
nonchromatographic methods, and are stereospecific (but not
necessarily enantioselective) (see, e.g., Angew. Chem. Int. Ed.,
2001, 40(11):2004-2021, which is incorporated herein by reference
in its entirety). In some cases, click chemistry can describe pairs
of functional groups that can selectively react with each other in
mild, aqueous conditions.
[1261] An example of a click chemistry reaction is the Huisgen
1,3-dipolar cycloaddition of an azide and an alkyne, i.e.,
copper-catalyzed reaction of an azide with an alkyne to form the
5-membered heteroatom ring 1,2,3-triazole. The reaction is also
known as a Cu(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC), a
Cu(I) click chemistry or a Cu+ click chemistry. Catalysts for the
click chemistry include, but are not limited to, Cu(I) salts, or
Cu(I) salts made in situ by reducing Cu(II) reagents to Cu(I)
reagents with a reducing reagent (Pharm Res. 2008, 25(10):
2216-2230, which is incorporated herein by reference in its
entirety). Known Cu(II) reagents for the click chemistry can
include, but are not limited to, the Cu(II)-(TBTA) complex and the
Cu(II) (THPTA) complex. TBTA, which is
tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known as
tris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for
Cu(I) salts. THPTA, which is
tris-(hydroxypropyltriazolylmethyl)amine, is another example of a
stabilizing agent for Cu(I). Other conditions can also be used to
construct the 1,2,3-triazole ring from an azide and an alkyne using
copper-free click chemistry, such as the Strain-promoted
Azide-Alkyne Click chemistry reaction (SPAAC) (see, e.g., Chem.
Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90,
each of which is incorporated herein by reference in its
entirety).
[1262] (xi) Receptor-Ligand Systems
[1263] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can be coupled to a ligand,
wherein the ligand is part of a receptor-ligand interaction on the
surface of a cell. For example, a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) can be coupled
to a ligand that interacts selectively with a cell surface receptor
thereby targeting the molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode) to a specific cell.
Non-limiting examples of receptor-ligand systems that can be used
include integrin receptor-ligand interactions, GPCR receptor-ligand
interactions, RTK receptor-ligand interactions, and TLR-ligand
interactions (see Juliano, Nucleic Acids Res., 44(14): 6518-6548
(2016), which is incorporated herein by reference in its entirety).
Any of the methods described herein for attaching a molecule (e.g.,
a nucleic acid molecule) having a barcode (e.g., a spatial barcode)
to a ligand (e.g., any of the methods described herein relating to
attaching a molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) to an antibody) can be used.
[1264] (xii) Covalent Binding Systems via Amine or Thiol
Functionalities
[1265] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can incorporate reactive
functional groups at sites within the molecule (e.g., with a
nucleic acid sequence). In such cases, the reactive functional
groups can facilitate conjugation to ligands and/or surfaces. In
some embodiments, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can include thiol modifiers
that are designed to react with a broad array of activated
accepting groups (e.g., maleimide and gold microspheres). For
example, a molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) having thiol modifiers can
interact with a maleimide-conjugated peptide thereby resulting in
labelling of the peptide. In some embodiments, maleimide-conjugated
peptides are present on the surface of a cell whereupon interaction
with the thiol-modified molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode), the molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
is coupled to the surface of the cell. Non-limiting examples of
thiol modifiers include: 5' thiol modifier C6 S--S, 3' thiol
modifier C3 S--S, dithiol, 3'thiol modifier oxa 6-S--S, and dithiol
serinol.
[1266] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can include amine modifiers,
e.g., amine modifiers that are designed to attach to another
molecule in the presence of an acylating agent. In some
embodiments, a molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) can include amine modifiers that
are designed to attach to a broad array of linkage groups (e.g.,
carbonyl amide, thiourea, sulfonamide, and carboxamide). For
example, a molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) and an amine modifier can
interact with a sulfonamide-conjugated peptide thereby resulting in
labelling of the peptide. In some embodiments,
sulfonamide-conjugated peptides are present on the surface of a
cell whereupon interaction with the amine-modified molecule (e.g.,
a nucleic acid molecule) having a barcode (e.g., a spatial
barcode), the molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) is coupled to the surface of the
cell. Non-limiting example of amine modifiers include:
DMS(O)MT-Amino-Modifier-C6, Amino-Modifier-C3-TFA,
Amino-Modifier-C12, Amino-Modifier-C6-TFA, Amino-dT,
Amino-Modifier-5, Amino-Modifier-C2-dT, Amino-Modifier-C6-dT, and
3'-Amino-Modifier-C7.
[1267] As another example, a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) can
incorporate reactive functional groups at sites within the molecule
(e.g., with a nucleic acid sequence) such as N-hydroxysuccinimide
(NHS). In some embodiments, amines (e.g., amine-containing
peptides) are present on the surface of a cell whereupon
interaction with the NHS-modified molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode), the molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode) is coupled to the surface of the cell. In some
embodiments, a molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) is reacted with a bifunctional
NHS linker to form an NHS-modified molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode).
[1268] In some embodiments, a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) can be coupled
to a biocompatible anchor for cell membrane (BAM). For example, a
BAM can include molecules that comprise an oleyl group and PEG. The
oleyl group can facilitate anchoring the molecule (e.g., a nucleic
acid molecule) having a barcode (e.g., a spatial barcode) to a
cell, and the PEG can increase water solubility. In some
embodiments, oleyl-PEG-NHS can be coupled to a molecule (e.g., a
nucleic acid molecule) having a barcode (e.g., a spatial barcode)
using NHS chemistry.
[1269] (xiii) Azide-Based Systems
[1270] In some embodiments, wherein the molecule (e.g., with a
nucleic acid sequence) incorporates reactive functional groups at
sites within the molecule, a molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) can be coupled
to an azide group on a cell surface. In some embodiments, the
reactive functional group is an alkynyl group. In some embodiments,
click chemistry as described herein can be used to attach the
alkynyl-modified molecule (e.g., a nucleic acid molecule) having a
barcode (e.g., a spatial barcode) to an azide group on the cell
surface. An azide group can be attached to the cell surface through
a variety of methods. For example, NHS chemistry can be used to
attach an azide group to the cell surface. In some embodiments,
N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), which contains
an azide group, can react with sialic acid on the surface of a cell
to attach azide to the cell surface. In some embodiments, azide is
attached to the cell surface by bio-orthogonal expression of azide.
For example, azide is incubated with the cells. In some
embodiments, the alkynyl-modified molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) can attach to
the surface of a cell via an azide group in the presence of copper.
In some embodiments, the alkynyl-modified molecule (e.g., a nucleic
acid molecule) having a barcode (e.g., a spatial barcode) can
attach to the surface of a cell via an azide group in the absence
of copper.
[1271] (xiv) Lectin-Based Systems
[1272] In some embodiments of any of the spatial analysis methods
described herein, a molecule (e.g., a nucleic acid molecule) having
a barcode (e.g., a spatial barcode) can be coupled to a lectin that
facilitates attachment of the molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) to a cell
surface. Lectin can bind to glycans, e.g., glycans on the surface
of cells. In some embodiments, the molecule (e.g., a nucleic acid
molecule) having a barcode (e.g., a spatial barcode) has an
incorporated reactive functional group such as an azide group. In
some embodiments, the molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode) and an azide group is
reacted with a modified lectin, e.g., a lectin modified using NHS
chemistry to introduce an azide reactive group. In some
embodiments, a live cell is labelled with a lectin-modified
molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a
spatial barcode). In some embodiments, a fixed cell is labelled
with a lectin-modified molecule (e.g., a nucleic acid molecule)
having a barcode (e.g., a spatial barcode). In some embodiments, a
permeabilized cell is labelled with a lectin-modified molecule
(e.g., a nucleic acid molecule) having a barcode (e.g., a spatial
barcode). In some embodiments, organelles in the secretory pathway
can be labelled with a lectin-modified molecule (e.g., a nucleic
acid molecule) having a barcode (e.g., a spatial barcode).
[1273] (b) Methods for Separating a Sample into Single Cells or
Cell Groups
[1274] Some embodiments of any of the methods described herein can
include separating a biological sample into single cells, cell
groups, types of cells, or a region or regions of interest. For
example, a biological sample can be separated into single cells,
cell groups, types of cells, or a region or regions of interest
before being contacted with one or more capture probes. In other
examples, a biological sample is first contacted with one or more
capture probes, and then separated into single cells, cell groups,
types of cells, or a region or regions of interest.
[1275] In some embodiments, a biological sample can be separated
into chucks using pixelation. Pixelation can include the steps of
providing a biological sample, and punching out one or more
portions of the biological sample. The punched out portions of the
biological sample can then be used to perform any of the methods
described herein. In some embodiments, the punched-out portions of
the biological sample can be in a random pattern or a designed
pattern. In some embodiments, the punched-out portions of the
biological sample can be focused on a region of interest or a
subcellular structure in the biological sample.
[1276] FIG. 20A is a workflow schematic illustrating exemplary,
non-limiting, non-exhaustive steps for "pixelating" a sample,
wherein the sample is cut, stamped, microdissected, or transferred
by hollow-needle or microneedle, moving a small portion of the
sample into an individual partition or well.
[1277] FIG. 20B is a schematic depicting multi-needle pixelation,
wherein an array of needles is punched through a sample on a
scaffold, into nanowells containing beads (e.g., gel beads) and
reagents. Once the needle is in the nanowell, the cell(s) are
ejected.
[1278] In some embodiments, a biological sample (e.g., a tissue
sample or tissue section) is divided into smaller portions as
compared to the original biological sample size ("chunks") before
performance of any of the spatial analysis methods described
herein. In some embodiments, the methods can include spatial
barcoding of FFPE "chunks" via barcodes applied in a spatially
well-defined pattern (e.g., array printing). In order to associate
a spatial barcode with a particular "chunk" of biological sample,
the barcode (e.g., a spatial barcode) can be of a sufficient length
to prevent diffusion of the barcode in subsequent steps, or the
spatial barcode can be covalently applied to the FFPE sample. In
some embodiments, the spatial barcode is unique to each FFPE chunk.
In some embodiments, spatial barcodes can be embedded onto an FFPE
slide (e.g., within a matrix, such as a wax or a hydrogel). In some
embodiments, the FFPE slide is heated (e.g., wax is heated) prior
to addition of the spatial barcodes. In some embodiments, after
addition of the spatial barcodes, the FFPE slide can be cooled and
cut or dissociated into chunks. Methods of chunking (e.g., cutting)
biological samples are known in the art. For example, in a
non-limiting example, chunking of biological samples can be done in
various ways such as laser microdissection, mechanical means,
acoustic (e.g., sonication) means, or any other method described
herein. In some embodiments, fluorophores/Qdots, etc. can be
embedded in the chunk to preserve spatial information about the
biological sample. Barcoding at this step enables massively
parallel encapsulation of chunks while retaining local spatial
information (e.g., tumor versus normal/healthy cells). In some
embodiments, chunking of a biological sample (e.g., a tissue
section) can result in single-cell chunks of the biological sample.
In other embodiments, chunking of a biological sample can be
performed to obtain chunks that correspond to diseased portions of
the biological sample. In another embodiment, chunking of
biological samples can be performed to obtain discrete chunks of
the biological sample that correspond to diseased or healthy
portions of the biological sample. In some embodiments, chunking of
biological samples can be performed to obtain chunks that
correspond to specific cell types (e.g., chunking based on
fluorescent or chemiluminescent imaging of antibodies bound to
target proteins) in the biological sample.
[1279] In some embodiments, the spatially-barcoded chunks can be
further processed. For example, the spatially-barcoded chunk can be
individually encapsulated (e.g., a matrix, emulsion, or hydrogel).
In some embodiments, the spatially-barcoded chunk can be
encapsulated in a partition (e.g., a well, droplet, channel, or
vesicle). In some embodiments, the spatially-barcoded chunk can be
encapsulated in a vesicle. In some embodiments, the vesicle can
comprise a lipid bilayer. In some embodiments, the
spatially-barcoded FFPE chunk can be encapsulated with a uniquely
barcoded bead. In some embodiments, the uniquely barcoded bead can
have a functional domain, a cleavage domain, a unique molecular
identifier, and a capture domain, or combinations thereof. In some
embodiments, the encapsulated spatially-barcoded FFPE chunk and the
uniquely barcoded bead can be heated to deparaffinize the FFPE
sample. In some embodiments, the encapsulated spatially-barcoded
FFPE chunk and the uniquely barcoded bead can be treated with
xylene to deparaffinize the FFPE sample. In some embodiments, the
deparaffinized sample can be treated to de-crosslink methylene
bridges in a single step. In some embodiments, additional steps can
be performed when, for example, de-crosslinking chemistry is
incompatible with barcoding or library preparation steps. In some
embodiments, after de-crosslinking methylene bridges, the nucleic
acids originating or present in the chunk can bind to the uniquely
barcoded bead. In some embodiments, after the spatial barcode binds
the uniquely barcoded bead, the encapsulation can be disrupted
(e.g., lysed, melted, or removed) and the barcoded beads can be
collected. In some embodiments, the collected barcoded beads can be
washed and re-encapsulated. In some embodiments, the nucleic acids
associated with the bead (e.g., spatial barcode, unique barcode,
analyte transcript) can be amplified (e.g., PCR amplified) and
processed (e.g., sequenced) according to any of the methods
described herein.
[1280] In some embodiments, a biological sample can be divided or
portioned using laser capture microdissection (e.g.,
highly-multiplexed laser capture microdissection).
[1281] (c) Release and Amplification of Analytes
[1282] In some embodiments, lysis reagents can be added to the
sample to facilitate release of analyte(s) from a sample. Examples
of lysis agents include, but are not limited to, bioactive reagents
such as lysis enzymes that are used for lysis of different cell
types, e.g., gram positive or negative bacteria, plants, yeast,
mammalian, such as lysozymes, achromopeptidase, lysostaphin,
labiase, kitalase, lyticase, and a variety of other commercially
available lysis enzymes. Other lysis agents can additionally or
alternatively be co-partitioned with the biological sample to cause
the release of the sample's contents into the partitions. In some
embodiments, surfactant-based lysis solutions can be used to lyse
cells, although these can be less desirable for emulsion-based
systems where the surfactants can interfere with stable emulsions.
Lysis solutions can include ionic surfactants such as, for example,
sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,
thermal, acoustic or mechanical cellular disruption can also be
used in certain embodiments, e.g., non-emulsion based partitioning
such as encapsulation of biological materials that can be in
addition to or in place of droplet partitioning, where any pore
volume of the encapsulate is sufficiently small to retain nucleic
acid fragments of a given size, following cellular disruption.
[1283] In addition to the permeabilization agents, other reagents
can also be added to interact with the biological sample,
including, for example, DNase and RNase inactivating agents or
inhibitors or chelating agents, such as EDTA, and other reagents to
allow for subsequent processing of analytes from the sample. In
other embodiments, nucleases, such as DNase or RNAse, or proteases,
such as pepsin or proteinase K, are added to the sample.
[1284] Further reagents that can be added to a sample, include, for
example, endonucleases to fragment DNA, DNA polymerase enzymes, and
dNTPs used to amplify nucleic acids. Other enzymes that can also be
added to the sample include, but are not limited to, polymerase,
transposase, ligase, proteinase K, and DNAse, etc. Additional
reagents can also include reverse transcriptase enzymes, including
enzymes with terminal transferase activity, primers, and switch
oligonucleotides. In some embodiments, template switching can be
used to increase the length of a cDNA, e.g., by appending a
predefined nucleic acid sequence to the cDNA.
[1285] If a tissue sample is not permeabilized sufficiently, the
amount of analyte captured on the substrate can be too low to
enable adequate analysis. Conversely, if the tissue sample is too
permeable, the analyte can diffuse away from its origin in the
tissue sample, such that the relative spatial relationship of the
analytes within the tissue sample is lost. Hence, a balance between
permeabilizing the tissue sample enough to obtain good signal
intensity while still maintaining the spatial resolution of the
analyte distribution in the tissue sample is desired.
[1286] In some embodiments, where the biological sample includes
live cells, permeabilization conditions can be modified so that the
live cells experience only brief permeabilization (e.g., through
short repetitive bursts of electric field application), thereby
allowing one or more analytes to migrate from the live cells to the
substrate while retaining cellular viability.
[1287] In some embodiments, after contacting a biological sample
with a substrate that include capture probes, a removal step is
performed to remove all or a portion of the biological sample from
the substrate. In some embodiments, the removal step includes
enzymatic or chemical degradation of the permeabilized cells of the
biological sample. For example, the removal step can include
treating the biological samples with an enzyme (e.g., proteinase K)
to remove at least a portion of the biological sample from the
first substrates. In some embodiments, the removal step can include
ablation of the tissue (e.g., laser ablation).
[1288] In some embodiments, where RNA is captured from cells in a
sample, one or more RNA species of interest can be selectively
enriched. For example, one or more species of RNA of interest can
be selected by addition of one or more oligonucleotides. One or
more species of RNA can be selectively down-selected (e.g.,
removed) using any of a variety of methods. For example, probes can
be administered to a sample that selectively hybridize to ribosomal
RNA (rRNA), thereby reducing the pool and concentration of rRNA in
the sample. Subsequent application of the capture probes to the
sample can result in improved RNA capture due to the reduction in
non-specific RNA present in the sample. In some embodiments, the
additional oligonucleotide is a sequence used for priming a
reaction by a polymerase. For example, one or more primer sequences
with sequence complementarity to one or more RNAs of interest, can
be used to amplify the one or more RNAs of interest, thereby
selectively enriching these RNAs. In some embodiments, an
oligonucleotide with sequence complementarity to the complementary
strand of captured RNA (e.g., cDNA) can bind to the cDNA. In one
non-limiting example, biotinylated oligonucleotides with sequence
complementary to one or more cDNA of interest binds to the cDNA and
can be selected using biotinylation-strepavidin affinity in any
number of methods known to the field (e.g., streptavidin
beads).
[1289] In some embodiments, any of the spatial analysis methods
described herein can include modulating the rate of interaction
between biological analytes from the biological sample and the
capture probes on the array. In some embodiments, modulating the
rate of interaction can occur by modulating the biological sample
(e.g., modulating temperature or pH). In some embodiments,
modulating the rate of interaction includes using external stimuli.
Non-limiting examples of external stimuli that can be used to
modulate the rate of interaction include light, temperature, small
molecules, enzymes, and/or an activating reagent. In one example,
light can be used to activate a polymerase in a nucleic acid
extension reaction. In another example, temperature can be used to
modulate hybridization between two complementary nucleic acid
molecules.
[1290] Nucleic acid analytes can be amplified using a polymerase
chain reaction (e.g., digital PCR, quantitative PCR, or real time
PCR), isothermal amplification, or any nucleic acid amplification
or extension reactions described herein, or known in the art.
[1291] (d) Partitioning
[1292] As discussed above, in some embodiments, the sample can
optionally be separated into single cells, cell groups (e.g., based
on cell sub-type or gene expression profile), or other
fragments/pieces that are smaller than the original sample. Each of
these smaller portions of the sample can be analyzed to obtain
spatially-resolved analyte information from the sample.
Non-limiting partitioning methods are described herein.
[1293] For samples that have been separated into smaller
fragments--and particularly, for samples that have been
disaggregated, dissociated, or otherwise separated into individual
cells--one method for analyzing the fragments involves partitioning
the fragments into individual partitions (e.g., fluid droplets),
and then analyzing the contents of the partitions. In general, each
partition maintains separation of its own contents from the
contents of other partitions. For example, the partition can be a
droplet in an emulsion.
[1294] The methods described herein provide for the
compartmentalization or partitioning of a cell (e.g., a cell) from
a sample into discrete compartments or voxels. As used herein, each
"voxel" represents a 3-dimensional volumetric unit. In some
embodiments, a voxel maintains separation of its own contents from
the contents of other voxels. A voxel can be one partition of an
array of partitions of volume. For example, a voxel can be one
partition of an array of discrete partitions into which a
3-dimensional object is divided. As another example, members of a
plurality of photo-crosslinkable polymer precursors can be
cross-linked into voxels that are part of an array of the
photo-crosslinked polymer covering the substrate or a portion of
the substrate. Unique identifiers, e.g., barcodes, may be
previously, subsequently, or concurrently delivered to the cell, in
order to allow for the later attribution of the characteristics of
the cell to the particular voxel. In some embodiments, a voxel has
defined dimensions. In some embodiments, a voxel comprises a single
cell.
[1295] For example, a substrate can be coated with a DTT-sensitive
hydrogel and then contacted with a biological sample. Optionally,
capture probes attached to the substrate are released from the
substrate such that the released capture probes are introduced into
the biological sample and at least one released capture probe
interacts with at least one biological analyte present in the
biological sample via the capture domain. The biological sample and
substrate can be assembled into a flow-cell and a
photo-crosslinkable polymer precursor added. The cells of the
biological sample can be then crosslinked into hydrogel-voxels of
defined dimensions using a light source. The flow-cell can be
dismantled and washed to remove unpolymerized polymer precursors.
The coating can be treated with DTT to yield single-cell partitions
for use in downstream applications. The capture probes/biological
analytes can be analyzed, and the spatial information of the
spatially-barcoded features can be used to determine the spatial
location of the captured biological analytes in the biological
sample.
[1296] In addition to analytes, a partition can include additional
components, and in particular, one or more beads. A partition can
include a single gel bead, a single cell bead, or both a single
cell bead and single gel bead.
[1297] A partition can also include one or more reagents. Unique
identifiers, such as barcodes, can be injected into the droplets
previous to, subsequent to, or concurrently with droplet
generation, such as via a microcapsule (e.g., bead). Microfluidic
channel networks (e.g., on a chip) can be utilized to generate
partitions. Alternative mechanisms can also be employed in the
partitioning of individual biological particles, including porous
membranes through which aqueous mixtures of cells are extruded into
non-aqueous fluids.
[1298] The partitions can be flowable within fluid streams. The
partitions can include, for example, micro-vesicles that have an
outer barrier surrounding an inner fluid center or core. In some
cases, the partitions can include a porous matrix that is capable
of entraining and/or retaining materials within its matrix. The
partitions can be droplets of a first phase within a second phase,
wherein the first and second phases are immiscible. For example,
the partitions can be droplets of aqueous fluid within a
non-aqueous continuous phase (e.g., oil phase). In another example,
the partitions can be droplets of a non-aqueous fluid within an
aqueous phase. In some examples, the partitions can be provided in
a water-in-oil emulsion or oil-in-water emulsion. A variety of
different vessels are described in, for example, U.S. Patent
Application Publication No. 2014/0155295, the entire contents of
which are incorporated herein by reference. Emulsion systems for
creating stable droplets in non-aqueous or oil continuous phases
are described, for example, in U.S. Patent Application Publication
No. 2010/0105112, the entire contents of which are incorporated
herein by reference.
[1299] For droplets in an emulsion, allocating individual particles
to discrete partitions can be accomplished, for example, by
introducing a flowing stream of particles in an aqueous fluid into
a flowing stream of a non-aqueous fluid, such that droplets are
generated at the junction of the two streams. Fluid properties
(e.g., fluid flow rates, fluid viscosities, etc.), particle
properties (e.g., volume fraction, particle size, particle
concentration, etc.), microfluidic architectures (e.g., channel
geometry, etc.), and other parameters can be adjusted to control
the occupancy of the resulting partitions (e.g., number of analytes
per partition, number of beads per partition, etc.) For example,
partition occupancy can be controlled by providing the aqueous
stream at a certain concentration and/or flow rate of analytes.
[1300] To generate single analyte partitions, the relative flow
rates of the immiscible fluids can be selected such that, on
average, the partitions can contain less than one analyte per
partition to ensure that those partitions that are occupied are
primarily singly occupied. In some cases, partitions among a
plurality of partitions can contain at most one analyte. In some
embodiments, the various parameters (e.g., fluid properties,
particle properties, microfluidic architectures, etc.) can be
selected or adjusted such that a majority of partitions are
occupied, for example, allowing for only a small percentage of
unoccupied partitions. The flows and channel architectures can be
controlled as to ensure a given number of singly occupied
partitions, less than a certain level of unoccupied partitions
and/or less than a certain level of multiply occupied
partitions.
[1301] The channel segments described herein can be coupled to any
of a variety of different fluid sources or receiving components,
including reservoirs, tubing, manifolds, or fluidic components of
other systems. As will be appreciated, the microfluidic channel
structure can have a variety of geometries. For example, a
microfluidic channel structure can have one or more than one
channel junction. As another example, a microfluidic channel
structure can have 2, 3, 4, or 5 channel segments each carrying
particles that meet at a channel junction. Fluid can be directed to
flow along one or more channels or reservoirs via one or more fluid
flow units. A fluid flow unit can include compressors (e.g.,
providing positive pressure), pumps (e.g., providing negative
pressure), actuators, and the like to control flow of the fluid.
Fluid can also or otherwise be controlled via applied pressure
differentials, centrifugal force, electrokinetic pumping, vacuum,
capillary, and/or gravity flow.
[1302] A partition can include one or more unique identifiers, such
as barcodes. Barcodes can be previously, subsequently, or
concurrently delivered to the partitions that hold the
compartmentalized or partitioned biological particle. For example,
barcodes can be injected into droplets previous to, subsequent to,
or concurrently with droplet generation. The delivery of the
barcodes to a particular partition allows for the later attribution
of the characteristics of the individual biological particle to the
particular partition. Barcodes can be delivered, for example on a
nucleic acid molecule (e.g., an oligonucleotide), to a partition
via any suitable mechanism. Barcoded nucleic acid molecules can be
delivered to a partition via a microcapsule. A microcapsule, in
some instances, can include a bead.
[1303] In some embodiments, barcoded nucleic acid molecules can be
initially associated with the microcapsule and then released from
the microcapsule. Release of the barcoded nucleic acid molecules
can be passive (e.g., by diffusion out of the microcapsule). In
addition or alternatively, release from the microcapsule can be
upon application of a stimulus which allows the barcoded nucleic
acid nucleic acid molecules to dissociate or to be released from
the microcapsule. Such stimulus can disrupt the microcapsule, an
interaction that couples the barcoded nucleic acid molecules to or
within the microcapsule, or both. Such stimulus can include, for
example, a thermal stimulus, photo-stimulus, chemical stimulus
(e.g., change in pH or use of a reducing agent(s)), a mechanical
stimulus, a radiation stimulus; a biological stimulus (e.g.,
enzyme), or any combination thereof.
[1304] In some embodiments, one more barcodes (e.g., spatial
barcodes, UMIs, or a combination thereof) can be introduced into a
partition as part of the analyte. As described previously, barcodes
can be bound to the analyte directly, or can form part of a capture
probe or analyte capture agent that is hybridized to, conjugated
to, or otherwise associated with an analyte, such that when the
analyte is introduced into the partition, the barcode(s) are
introduced as well.
[1305] FIG. 21 depicts an exemplary workflow, where a sample is
contacted with a spatially-barcoded capture probe array and the
sample is fixed, stained, and imaged 2101, as described elsewhere
herein. The capture probes can be cleaved from the array 2102 using
any method as described herein. The capture probes can diffuse
toward the cells by either passive or active migration as described
elsewhere herein. The capture probes may then be introduced to the
sample 2103 as described elsewhere herein, wherein the capture
probe is able to gain entry into the cell in the absence of cell
permeabilization, using one of the cell penetrating peptides or
lipid delivery systems described herein. The sample can then be
optionally imaged in order to confirm probe uptake, via a reporter
molecule incorporated within the capture probe 2104. The sample can
then be separated from the array and undergo dissociation 2105,
wherein the sample is separated into single cells or small groups
of cells. Once the sample is dissociated, the single cells can be
introduced to an oil-in water droplet 2106, wherein a single cell
is combined with reagents within the droplet and processed so that
the spatial barcode that penetrated the cell labels the contents of
that cell within the droplet. Other cells undergo separately
partitioned reactions concurrently. The contents of the droplet is
then sequenced 2107 in order to associate a particular cell or
cells with a particular spatial location within the sample
2108.
[1306] As described above, FIG. 16 shows an example of a
microfluidic channel structure for partitioning individual analytes
(e.g., cells) into discrete partitions. FIGS. 17A and 17C also show
other examples of microfluidic channel structures that can be used
for delivering beads to droplets.
[1307] A variety of different beads can be incorporated into
partitions as described above. In some embodiments, for example,
non-barcoded beads can be incorporated into the partitions. For
example, where the biological particle (e.g., a cell) that is
incorporated into the partitions carries one or more barcodes
(e.g., spatial barcode(s), UMI(s), and combinations thereof), the
bead can be a non-barcoded bead.
[1308] In some embodiments, a barcode carrying bead can be
incorporated into partitions. For example, a nucleic acid molecule,
such as an oligonucleotide, can be coupled to a bead by a
releasable linkage, such as, for example, a disulfide linker. The
same bead can be coupled (e.g., via releasable linkage) to one or
more other nucleic acid molecules. The nucleic acid molecule can be
or include a barcode. As noted elsewhere herein, the structure of
the barcode can include a number of sequence elements.
[1309] The nucleic acid molecule can include a functional domain
that can be used in subsequent processing. For example, the
functional domain can include one or more of a sequencer specific
flow cell attachment sequence (e.g., a P5 sequence for
Illumina.RTM. sequencing systems) and a sequencing primer sequence
(e.g., a R1 primer for Illumina.RTM. sequencing systems). The
nucleic acid molecule can include a barcode sequence for use in
barcoding the sample (e.g., DNA, RNA, protein, etc.). In some
cases, the barcode sequence can be bead-specific such that the
barcode sequence is common to all nucleic acid molecules coupled to
the same bead. Alternatively or in addition, the barcode sequence
can be partition-specific such that the barcode sequence is common
to all nucleic acid molecules coupled to one or more beads that are
partitioned into the same partition. The nucleic acid molecule can
include a specific priming sequence, such as an mRNA specific
priming sequence (e.g., poly (T) sequence), a targeted priming
sequence, and/or a random priming sequence. The nucleic acid
molecule can include an anchoring sequence to ensure that the
specific priming sequence hybridizes at the sequence end (e.g., of
the mRNA). For example, the anchoring sequence can include a random
short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or
longer sequence, which can ensure that a poly(T) segment is more
likely to hybridize at the sequence end of the poly(A) tail of the
mRNA.
[1310] The nucleic acid molecule can include a unique molecular
identifying sequence (e.g., unique molecular identifier (UMI)). In
some embodiments, the unique molecular identifying sequence can
include from about 5 to about 8 nucleotides. Alternatively, the
unique molecular identifying sequence can include less than about 5
or more than about 8 nucleotides. The unique molecular identifying
sequence can be a unique sequence that varies across individual
nucleic acid molecules coupled to a single bead.
[1311] In some embodiments, the unique molecular identifying
sequence can be a random sequence (e.g., such as a random N-mer
sequence). For example, the UMI can provide a unique identifier of
the starting mRNA molecule that was captured, in order to allow
quantitation of the number of original expressed RNA.
[1312] In general, an individual bead can be coupled to any number
of individual nucleic acid molecules, for example, from one to tens
to hundreds of thousands or even millions of individual nucleic
acid molecules. The respective barcodes for the individual nucleic
acid molecules can include both common sequence segments or
relatively common sequence segments and variable or unique sequence
segments between different individual nucleic acid molecules
coupled to the same bead.
[1313] Within any given partition, all of the cDNA transcripts of
the individual mRNA molecules can include a common barcode sequence
segment. However, the transcripts made from the different mRNA
molecules within a given partition can vary at the unique molecular
identifying sequence segment (e.g., UMI segment). Beneficially,
even following any subsequent amplification of the contents of a
given partition, the number of different UMIs can be indicative of
the quantity of mRNA originating from a given partition. As noted
above, the transcripts can be amplified, cleaned up, and sequenced
to identify the sequence of the cDNA transcript of the mRNA, as
well as to sequence the barcode segment and the UMI segment. While
a poly(T) primer sequence is described, other targeted or random
priming sequences can also be used in priming the reverse
transcription reaction. Likewise, although described as releasing
the barcoded oligonucleotides into the partition, in some cases,
the nucleic acid molecules bound to the bead can be used to
hybridize and capture the mRNA on the solid phase of the bead, for
example, in order to facilitate the separation of the RNA from
other cell contents.
[1314] In some embodiments, precursors that include a functional
group that is reactive or capable of being activated such that it
becomes reactive can be polymerized with other precursors to
generate gel beads that include the activated or activatable
functional group. The functional group can then be used to attach
additional species (e.g., disulfide linkers, primers, other
oligonucleotides, etc.) to the gel beads. For example, some
precursors featuring a carboxylic acid (COOH) group can
co-polymerize with other precursors to form a bead that also
includes a COOH functional group. In some cases, acrylic acid (a
species comprising free COOH groups), acrylamide, and
bis(acryloyl)cystamine can be co-polymerized together to generate a
bead with free COOH groups. The COOH groups of the bead can be
activated (e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) and N-Hydroxysuccinimide (NHS) or
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM)) such that they are reactive (e.g., reactive to amine
functional groups where EDC/NHS or DMTMM are used for activation).
The activated COOH groups can then react with an appropriate
species (e.g., a species comprising an amine functional group where
the carboxylic acid groups are activated to be reactive with an
amine functional group) comprising a moiety to be linked to the
bead.
[1315] In some embodiments, a degradable bead can be introduced
into a partition, such that the bead degrades within the partition
and any associated species (e.g., oligonucleotides) are released
within the droplet when the appropriate stimulus is applied. The
free species (e.g., oligonucleotides, nucleic acid molecules) can
interact with other reagents contained in the partition. For
example, a polyacrylamide bead featuring cystamine and linked, via
a disulfide bond, to a barcode sequence, can be combined with a
reducing agent within a droplet of a water-in-oil emulsion. Within
the droplet, the reducing agent can break the various disulfide
bonds, resulting in bead degradation and release of the barcode
sequence into the aqueous, inner environment of the droplet. In
another example, heating of a droplet with a bead-bound barcode
sequence in basic solution can also result in bead degradation and
release of the attached barcode sequence into the aqueous, inner
environment of the droplet.
[1316] Any suitable number of species (e.g., primer, barcoded
oligonucleotide) can be associated with a bead such that, upon
release from the bead, the species (e.g., primer, e.g., barcoded
oligonucleotide) are present in the partition at a pre-defined
concentration. Such pre-defined concentration can be selected to
facilitate certain reactions for generating a sequencing library,
e.g., amplification, within the partition. In some cases, the
pre-defined concentration of the primer can be limited by the
process of producing nucleic acid molecule (e.g., oligonucleotide)
bearing beads.
[1317] A degradable bead can include one or more species with a
labile bond such that, when the bead/species is exposed to the
appropriate stimulus, the bond is broken and the bead degrades. The
labile bond can be a chemical bond (e.g., covalent bond, ionic
bond) or can be another type of physical interaction (e.g., van der
Waals interactions, dipole-dipole interactions, etc.). In some
embodiments, a cross-linker used to generate a bead can include a
labile bond. Upon exposure to the appropriate conditions, the
labile bond can be broken and the bead degraded. For example, upon
exposure of a polyacrylamide gel bead that includes cystamine
cross-linkers to a reducing agent, the disulfide bonds of the
cystamine can be broken and the bead degraded.
[1318] A degradable bead can be useful in more quickly releasing an
attached species (e.g., a nucleic acid molecule, a barcode
sequence, a primer, etc.) from the bead when the appropriate
stimulus is applied to the bead as compared to a bead that does not
degrade. For example, for a species bound to an inner surface of a
porous bead or in the case of an encapsulated species, the species
can have greater mobility and accessibility to other species in
solution upon degradation of the bead. In some embodiments, a
species can also be attached to a degradable bead via a degradable
linker (e.g., disulfide linker). The degradable linker can respond
to the same stimuli as the degradable bead or the two degradable
species can respond to different stimuli. For example, a barcode
sequence can be attached, via a disulfide bond, to a polyacrylamide
bead comprising cystamine. Upon exposure of the barcoded-bead to a
reducing agent, the bead degrades and the barcode sequence is
released upon breakage of both the disulfide linkage between the
barcode sequence and the bead and the disulfide linkages of the
cystamine in the bead.
[1319] As will be appreciated from the above description, while
referred to as degradation of a bead, in many embodiments,
degradation can refer to the disassociation of a bound or entrained
species from a bead, both with and without structurally degrading
the physical bead itself. For example, entrained species can be
released from beads through osmotic pressure differences due to,
for example, changing chemical environments. By way of example,
alteration of bead pore volumes due to osmotic pressure differences
can generally occur without structural degradation of the bead
itself. In some cases, an increase in pore volume due to osmotic
swelling of a bead can permit the release of entrained species
within the bead. In some embodiments, osmotic shrinking of a bead
can cause a bead to better retain an entrained species due to pore
volume contraction.
[1320] Numerous chemical triggers can be used to trigger the
degradation of beads within partitions. Examples of these chemical
changes can include, but are not limited to pH-mediated changes to
the integrity of a component within the bead, degradation of a
component of a bead via cleavage of cross-linked bonds, and
depolymerization of a component of a bead.
[1321] In some embodiments, a bead can be formed from materials
that include degradable chemical cross-linkers, such as BAC or
cystamine. Degradation of such degradable cross-linkers can be
accomplished through a number of mechanisms. In some examples, a
bead can be contacted with a chemical degrading agent that can
induce oxidation, reduction or other chemical changes. For example,
a chemical degrading agent can be a reducing agent, such as
dithiothreitol (DTT). Additional examples of reducing agents can
include .beta.-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane
(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP),
or combinations thereof. A reducing agent can degrade the disulfide
bonds formed between gel precursors forming the bead, and thus,
degrade the bead.
[1322] In certain embodiments, a change in pH of a solution, such
as an increase in pH, can trigger degradation of a bead. In other
embodiments, exposure to an aqueous solution, such as water, can
trigger hydrolytic degradation, and thus degradation of the bead.
In some cases, any combination of stimuli can trigger degradation
of a bead. For example, a change in pH can enable a chemical agent
(e.g., DTT) to become an effective reducing agent.
[1323] Beads can also be induced to release their contents upon the
application of a thermal stimulus. A change in temperature can
cause a variety of changes to a bead. For example, heat can cause a
solid bead to liquefy. A change in heat can cause melting of a bead
such that a portion of the bead degrades. In other cases, heat can
increase the internal pressure of the bead components such that the
bead ruptures or explodes. Heat can also act upon heat-sensitive
polymers used as materials to construct beads.
[1324] In addition to beads and analytes, partitions that are
formed can include a variety of different reagents and species. For
example, when lysis reagents are present within the partitions, the
lysis reagents can facilitate the release of analytes within the
partition. Examples of lysis agents include bioactive reagents,
such as lysis enzymes that are used for lysis of different cell
types, e.g., gram positive or negative bacteria, plants, yeast,
mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin,
labiase, kitalase, lyticase, and a variety of other lysis enzymes
available from, e.g., Sigma-Aldrich, Inc. (St. Louis, Mo.), as well
as other commercially available lysis enzymes. Other lysis agents
can additionally or alternatively be co-partitioned to cause the
release analytes into the partitions. For example, in some cases,
surfactant-based lysis solutions can be used to lyse cells,
although these can be less desirable for emulsion based systems
where the surfactants can interfere with stable emulsions. In some
embodiments, lysis solutions can include non-ionic surfactants such
as, for example, TritonX-100 and Tween 20. In some embodiments,
lysis solutions can include ionic surfactants such as, for example,
sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,
thermal, acoustic or mechanical cellular disruption can also be
used in certain embodiments, e.g., non-emulsion based partitioning
such as encapsulation of analytes that can be in addition to or in
place of droplet partitioning, where any pore volume of the
encapsulate is sufficiently small to retain nucleic acid fragments
of a given size, following cellular disruption.
[1325] Examples of other species that can be co-partitioned with
analytes in the partitions include, but are not limited to, DNase
and RNase inactivating agents or inhibitors or chelating agents,
such as EDTA, and other reagents employed in removing or otherwise
reducing negative activity or impact of different cell lysate
components on subsequent processing of nucleic acids. Additional
reagents can also be co-partitioned, including endonucleases to
fragment DNA, DNA polymerase enzymes and dNTPs used to amplify
nucleic acid fragments and to attach the barcode molecular tags to
the amplified fragments.
[1326] Additional reagents can also include reverse transcriptase
enzymes, including enzymes with terminal transferase activity,
primers and oligonucleotides, and switch oligonucleotides (also
referred to herein as "switch oligos" or "template switching
oligonucleotides") which can be used for template switching. In
some embodiments, template switching can be used to increase the
length of a cDNA. Template switching can be used to append a
predefined nucleic acid sequence to the cDNA. In an example of
template switching, cDNA can be generated from reverse
transcription of a template, e.g., cellular mRNA, where a reverse
transcriptase with terminal transferase activity can add additional
nucleotides, e.g., poly(C), to the cDNA in a template independent
manner. Switch oligos can include sequences complementary to the
additional nucleotides, e.g., poly(G). The additional nucleotides
(e.g., poly(C)) on the cDNA can hybridize to the additional
nucleotides (e.g., poly(G)) on the switch oligo, whereby the switch
oligo can be used by the reverse transcriptase as template to
further extend the cDNA.
[1327] Template switching oligonucleotides can include a
hybridization region and a template region. The hybridization
region can include any sequence capable of hybridizing to the
target. In some cases, the hybridization region includes a series
of G bases to complement the overhanging C bases at the 3' end of a
cDNA molecule. The series of G bases can include 1 G base, 2 G
bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases. The
template sequence can include any sequence to be incorporated into
the cDNA. In some cases, the template region includes at least 1
(e.g., at least 2, 3, 4, 5 or more) tag sequences and/or functional
sequences. Switch oligos can include deoxyribonucleic acids;
ribonucleic acids; modified nucleic acids including 2-Aminopurine,
2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,
2'-deoxylnosine, Super T (5-hydroxybutynl-2'-deoxyuridine), Super G
(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked
nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG,
Iso-dC, 2' Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and
Fluoro G), and combinations of the foregoing.
[1328] In some embodiments, beads that are partitioned with the
analyte can include different types of oligonucleotides bound to
the bead, where the different types of oligonucleotides bind to
different types of analytes. For example, a bead can include one or
more first oligonucleotides (which can be capture probes, for
example) that can bind or hybridize to a first type of analyte,
such as mRNA for example, and one or more second oligonucleotides
(which can be capture probes, for example) that can bind or
hybridize to a second type of analyte, such as gDNA for example.
Partitions can also include lysis agents that aid in releasing
nucleic acids from the co-partitioned cell, and can also include an
agent (e.g., a reducing agent) that can degrade the bead and/or
break covalent linkages between the oligonucleotides and the bead,
releasing the oligonucleotides into the partition. The released
barcoded oligonucleotides (which can also be barcoded) can
hybridize with mRNA released from the cell and also with gDNA
released from the cell.
[1329] Barcoded constructs thus formed from hybridization can
include a first type of construct that includes a sequence
corresponding to an original barcode sequence from the bead and a
sequence corresponding to a transcript from the cell, and a second
type of construct that includes a sequence corresponding to the
original barcode sequence from the bead and a sequence
corresponding to genomic DNA from the cell. The barcoded constructs
can then be released/removed from the partition and, in some
embodiments, further processed to add any additional sequences. The
resulting constructs can then be sequenced, the sequencing data
processed, and the results used to spatially characterize the mRNA
and the gDNA from the cell.
[1330] In another example, a partition includes a bead that
includes a first type of oligonucleotide (e.g., a first capture
probe) with a first barcode sequence, a poly(T) priming sequence
that can hybridize with the poly(A) tail of an mRNA transcript, and
a UMI barcode sequence that can uniquely identify a given
transcript. The bead also includes a second type of oligonucleotide
(e.g., a second capture probe) with a second barcode sequence, a
targeted priming sequence that is capable of specifically
hybridizing with a third barcoded oligonucleotide (e.g., an analyte
capture agent) coupled to an antibody that is bound to the surface
of the partitioned cell. The third barcoded oligonucleotide
includes a UMI barcode sequence that uniquely identifies the
antibody (and thus, the particular cell surface feature to which it
is bound).
[1331] In this example, the first and second barcoded
oligonucleotides include the same spatial barcode sequence (e.g.,
the first and second barcode sequences are the same), which permits
downstream association of barcoded nucleic acids with the
partition. In some embodiments, however, the first and second
barcode sequences are different.
[1332] The partition also includes lysis agents that aid in
releasing nucleic acids from the cell and can also include an agent
(e.g., a reducing agent) that can degrade the bead and/or break a
covalent linkage between the barcoded oligonucleotides and the
bead, releasing them into the partition. The first type of released
barcoded oligonucleotide can hybridize with mRNA released from the
cell and the second type of released barcoded oligonucleotide can
hybridize with the third type of barcoded oligonucleotide, forming
barcoded constructs.
[1333] The first type of barcoded construct includes a spatial
barcode sequence corresponding to the first barcode sequence from
the bead and a sequence corresponding to the UMI barcode sequence
from the first type of oligonucleotide, which identifies cell
transcripts. The second type of barcoded construct includes a
spatial barcode sequence corresponding to the second barcode
sequence from the second type of oligonucleotide, and a UMI barcode
sequence corresponding to the third type of oligonucleotide (e.g.,
the analyte capture agent) and used to identify the cell surface
feature. The barcoded constructs can then be released/removed from
the partition and, in some embodiments, further processed to add
any additional sequences. The resulting constructs are then
sequenced, sequencing data processed, and the results used to
characterize the mRNA and cell surface feature of the cell.
[1334] The foregoing discussion involves two specific examples of
beads with oligonucleotides for analyzing two different analytes
within a partition. More generally, beads that are partitioned can
have any of the structures described previously, and can include
any of the described combinations of oligonucleotides for analysis
of two or more (e.g., three or more, four or more, five or more,
six or more, eight or more, ten or more, 12 or more, 15 or more, 20
or more, 25 or more, 30 or more, 40 or more, 50 or more) different
types of analytes within a partition. Examples of beads with
combinations of different types of oligonucleotides (e.g., capture
probes) for concurrently analyzing different combinations of
analytes within partitions include, but are not limited to: (a)
genomic DNA and cell surface features (e.g., using the analyte
capture agents described herein); (b) mRNA and a lineage tracing
construct; (c) mRNA and cell methylation status; (d) mRNA and
accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq);
(e) mRNA and cell surface or intracellular proteins and/or
metabolites; (f) a barcoded analyte capture agent (e.g., the MHC
multimers described herein) and a V(D)J sequence of an immune cell
receptor (e.g., T-cell receptor); and (g) mRNA and a perturbation
agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease,
and/or antisense oligonucleotide as described herein). In some
embodiments, a perturbation agent can be a small molecule, an
antibody, a drug, an aptamer, a nucleic acid (e.g., miRNA), a
physical environmental (e.g., temperature change), or any other
known perturbation agents.
[1335] (e) Sequencing Analysis
[1336] After analytes from the sample have hybridized or otherwise
been associated with capture probes, analyte capture agents, or
other barcoded oligonucleotide sequences according to any of the
methods described above in connection with the general spatial
cell-based analytical methodology, the barcoded constructs that
result from hybridization/association are analyzed via sequencing
to identify the analytes.
[1337] In some embodiments, where a sample is barcoded directly via
hybridization with capture probes or analyte capture agents
hybridized, bound, or associated with either the cell surface, or
introduced into the cell, as described above, sequencing can be
performed on the intact sample. Alternatively, if the barcoded
sample has been separated into fragments, cell groups, or
individual cells, as described above, sequencing can be performed
on individual fragments, cell groups, or cells. For analytes that
have been barcoded via partitioning with beads, as described above,
individual analytes (e.g., cells, or cellular contents following
lysis of cells) can be extracted from the partitions by breaking
the partitions, and then analyzed by sequencing to identify the
analytes.
[1338] A wide variety of different sequencing methods can be used
to analyze barcoded analyte constructs. In general, sequenced
polynucleotides can be, for example, nucleic acid molecules such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including
variants or derivatives thereof (e.g., single stranded DNA or
DNA/RNA hybrids, and nucleic acid molecules with a nucleotide
analog).
[1339] Sequencing of polynucleotides can be performed by various
commercial systems. More generally, sequencing can be performed
using nucleic acid amplification, polymerase chain reaction (PCR)
(e.g., digital PCR and droplet digital PCR (ddPCR), quantitative
PCR, real time PCR, multiplex PCR, PCR-based singleplex methods,
emulsion PCR), and/or isothermal amplification.
[1340] Other examples of methods for sequencing genetic material
include, but are not limited to, DNA hybridization methods (e.g.,
Southern blotting), restriction enzyme digestion methods, Sanger
sequencing methods, next-generation sequencing methods (e.g.,
single-molecule real-time sequencing, nanopore sequencing, and
Polony sequencing), ligation methods, and microarray methods.
Additional examples of sequencing methods that can be used include
targeted sequencing, single molecule real-time sequencing, exon
sequencing, electron microscopy-based sequencing, panel sequencing,
transistor-mediated sequencing, direct sequencing, random shotgun
sequencing, Sanger dideoxy termination sequencing, whole-genome
sequencing, sequencing by hybridization, pyrosequencing, capillary
electrophoresis, gel electrophoresis, duplex sequencing, cycle
sequencing, single-base extension sequencing, solid-phase
sequencing, high-throughput sequencing, massively parallel
signature sequencing, co-amplification at lower denaturation
temperature-PCR (COLD-PCR), sequencing by reversible dye
terminator, paired-end sequencing, near-term sequencing,
exonuclease sequencing, sequencing by ligation, short-read
sequencing, single-molecule sequencing, sequencing-by-synthesis,
real-time sequencing, reverse-terminator sequencing, nanopore
sequencing, 454 sequencing, Solexa Genome Analyzer sequencing,
SOLiD.TM. sequencing, MS-PET sequencing, and any combinations
thereof.
[1341] Sequence analysis of the nucleic acid molecules (including
barcoded nucleic acid molecules or derivatives thereof) can be
direct or indirect. Thus, the sequence analysis substrate (which
can be viewed as the molecule which is subjected to the sequence
analysis step or process) can directly be the barcoded nucleic acid
molecule or it can be a molecule which is derived therefrom (e.g.,
a complement thereof). Thus, for example, in the sequence analysis
step of a sequencing reaction, the sequencing template can be the
barcoded nucleic acid molecule or it can be a molecule derived
therefrom. For example, a first and/or second strand DNA molecule
can be directly subjected to sequence analysis (e.g., sequencing),
i.e., can directly take part in the sequence analysis reaction or
process (e.g., the sequencing reaction or sequencing process, or be
the molecule which is sequenced or otherwise identified).
Alternatively, the barcoded nucleic acid molecule can be subjected
to a step of second strand synthesis or amplification before
sequence analysis (e.g., sequencing or identification by another
technique). The sequence analysis substrate (e.g., template) can
thus be an amplicon or a second strand of a barcoded nucleic acid
molecule.
[1342] In some embodiments, both strands of a double stranded
molecule can be subjected to sequence analysis (e.g., sequenced).
In some embodiments, single stranded molecules (e.g., barcoded
nucleic acid molecules) can be analyzed (e.g., sequenced). To
perform single molecule sequencing, the nucleic acid strand can be
modified at the 3' end.
[1343] In some embodiments, massively parallel pyrosequencing
techniques can be used for sequencing nucleic acids. In
pyrosequencing, the nucleic acid is amplified inside water droplets
in an oil solution (emulsion PCR), with each droplet containing a
single nucleic acid template attached to a single primer-coated
bead that then forms a clonal colony. The sequencing system
contains many picolitre-volume wells each containing a single bead
and sequencing enzymes. Pyrosequencing uses luciferase to generate
light for detection of the individual nucleotides added to the
nascent nucleic acid and the combined data are used to generate
sequence reads.
[1344] As another example application of pyrosequencing, released
PPi can be detected by being immediately converted to adenosine
triphosphate (ATP) by ATP sulfurylase, and the level of ATP
generated can be detected via luciferase-produced photons, such as
described in Ronaghi, et al., Anal. Biochem. 242(1), 84-9 (1996);
Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281
(5375), 363 (1998); and U.S. Pat. Nos. 6,210,891, 6,258,568, and
6,274,320, the entire contents of each of which are incorporated
herein by reference.
[1345] Massively parallel sequencing techniques can be used for
sequencing nucleic acids, as described above. In one embodiment, a
massively parallel sequencing technique can be based on reversible
dye-terminators. As an example, DNA molecules are first attached to
primers on, e.g., a glass or silicon substrate, and amplified so
that local clonal colonies are formed (e.g., by bridge
amplification). Four types of ddNTPs are added, and
non-incorporated nucleotides are washed away. Unlike
pyrosequencing, the DNA is only extended one nucleotide at a time
due to a blocking group (e.g., 3' blocking group present on the
sugar moiety of the ddNTP). A detector acquires images of the
fluorescently labelled nucleotides, and then the dye along with the
terminal 3' blocking group is chemically removed from the DNA, as a
precursor to a subsequent cycle. This process can be repeated until
the required sequence data is obtained.
[1346] In some embodiments, sequencing is performed by detection of
hydrogen ions that are released during the polymerization of DNA. A
microwell containing a template DNA strand to be sequenced can be
flooded with a single type of nucleotide. If the introduced
nucleotide is complementary to the leading template nucleotide, it
is incorporated into the growing complementary strand. This causes
the release of a hydrogen ion that triggers a hypersensitive ion
sensor, which indicates that a reaction has occurred. If
homopolymer repeats are present in the template sequence, multiple
nucleotides will be incorporated in a single cycle. This leads to a
corresponding number of released hydrogen ions and a proportionally
higher electronic signal.
[1347] In some embodiments, sequencing can be performed in situ. In
situ sequencing methods are particularly useful, for example, when
the biological sample remains intact after analytes on the sample
surface (e.g., cell surface analytes) or within the sample (e.g.,
intracellular analytes) have been barcoded. In situ sequencing
typically involves incorporation of a labeled nucleotide (e.g.,
fluorescently labeled mononucleotides or dinucleotides) in a
sequential, template-dependent manner or hybridization of a labeled
primer (e.g., a labeled random hexamer) to a nucleic acid template
such that the identities (i.e., nucleotide sequence) of the
incorporated nucleotides or labeled primer extension products can
be determined, and consequently, the nucleotide sequence of the
corresponding template nucleic acid. Aspects of in situ sequencing
are described, for example, in Mitra et al., (2003) Anal. Biochem.
320, 55-65, and Lee et al., (2014) Science, 343(6177), 1360-1363,
the entire contents of each of which are incorporated herein by
reference.
[1348] In addition, examples of methods and systems for performing
in situ sequencing are described in PCT Patent Application
Publication Nos. WO2014/163886, WO2018/045181, WO2018/045186, and
in U.S. Pat. Nos. 10,138,509 and 10,179,932, the entire contents of
each of which are incorporated herein by reference. Example
techniques for in situ sequencing include, but are not limited to,
STARmap (described for example in Wang et al., (2018) Science,
361(6499) 5691), MERFISH (described for example in Moffitt, (2016)
Methods in Enzymology, 572, 1-49), and FISSEQ (described for
example in U.S. Patent Application Publication No. 2019/0032121).
The entire contents of each of the foregoing references are
incorporated herein by reference.
[1349] For analytes that have been barcoded via partitioning,
barcoded nucleic acid molecules or derivatives thereof (e.g.,
barcoded nucleic acid molecules to which one or more functional
sequences have been added, or from which one or more features have
been removed) can be pooled and processed together for subsequent
analysis such as sequencing on high throughput sequencers.
Processing with pooling can be implemented using barcode sequences.
For example, barcoded nucleic acid molecules of a given partition
can have the same barcode, which is different from barcodes of
other spatial partitions. Alternatively, barcoded nucleic acid
molecules of different partitions can be processed separately for
subsequent analysis (e.g., sequencing).
[1350] In some embodiments, where capture probes do not contain a
spatial barcode, the spatial barcode can be added after the capture
probe captures analytes from a biological sample and before
analysis of the analytes. When a spatial barcode is added after an
analyte is captured, the barcode can be added after amplification
of the analyte (e.g., reverse transcription and polymerase
amplification of RNA). In some embodiments, analyte analysis uses
direct sequencing of one or more captured analytes, such as direct
sequencing of hybridized RNA. In some embodiments, direct
sequencing is performed after reverse transcription of hybridized
RNA. In some embodiments direct sequencing is performed after
amplification of reverse transcription of hybridized RNA.
[1351] In some embodiments, direct sequencing of captured RNA is
performed by sequencing-by-synthesis (SBS). In some embodiments, a
sequencing primer is complementary to a sequence in one or more of
the domains of a capture probe (e.g., functional domain). In such
embodiments, sequencing-by-synthesis can include reverse
transcription and/or amplification in order to generate a template
sequence (e.g., functional domain) from which a primer sequence can
bind.
[1352] SBS can involve hybridizing an appropriate primer, sometimes
referred to as a sequencing primer, with the nucleic acid template
to be sequenced, extending the primer, and detecting the
nucleotides used to extend the primer. Preferably, the nucleic acid
used to extend the primer is detected before a further nucleotide
is added to the growing nucleic acid chain, thus allowing
base-by-base in situ nucleic acid sequencing. The detection of
incorporated nucleotides is facilitated by including one or more
labelled nucleotides in the primer extension reaction. To allow the
hybridization of an appropriate sequencing primer to the nucleic
acid template to be sequenced, the nucleic acid template should
normally be in a single stranded form. If the nucleic acid
templates making up the nucleic acid features are present in a
double stranded form these can be processed to provide single
stranded nucleic acid templates using methods well known in the
art, for example by denaturation, cleavage, etc. The sequencing
primers which are hybridized to the nucleic acid template and used
for primer extension are preferably short oligonucleotides, for
example, 15 to 25 nucleotides in length. The sequencing primers can
be greater than 25 nucleotides in length as well. For example,
sequencing primers can be about 20 to about 60 nucleotides in
length, or more than 60 nucleotides in length. The sequencing
primers can be provided in solution or in an immobilized form. Once
the sequencing primer has been annealed to the nucleic acid
template to be sequenced by subjecting the nucleic acid template
and sequencing primer to appropriate conditions, primer extension
is carried out, for example using a nucleic acid polymerase and a
supply of nucleotides, at least some of which are provided in a
labelled form, and conditions suitable for primer extension if a
suitable nucleotide is provided.
[1353] Preferably after each primer extension step, a washing step
is included in order to remove unincorporated nucleotides which can
interfere with subsequent steps. Once the primer extension step has
been carried out, the nucleic acid colony is monitored to determine
whether a labelled nucleotide has been incorporated into an
extended primer. The primer extension step can then be repeated to
determine the next and subsequent nucleotides incorporated into an
extended primer. If the sequence being determined is unknown, the
nucleotides applied to a given colony are usually applied in a
chosen order which is then repeated throughout the analysis, for
example dATP, dTTP, dCTP, dGTP.
[1354] SBS techniques which can be used are described for example,
but not limited to, those in U.S. Patent App. Pub. No.
2007/0166705, U.S. Patent App. Pub. No. 2006/0188901, U.S. Pat. No.
7,057,026, U.S. Patent App. Pub. No. 2006/0240439, U.S. Patent App.
Pub. No. 2006/0281109, PCT Patent App. Pub. No. WO 05/065814, U.S.
Patent App. Pub. No. 2005/0100900, PCT Patent App. Pub. No. WO
06/064199, PCT Patent App. Pub. No. WO07/010,251, U.S. Patent App.
Pub. No. 2012/0270305, U.S. Patent App. Pub. No. 2013/0260372, and
U.S. Patent App. Pub. No. 2013/0079232, the entire contents of each
of which are incorporated herein by reference.
[1355] In some embodiments, direct sequencing of captured RNA is
performed by sequential fluorescence hybridization (e.g.,
sequencing by hybridization). In some embodiments, a hybridization
reaction where RNA is hybridized to a capture probe is performed in
situ. In some embodiments, captured RNA is not amplified prior to
hybridization with a sequencing probe. In some embodiments, RNA is
amplified prior to hybridization with sequencing probes (e.g.,
reverse transcription to cDNA and amplification of cDNA). In some
embodiments, amplification is performed using single-molecule
hybridization chain reaction. In some embodiments, amplification is
performed using rolling chain amplification.
[1356] Sequential fluorescence hybridization can involve sequential
hybridization of probes including degenerate primer sequences and a
detectable label. A degenerate primer sequence is a short
oligonucleotide sequence which is capable of hybridizing to any
nucleic acid fragment independent of the sequence of said nucleic
acid fragment. For example, such a method could include the steps
of: (a) providing a mixture including four probes, each of which
includes either A, C, G, or T at the 5'-terminus, further including
degenerate nucleotide sequence of 5 to 11 nucleotides in length,
and further including a functional domain (e.g., fluorescent
molecule) that is distinct for probes with A, C, G, or T at the
5'-terminus; (b) associating the probes of step (a) to the target
polynucleotide sequences, whose sequence needs will be determined
by this method; (c) measuring the activities of the four functional
domains and recording the relative spatial location of the
activities; (d) removing the reagents from steps (a)-(b) from the
target polynucleotide sequences; and repeating steps (a)-(d) for n
cycles, until the nucleotide sequence of the spatial domain for
each bead is determined, with modification that the
oligonucleotides used in step (a) are complementary to part of the
target polynucleotide sequences and the positions 1 through n
flanking the part of the sequences. Because the barcode sequences
are different, in some embodiments, these additional flanking
sequences are degenerate sequences. The fluorescent signal from
each spot on the array for cycles 1 through n can be used to
determine the sequence of the target polynucleotide sequences.
[1357] In some embodiments, direct sequencing of captured RNA using
sequential fluorescence hybridization is performed in vitro. In
some embodiments, captured RNA is amplified prior to hybridization
with a sequencing probe (e.g., reverse transcription to cDNA and
amplification of cDNA). In some embodiments, a capture probe
containing captured RNA is exposed to the sequencing probe
targeting coding regions of RNA. In some embodiments, one or more
sequencing probes are targeted to each coding region. In some
embodiments, the sequencing probe is designed to hybridize with
sequencing reagents (e.g., a dye-labeled readout oligonucleotides).
A sequencing probe can then hybridize with sequencing reagents. In
some embodiments, output from the sequencing reaction is imaged. In
some embodiments, a specific sequence of cDNA is resolved from an
image of a sequencing reaction. In some embodiments, reverse
transcription of captured RNA is performed prior to hybridization
to the sequencing probe. In some embodiments, the sequencing probe
is designed to target complementary sequences of the coding regions
of RNA (e.g., targeting cDNA).
[1358] In some embodiments, a captured RNA is directly sequenced
using a nanopore-based method. In some embodiments, direct
sequencing is performed using nanopore direct RNA sequencing in
which captured RNA is translocated through a nanopore. A nanopore
current can be recorded and converted into a base sequence. In some
embodiments, captured RNA remains attached to a substrate during
nanopore sequencing. In some embodiments, captured RNA is released
from the substrate prior to nanopore sequencing. In some
embodiments, where the analyte of interest is a protein, direct
sequencing of the protein can be performed using nanopore-based
methods. Examples of nanopore-based sequencing methods that can be
used are described in Deamer et al., Trends Biotechnol. 18, 14
7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li
et al., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,
1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007);
Cockroft et al., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S.
Pat. No. 7,001,792. The entire contents of each of the foregoing
references are incorporated herein by reference.
[1359] In some embodiments, direct sequencing of captured RNA is
performed using single molecule sequencing by ligation. Such
techniques utilize DNA ligase to incorporate oligonucleotides and
identify the incorporation of such oligonucleotides. The
oligonucleotides typically have different labels that are
correlated with the identity of a particular nucleotide in a
sequence to which the oligonucleotides hybridize. Aspects and
features involved in sequencing by ligation are described, for
example, in Shendure et al. Science (2005), 309: 1728-1732, and in
U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488; 6,172,218; and
6,306,597, the entire contents of each of which are incorporated
herein by reference.
[1360] In some embodiments, nucleic acid hybridization can be used
for sequencing. These methods utilize labeled nucleic acid decoder
probes that are complementary to at least a portion of a barcode
sequence. Multiplex decoding can be performed with pools of many
different probes with distinguishable labels. Non-limiting examples
of nucleic acid hybridization sequencing are described for example
in U.S. Pat. No. 8,460,865, and in Gunderson et al., Genome
Research 14:870-877 (2004), the entire contents of each of which
are incorporated herein by reference.
[1361] In some embodiments, commercial high-throughput digital
sequencing techniques can be used to analyze barcode sequences, in
which DNA templates are prepared for sequencing not one at a time,
but in a bulk process, and where many sequences are read out
preferably in parallel, or alternatively using an ultra-high
throughput serial process that itself may be parallelized. Examples
of such techniques include Illumina.RTM. sequencing (e.g., flow
cell-based sequencing techniques), sequencing by synthesis using
modified nucleotides (such as commercialized in TruSeq.TM. and
HiSeq.TM. technology by Illumina, Inc., San Diego, Calif.),
HeliScope.TM. by Helicos Biosciences Corporation, Cambridge, Mass.,
and PacBio RS by Pacific Biosciences of California, Inc., Menlo
Park, Calif.), sequencing by ion detection technologies (Ion
Torrent, Inc., South San Francisco, Calif.), and sequencing of DNA
nanoballs (Complete Genomics, Inc., Mountain View, Calif.).
[1362] In some embodiments, detection of a proton released upon
incorporation of a nucleotide into an extension product can be used
in the methods described herein. For example, the sequencing
methods and systems described in U.S. Patent Application
Publication Nos. 2009/0026082, 2009/0127589, 2010/0137143, and
2010/0282617, can be used to directly sequence barcodes. The entire
contents of each of the foregoing references are incorporated
herein by reference.
[1363] In some embodiments, real-time monitoring of DNA polymerase
activity can be used during sequencing. For example, nucleotide
incorporations can be detected through fluorescence resonance
energy transfer (FRET), as described for example in Levene et al.,
Science (2003), 299, 682-686, Lundquist et al., Opt. Lett. (2008),
33, 1026-1028, and Korlach et al., Proc. Natl. Acad. Sci. USA
(2008), 105, 1176-1181. The entire contents of each of the
foregoing references are herein incorporated by reference.
IV. Multiplexing
[1364] (a) Multiplexing Generally
[1365] In various embodiments of spatial analysis as described
herein, features can include different types of capture probes for
analyzing both intrinsic and extrinsic information for individual
cells. For example, a feature can include one or more of the
following: 1) a capture probe featuring a capture domain that binds
to one or more endogenous nucleic acids in the cell; 2) a capture
probe featuring a capture domain that binds to one or more
exogenous nucleic acids in the cell (e.g., nucleic acids from a
microorganism (e.g., a virus, a bacterium)) that infects the cell,
nucleic acids introduced into the cell (e.g., such as plasmids or
nucleic acid derived therefrom), nucleic acids for gene editing
(e.g., CRISPR-related RNA such as crRNA, guide RNA); 3) a capture
probe featuring a capture domain that binds to an analyte capture
agent (e.g., an antibody coupled to a oligonucleotide that includes
a capture agent barcode domain having an analyte capture sequence
that binds the capture domain), and 4) a capture moiety featuring a
domain that binds to a protein (e.g., an exogenous protein
expressed in the cell, a protein from a microorganism (e.g., a
virus, a bacterium)) that infects the cell, or a binding partner
for a protein of the cell (e.g., an antigen for an immune cell
receptor).
[1366] In some embodiments of any of the spatial analysis methods
as described herein, spatial profiling includes concurrent analysis
of two different types of analytes. A feature can be a gel bead,
which is coupled (e.g., reversibly coupled) to one or more capture
probes. The capture probes can include a spatial barcode sequence
and a poly(T) priming sequence that can hybridize with the poly(A)
tail of an mRNA transcript. The capture probe can also include a
UMI sequence that can uniquely identify a given transcript. The
capture probe can also include a spatial barcode sequence and a
random N-mer priming sequence that is capable of randomly
hybridizing with gDNA. In this configuration, capture probes can
include the same spatial barcode sequence, which permits
association of downstream sequencing reads with the feature.
[1367] In some embodiments of any of the spatial analysis methods
as described herein, a feature can be a gel bead, which is coupled
(e.g., reversibly coupled) to capture probes. The Capture probe can
include a spatial barcode sequence and a poly(T) priming sequence
that can hybridize with the poly(A) tail of an mRNA transcript. The
capture probe can also include a UMI sequence that can uniquely
identify a given transcript. The capture probe can include a
spatial barcode sequence and a capture domain that is capable of
specifically hybridizing with an analyte capture agent. The analyte
capture agent can includes an oligonucleotide that includes an
analyte capture sequence that interacts with the capture domain
coupled to the feature. The oligonucleotide of the analyte capture
agent can be coupled to an antibody that is bound to the surface of
a cell. The oligonucleotide includes a barcode sequence (e.g., an
analyte binding moiety barcode) that uniquely identifies the
antibody (and thus, the particular cell surface feature to which it
is bound). In this configuration, the capture probes include the
same spatial barcode sequence, which permit downstream association
of barcoded nucleic acids with the location on the spatial array.
In some embodiments of any of the spatial profiling methods
described herein, the analyte capture agents can be can be produced
by any suitable route, including via example coupling schemes
described elsewhere herein.
[1368] In some embodiments of any of the spatial analysis methods
described herein, other combinations of two or more biological
analytes that can be concurrently measured include, without
limitation: (a) genomic DNA and cell surface features (e.g., via
analyte capture agents that bind to a cell surface feature), (b)
mRNA and a lineage tracing construct, (c) mRNA and cell methylation
status, (d) mRNA and accessible chromatin (e.g., ATAC-seq,
DNase-seq, and/or MNase-seq), (e) mRNA and cell surface or
intracellular proteins and/or metabolites, (f) mRNA and chromatin
(spatial organization of chromatin in a cell), (g) an analyte
capture agent (e.g., any of the MHC multimers described herein) and
a V(D)J sequence of an immune cell receptor (e.g., T-cell
receptor), (h) mRNA and a perturbation agent (e.g., a CRISPR
crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense
oligonucleotide as described herein), (i) genomic DNA and a
perturbation agent, (j) an analyte capture agent and a perturbation
agents, (k) accessible chromatin and a perturbation agent, (l)
chromatin (e.g., spatial organization of chromatin in a cell) and a
perturbation agent, and (m) cell surface or intracellular proteins
and/or metabolites and a perturbation agent (e.g., any of the
perturbation agents described herein), or any combination
thereof.
[1369] In some embodiments of any of the spatial analysis methods
described herein, the first analyte can include a nucleic acid
molecule with a nucleic acid sequence (e.g., mRNA, complementary
DNA derived from reverse transcription of mRNA) encoding at least a
portion of a V(D)J sequence of an immune cell receptor (e.g., a TCR
or BCR). In some embodiments, the nucleic acid molecule with a
nucleic acid sequence encoding at least a portion of a V(D)J
sequence of an immune cell receptor is cDNA first generated from
reverse transcription of the corresponding mRNA, using a poly(T)
containing primer. The cDNA that is generated can then be barcoded
using a primer, featuring a spatial barcode sequence (and
optionally, a UMI sequence) that hybridizes with at least a portion
of the cDNA that is generated. In some embodiments, a template
switching oligonucleotide in conjunction a terminal transferase or
a reverse transcriptase having terminal transferase activity can be
employed to generate a priming region on the cDNA to which a
barcoded primer can hybridize during cDNA generation. Terminal
transferase activity can, for example, add a poly(C) tail to a 3'
end of the cDNA such that the template switching oligonucleotide
can bind via a poly(G) priming sequence and the 3' end of the cDNA
can be further extended. The original mRNA template and template
switching oligonucleotide can then be denatured from the cDNA and
the barcoded primer comprising a sequence complementary to at least
a portion of the generated priming region on the cDNA can then
hybridize with the cDNA and a barcoded construct comprising the
barcode sequence (and any optional UMI sequence) and a complement
of the cDNA generated. Additional methods and compositions suitable
for barcoding cDNA generated from mRNA transcripts including those
encoding V(D)J regions of an immune cell receptor and/or barcoding
methods and composition including a template switch oligonucleotide
are described, for example, in PCT Patent Application Publication
No. WO 2018/075693, and in U.S. Patent Application Publication No.
2018/0105808, the entire contents of each of which are incorporated
herein by reference.
[1370] In some embodiments, V(D)J analysis can be performed using
methods similar to those described herein. For example, V(D)J
analysis can be completed with the use of one or more analyte
capture agents that bind to particular surface features of immune
cells and are associated with barcode sequences (e.g., analyte
binding moiety barcodes). The one or more analyte capture agents
can include an MHC or MHC multimer. A barcoded oligonucleotide
coupled to a bead that can be used for V(D)J analysis. The
oligonucleotide is coupled to a bead by a releasable linkage, such
as a disulfide linker. The oligonucleotide can include functional
sequences that are useful for subsequent processing, such as
functional sequence, which can include a sequencer specific flow
cell attachment sequence, e.g., a P5 sequence, as well as
functional sequence, which can include sequencing primer sequences,
e.g., a R1 primer binding site. In some embodiments, the sequence
can include a P7 sequence and a R2 primer binding site. A barcode
sequence can be included within the structure for use in barcoding
the template polynucleotide. The functional sequences can be
selected for compatibility with a variety of different sequencing
systems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina
X10, etc., and the requirements thereof. In some embodiments, the
barcode sequence, functional sequences (e.g., flow cell attachment
sequence) and additional sequences (e.g., sequencing primer
sequences) can be common to all of the oligonucleotides attached to
a given bead. The barcoded oligonucleotide can also include a
sequence to facilitate template switching (e.g., a poly(G)
sequence). In some embodiments, the additional sequence provides a
unique molecular identifier (UMI) sequence segment, as described
elsewhere herein.
[1371] In an exemplary method of cellular polynucleotide analysis
using a barcode oligonucleotide, a cell is co-partitioned along
with a bead bearing a barcoded oligonucleotide and additional
reagents such as a reverse transcriptase, primers, oligonucleotides
(e.g., template switching oligonucleotides), dNTPs, and a reducing
agent into a partition (e.g., a droplet in an emulsion). Within the
partition, the cell can be lysed to yield a plurality of template
polynucleotides (e.g., DNA such as genomic DNA, RNA such as mRNA,
etc.).
[1372] A reaction mixture featuring a template polynucleotide from
a cell and (i) the primer having a sequence towards a 3' end that
hybridizes to the template polynucleotide (e.g., poly(T)) and (ii)
a template switching oligonucleotide that includes a first
oligonucleotide towards a 5' end can be subjected to an
amplification reaction to yield a first amplification product. In
some embodiments, the template polynucleotide is an mRNA with a
poly(A) tail and the primer that hybridizes to the template
polynucleotide includes a poly(T) sequence towards a 3' end, which
is complementary to the poly(A) segment. The first oligonucleotide
can include at least one of an adaptor sequence, a barcode
sequence, a unique molecular identifier (UMI) sequence, a primer
binding site, and a sequencing primer binding site or any
combination thereof. In some cases, a first oligonucleotide is a
sequence that can be common to all partitions of a plurality of
partitions. For example, the first oligonucleotide can include a
flow cell attachment sequence, an amplification primer binding
site, or a sequencing primer binding site and the first
amplification reaction facilitates the attachment the
oligonucleotide to the template polynucleotide from the cell. In
some embodiments, the first oligonucleotide includes a primer
binding site. In some embodiments, the first oligonucleotide
includes a sequencing primer binding site.
[1373] The sequence towards a 3' end (e.g., poly(T)) of the primer
hybridizes to the template polynucleotide. In a first amplification
reaction, extension reaction reagents, e.g., reverse transcriptase,
nucleoside triphosphates, co-factors (e.g., Mg.sup.2+ or
Mn.sup.2+), that are also co-partitioned, can extend the primer
sequence using the cell's nucleic acid as a template, to produce a
transcript, e.g., cDNA, having a fragment complementary to the
strand of the cell's nucleic acid to which the primer annealed. In
some embodiments, the reverse transcriptase has terminal
transferase activity and the reverse transcriptase adds additional
nucleotides, e.g., poly(C), to the cDNA in a template independent
manner.
[1374] The template switching oligonucleotide, for example a
template switching oligonucleotide which includes a poly(G)
sequence, can hybridize to the cDNA and facilitate template
switching in the first amplification reaction. The transcript,
therefore, can include the sequence of the primer, a sequence
complementary to the template polynucleotide from the cell, and a
sequence complementary to the template switching
oligonucleotide.
[1375] In some embodiments of any of the spatial analysis methods
described herein, subsequent to the first amplification reaction,
the first amplification product or transcript can be subjected to a
second amplification reaction to generate a second amplification
product. In some embodiments, additional sequences (e.g.,
functional sequences such as flow cell attachment sequence,
sequencing primer binding sequences, barcode sequences, etc.) are
attached. The first and second amplification reactions can be
performed in the same volume, such as for example in a droplet. In
some embodiments, the first amplification product is subjected to a
second amplification reaction in the presence of a barcoded
oligonucleotide to generate a second amplification product having a
barcode sequence. The barcode sequence can be unique to a
partition, that is, each partition can have a unique barcode
sequence. The barcoded oligonucleotide can include a sequence of at
least a segment of the template switching oligonucleotide and at
least a second oligonucleotide. The segment of the template
switching oligonucleotide on the barcoded oligonucleotide can
facilitate hybridization of the barcoded oligonucleotide to the
transcript, e.g., cDNA, to facilitate the generation of a second
amplification product. In addition to a barcode sequence, the
barcoded oligonucleotide can include a second oligonucleotide such
as at least one of an adaptor sequence, a unique molecular
identifier (UMI) sequence, a primer binding site, and a sequencing
primer binding site, or any combination thereof.
[1376] In some embodiments of any of the spatial analysis methods
described herein, the second amplification reaction uses the first
amplification product as a template and the barcoded
oligonucleotide as a primer. In some embodiments, the segment of
the template switching oligonucleotide on the barcoded
oligonucleotide can hybridize to the portion of the cDNA or
complementary fragment having a sequence complementary to the
template switching oligonucleotide or that which was copied from
the template switching oligonucleotide. In the second amplification
reaction, extension reaction reagents, e.g., polymerase, nucleoside
triphosphates, co-factors (e.g., Mg.sup.2+ or Mn.sup.2+), that are
also co-partitioned, can extend the primer sequence using the first
amplification product as template. The second amplification product
can include a second oligonucleotide, a sequence of a segment of
the template polynucleotide (e.g., mRNA), and a sequence
complementary to the primer.
[1377] In some embodiments of any of the spatial analysis methods
described herein, the second amplification product uses the
barcoded oligonucleotide as a template and at least a portion of
the first amplification product as a primer. The segment of the
first amplification product (e.g., cDNA) having a sequence
complementary to the template switching oligonucleotide can
hybridize to the segment of the barcoded oligonucleotide comprising
a sequence of at least a segment of the template switching
oligonucleotide. In the second amplification reaction, extension
reaction reagents, e.g., polymerase, nucleoside triphosphates,
co-factors (e.g., Mg.sup.2+ or Mn.sup.2+), that are also
co-partitioned, can extend the primer sequence (e.g., first
amplification product) using the barcoded oligonucleotide as
template. The second amplification product can include the sequence
of the primer, a sequence which is complementary to the sequence of
the template polynucleotide (e.g., mRNA), and a sequence
complementary to the second oligonucleotide.
[1378] In some embodiments of any of the spatial analysis methods
described herein, three or more classes of biological analytes can
be concurrently measured. For example, a feature can include
capture probes that can participate in an assay of at least three
different types of analytes via three different capture domains. A
bead can be coupled to a barcoded oligonucleotide that includes a
capture domain that includes a poly(T) priming sequence for mRNA
analysis; a barcoded oligonucleotide that includes a capture domain
that includes a random N-mer priming sequence for gDNA analysis;
and a barcoded oligonucleotide that includes a capture domain that
can specifically bind a an analyte capture agent (e.g., an antibody
with a spatial barcode), via its analyte capture sequence.
[1379] In some embodiments of any of the spatial analysis methods
described herein, other combinations of three or more biological
analytes that can be concurrently measured include, without
limitation: (a) mRNA, a lineage tracing construct, and cell surface
and/or intracellular proteins and/or metabolites; (b) mRNA,
accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq),
and cell surface and/or intracellular proteins and/or metabolites;
(c) mRNA, genomic DNA, and a perturbation reagent (e.g., a CRISPR
crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense
oligonucleotide as described herein); (d) mRNA, accessible
chromatin, and a perturbation reagent; (e) mRNA, an analyte capture
agent (e.g., any of the MHC multimers described herein), and a
perturbation reagent; (f) mRNA, cell surface and/or intracellular
proteins and/or metabolites, and a perturbation agent; (g) mRNA, a
V(D)J sequence of an immune cell receptor (e.g., T-cell receptor),
and a perturbation reagent; (h) mRNA, an analyte capture agent, and
a V(D)J sequence of an immune cell receptor; (i) cell surface
and/or intracellular proteins and/or metabolites, a an analyte
capture agent (e.g., the MHC multimers described herein), and a
V(D)J sequence of an immune cell receptor; (j) methylation status,
mRNA, and cell surface and/or intracellular proteins and/or
metabolites; (k) mRNA, chromatin (e.g., spatial organization of
chromatin in a cell), and a perturbation reagent; (l) a V(D)J
sequence of an immune cell receptor, chromatin (e.g., spatial
organization of chromatin in a cell); and a perturbation reagent;
and (m) mRNA, a V(D)J sequence of an immune cell receptor, and
chromatin (e.g., spatial organization of chromatin in a cell), or
any combination thereof.
[1380] In some embodiments of any of the spatial analysis methods
described herein, four or more classes biological analytes can be
concurrently measured. A feature can be a bead that is coupled to
barcoded primers that can each participate in an assay of a
different type of analyte. The feature is coupled (e.g., reversibly
coupled) to a capture probe that includes a capture domain that
includes a poly(T) priming sequence for mRNA analysis and is also
coupled (e.g., reversibly coupled) to capture probe that includes a
capture domain that includes a random N-mer priming sequence for
gDNA analysis. Moreover, the feature is also coupled (e.g.,
reversibly coupled) to a capture probe that binds an analyte
capture sequence of an analyte capture agent via its capture
domain. The feature can also be coupled (e.g., reversibly coupled)
to a capture probe that can specifically bind a nucleic acid
molecule that can function as a perturbation agent (e.g., a CRISPR
crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense
oligonucleotide as described herein), via its capture domain.
[1381] In some embodiments of any of the spatial analysis methods
described herein, each of the various spatially-barcoded capture
probes present at a given feature or on a given bead include the
same spatial barcode sequence. In some embodiments, each barcoded
capture probe can be released from the feature in a manner suitable
for analysis of its respective analyte. For example, barcoded
constructs A, B, C and D can be generated as described elsewhere
herein and analyzed. Barcoded construct A can include a sequence
corresponding to the barcode sequence from the bead (e.g., a
spatial barcode) and a DNA sequence corresponding to a target mRNA.
Barcoded construct B can include a sequence corresponding to the
barcode sequence from the bead (e.g., a spatial barcode) and a
sequence corresponding to genomic DNA. Barcoded construct C can
include a sequence corresponding to the barcode sequence from the
bead (e.g., a spatial barcode) and a sequence corresponding to
barcode sequence associated with an analyte capture agent (e.g., an
analyte binding moiety barcode). Barcoded construct D can include a
sequence corresponding to the barcode sequence from the bead (e.g.,
a spatial barcode) and a sequence corresponding to a CRISPR nucleic
acid (which, in some embodiments, also includes a barcode
sequence). Each construct can be analyzed (e.g., via any of a
variety of sequencing methods) and the results can be associated
with the given cell from which the various analytes originated.
Barcoded (or even non-barcoded) constructs can be tailored for
analyses of any given analyte associated with a nucleic acid and
capable of binding with such a construct.
[1382] In some embodiments of any of the spatial analysis methods
described herein, other combinations of four or more biological
analytes that can be concurrently measured include, without
limitation: (a) mRNA, a lineage tracing construct, cell surface
and/or intracellular proteins and/or metabolites, and gDNA; (b)
mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or
MNase-seq), cell surface and/or intracellular proteins and/or
metabolites, and a perturbation agent (e.g., a CRISPR crRNA/sgRNA,
TALEN, zinc finger nuclease, and/or antisense oligonucleotide as
described herein); (c) mRNA, cell surface and/or intracellular
proteins and/or metabolites, an analyte capture agent (e.g., the
MHC multimers described herein), and a V(D)J sequence of an immune
cell receptor (e.g., T-cell receptor); (d) mRNA, genomic DNA, a
perturbation reagent, and accessible chromatin; (e) mRNA, cell
surface and/or intracellular proteins and/or metabolites, an
analyte capture agent (e.g., the MHC multimers described herein),
and a perturbation reagent; (f) mRNA, cell surface and/or
intracellular proteins and/or metabolites, a perturbation reagent,
and a V(D)J sequence of an immune cell receptor (e.g., T-cell
receptor); (g) mRNA, a perturbation reagent, an analyte capture
agent (e.g., the MHC multimers described herein), and a V(D)J
sequence of an immune cell receptor (e.g., T-cell receptor); (h)
mRNA, chromatin (e.g., spatial organization of chromatin in a
cell), and a perturbation reagent; (i) a V(D)J sequence of an
immune cell receptor, chromatin (e.g., spatial organization of
chromatin in a cell); and a perturbation reagent; (j) mRNA, a V(D)J
sequence of an immune cell receptor, chromatin (e.g., spatial
organization of chromatin in a cell), and genomic DNA; (k) mRNA, a
V(D)J sequence of an immune cell receptor, chromatin (e.g., spatial
organization of chromatin in a cell), and a perturbation reagent,
or any combination thereof.
[1383] (b) Construction of Spatial Arrays for Multi-Analyte
Analysis
[1384] This disclosure also provides methods and materials for
constructing a spatial array capable of multi-analyte analysis. In
some embodiments, a spatial array includes a plurality of features
on a substrate where one or more members of the plurality of
features include a plurality of oligonucleotides having a first
type functional sequence and oligonucleotides having a second,
different type of functional sequence. In some embodiments, a
feature can include oligonucleotides with two types of functional
sequences. A feature can be coupled to oligonucleotides comprising
a TruSeq functional sequence and also to oligonucleotides
comprising a Nextera functional sequence. In some embodiments, one
or more members of the plurality of features comprises both types
of functional sequences. In some embodiments, one or more members
of the plurality features includes a first type of functional
sequence. In some embodiments, one or more members of the plurality
of features includes a second type of functional sequence. In some
embodiments, an additional oligonucleotide can be added to the
functional sequence to generate a full oligonucleotide where the
full oligonucleotide includes a spatial barcode sequence, an
optional UMI sequence, a priming sequence, and a capture domain.
Attachment of these sequences can be via ligation (including via
splint ligation as is described in U.S. Patent Application
Publication No. 20140378345, the entire contents of which are
incorporated herein by reference), or any other suitable route. As
discussed herein, oligonucleotides can be hybridized with splint
sequences that can be helpful in constructing complete full
oligonucleotides (e.g., oligonucleotides that are capable of
spatial analysis).
[1385] In some embodiments, the oligonucleotides that hybridize to
the functional sequences (e.g., TruSeq and Nextera) located on the
features include capture domains capable of capturing different
types of analytes (e.g., mRNA, genomic DNA, cell surface proteins,
or accessible chromatin). In some examples, oligonucleotides that
can bind to the TruSeq functional sequences can include capture
domains that include poly(T) capture sequences. In addition to the
poly(T) capture sequences, the oligonucleotides that can bind the
TruSeq functional groups can also include a capture domain that
includes a random N-mer sequence for capturing genomic DNA (e.g.,
or any other sequence or domain as described herein capable of
capturing any of the biological analytes described herein). In such
cases, the spatial arrays can be constructed by applying ratios of
TruSeq-poly(T) and TruSeq-N-mer oligonucleotides to the features
comprising the functional TruSeq sequences. This can produce
spatial arrays where a portion of the oligonucleotides can capture
mRNA and a different portion of oligonucleotides can capture
genomic DNA. In some embodiments, one or more members of a
plurality of features include both TruSeq and Nextera functional
sequences. In such cases, a feature including both types of
functional sequences is capable of binding oligonucleotides
specific to each functional sequence. For example, an
oligonucleotide capable of binding to a TruSeq functional sequence
could be used to deliver an oligonucleotide including a poly(T)
capture domain and an oligonucleotide capable of binding to a
Nextera functional sequence could be used to deliver an
oligonucleotide including an N-mer capture domain for capturing
genomic DNA. It will be appreciated by a person of ordinary skill
in the art that any combination of capture domains (e.g., capture
domains having any of the variety of capture sequences described
herein capable of binding to any of the different types of analytes
as described herein) could be combined with oligonucleotides
capable of binding to TruSeq and Nextera functional sequences to
construct a spatial array.
[1386] In some embodiments, an oligonucleotide that includes a
capture domain (e.g., an oligonucleotide capable of coupling to an
analyte) or an analyte capture agent can include an oligonucleotide
sequence that is capable of binding or ligating to an assay primer.
The adapter can allow the capture probe or the analyte capture
agent to be attached to any suitable assay primers and used in any
suitable assays. The assay primer can include a priming region and
a sequence that is capable of binding or ligating to the adapter.
In some embodiments, the adapter can be a non-specific primer
(e.g., a 5' overhang) and the assay primer can include a 3'
overhang that can be ligated to the 5' overhang. The priming region
on the assay primer can be any primer described herein, e.g., a
poly (T) primer, a random N-mer primer, a target-specific primer,
or an analyte capture agent capture sequence.
[1387] In some examples, an oligonucleotide can includes an
adapter, e.g., a 5' overhang with 10 nucleotides. The adapter can
be ligated to assay primers, each of which includes a 3' overhang
with 10 nucleotides that complementary to the 5' overhang of the
adapter. The capture probe can be used in any assay by attaching to
the assay primer designed for that assay.
[1388] Adapters and assay primers can be used to allow the capture
probe or the analyte capture agent to be attached to any suitable
assay primers and used in any suitable assays. A capture probe that
includes a spatial barcode can be attached to a bead that includes
a poly(dT) sequence. A capture probe including a spatial barcode
and a poly(T) sequence can be used to assay multiple biological
analytes as generally described herein (e.g., the biological
analyte includes a poly(A) sequence or is coupled to or otherwise
is associated with an analyte capture agent comprising a poly(A)
sequence as the analyte capture sequence).
[1389] A splint oligonucleotide with a poly(A) sequence can be used
to facilitate coupling to a capture probe that includes a spatial
barcode and a second sequence that facilitates coupling with an
assay primer. Assay primers include a sequence complementary to the
splint oligo second sequence and an assay-specific sequence that
determines assay primer functionality (e.g., a poly(T) primer, a
random N-mer primer, a target-specific primer, or an analyte
capture agent capture sequence as described herein).
[1390] In some embodiments of any of the spatial profiling methods
described herein, a feature can include a capture probe that
includes a spatial barcode comprising a switch oligonucleotide,
e.g., with a 3' end 3rG. For example, a feature (e.g., a gel bead)
with a spatial barcode functionalized with a 3rG sequence can be
used that enables template switching (e.g., reverse transcriptase
template switching), but is not specific for any particular assay.
In some embodiments, the assay primers added to the reaction can
determine which type of analytes are analyzed. For example, the
assay primers can include binding domains capable of binding to
target biological analytes (e.g., poly (T) for mRNA, N-mer for
genomic DNA, etc.). A capture probe (e.g., an oligonucleotide
capable of spatial profiling) can be generated by using a reverse
transcriptase enzyme/polymerase to extend, which is followed by
template switching onto the barcoded adapter oligonucleotide to
incorporate the barcode and other functional sequences. In some
embodiments, the assay primers include capture domains capable of
binding to a poly(T) sequence for mRNA analysis, random primers for
genomic DNA analysis, or a capture sequence that can bind a nucleic
acid molecule coupled to an analyte binding moiety (e.g., a an
analyte capture sequence of an analyte capture agent) or a nucleic
acid molecule that can function in as a perturbation reagent (e.g.,
a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense
oligonucleotide as described herein).
V. Systems for Sample Analysis
[1391] The methods described above for analyzing biological samples
can be implemented using a variety of hardware components. In this
section, examples of such components are described. However, it
should be understood that in general, the various steps and
techniques discussed herein can be performed using a variety of
different devices and system components, not all of which are
expressly set forth.
[1392] FIG. 22A is a schematic diagram showing an example sample
handling apparatus 2200. Sample handling apparatus 2200 includes a
sample chamber 2202 that, when closed or sealed, is fluid-tight.
Within chamber 2202, a first holder 2204 holds a first substrate
2206 on which a sample 2208 is positioned. Sample chamber 2202 also
includes a second holder 2210 that holds a second substrate 2212
with an array of features 2214, as described above.
[1393] A fluid reservoir 2216 is connected to the interior volume
of sample chamber 2202 via a fluid inlet 2218. Fluid outlet 2220 is
also connected to the interior volume of sample chamber 2202, and
to valve 2222. In turn, valve 2222 is connected to waste reservoir
2224 and, optionally, to analysis apparatus 2226. A control unit
2228 is electrically connected to second holder 2210, to valve
2222, to waste reservoir 2224, and to fluid reservoir 2216.
[1394] During operation of apparatus 2200, any of the reagents,
solutions, and other biochemical components described above can be
delivered into sample chamber 2202 from fluid reservoir 2216 via
fluid inlet 2218. Control unit 2228, connected to fluid reservoir
2216, can control the delivery of reagents, solutions, and
components, and adjust the volumes and flow rates according to
programmed analytical protocols for various sample types and
analysis procedures. In some embodiments, fluid reservoir 2216
includes a pump, which can be controlled by control unit 2228, to
facilitate delivery of substances into sample chamber 2202.
[1395] In certain embodiments, fluid reservoir 2216 includes a
plurality of chambers, each of which is connected to fluid inlet
2218 via a manifold (not shown). Control unit 2228 can selectively
deliver substances from any one or more of the multiple chambers
into sample chamber 2202 by adjusting the manifold to ensure that
the selected chambers are fluidically connected to fluid inlet
2218.
[1396] In general, control unit 2228 can be configured to introduce
substances from fluid reservoir 2216 into sample chamber 2202
before, after, or both before and after, sample 2208 on first
substrate 2206 has interacted with the array of features 2214 on
first substrate 2212. Many examples of such substances have been
described previously. Examples of such substances include, but are
not limited to, permeabilizing agents, buffers, fixatives, staining
solutions, washing solutions, and solutions of various biological
reagents (e.g., enzymes, peptides, oligonucleotides, primers).
[1397] To initiate interaction between sample 2208 and feature
array 2214, the sample and array are brought into spatial
proximity. To facilitate this step, second holder 2210--under the
control of control unit 2228--can translate second substrate 2212
in any of the x-, y-, and z-coordinate directions. In particular,
control unit 2228 can direct second holder 2210 to translate second
substrate 2212 in the z-direction so that sample 2208 contacts, or
nearly contacts, feature array 2214.
[1398] In some embodiments, apparatus 2200 can optionally include
an alignment sub-system 2230, which can be electrically connected
to control unit 2228. Alignment sub-system 2230 functions to ensure
that sample 2208 and feature array 2214 are aligned in the x-y
plane prior to translating second substrate 2212 in the z-direction
so that sample 2208 contacts, or nearly contacts, feature array
2214.
[1399] Alignment sub-system 2230 can be implemented in a variety of
ways. In some embodiments, for example, alignment sub-system 2230
includes an imaging unit that obtains one or more images showing
fiducial markings on first substrate 2206 and/or second substrate
2212. Control unit 2218 analyzes the image(s) to determine
appropriate translations of second substrate 2212 in the x- and/or
y-coordinate directions to ensure that sample 2208 and feature
array 2214 are aligned prior to translation in the z-coordinate
direction.
[1400] In certain embodiments, control unit 2228 can optionally
regulate the removal of substances from sample chamber 2202. For
example, control unit 2228 can selectively adjust valve 2222 so
that substances introduced into sample chamber 2202 from fluid
reservoir 2216 are directed into waste reservoir 2224. In some
embodiments, waste reservoir 2224 can include a reduced-pressure
source (not shown) electrically connected to control unit 2228.
Control unit 2228 can adjust the fluid pressure in fluid outlet
2220 to control the rate at which fluids are removed from sample
chamber 2202 into waste reservoir 2224.
[1401] In some embodiments, analytes from sample 2208 or from
feature array 2214 can be selectively delivered to analysis
apparatus 2226 via suitable adjustment of valve 2222 by control
unit 2228. As described above, in some embodiments, analysis
apparatus 2226 includes a reduced-pressure source (not shown)
electrically connected to control unit 2228, so that control unit
2228 can adjust the rate at which analytes are delivered to
analysis apparatus 2226. As such, fluid outlet 2220 effectively
functions as an analyte collector, while analysis of the analytes
is performed by analysis apparatus 2226. It should be noted that
not all of the workflows and methods described herein are
implemented via analysis apparatus 2226. For example, in some
embodiments, analytes that are captured by feature array 2214
remain bound to the array (i.e., are not cleaved from the array),
and feature array 2214 is directly analyzed to identify
specifically-bound sample components.
[1402] In addition to the components described above, apparatus
2200 can optionally include other features as well. In some
embodiments, for example, sample chamber 2202 includes a heating
sub-system 2232 electrically connected to control unit 2228.
Control unit 2228 can activate heating sub-system 2232 to heat
sample 2208 and/or feature array 2214, which can help to facilitate
certain steps of the methods described herein.
[1403] In certain embodiments, sample chamber 2202 includes an
electrode 2234 electrically connected to control unit 2228. Control
unit 2228 can optionally activate electrode 2234, thereby
establishing an electric field between the first and second
substrates. Such fields can be used, for example, to facilitate
migration of analytes from sample 2208 toward feature array
2214.
[1404] In some of the methods described herein, one or more images
of a sample and/or a feature array are acquired. Imaging apparatus
that is used to obtain such images can generally be implemented in
a variety of ways. FIG. 22B shows one example of an imaging
apparatus 2250. Imaging apparatus 2250 includes a light source
2252, light conditioning optics 2254, light delivery optics 2256,
light collection optics 2260, light adjusting optics 2262, and a
detection sub-system 2264. Each of the foregoing components can
optionally be connected to control unit 2228, or alternatively, to
another control unit. For purposes of explanation below, it will be
assumed that control unit 2228 is connected to the components of
imaging apparatus 2250.
[1405] During operation of imaging apparatus 2250, light source
2252 generates light. In general, the light generated by source
2252 can include light in any one or more of the ultraviolet,
visible, and/or infrared regions of the electromagnetic spectrum. A
variety of different light source elements can be used to generate
the light, including (but not limited to) light emitting diodes,
laser diodes, laser sources, fluorescent sources, incandescent
sources, and glow-discharge sources.
[1406] The light generated by light source 2252 is received by
light conditioning optics 2254. In general, light conditioning
optics 2254 modify the light generated by light source 2252 for
specific imaging applications. For example, in some embodiments,
light conditioning optics 2254 modify the spectral properties of
the light, e.g., by filtering out certain wavelengths of the light.
For this purpose, light conditioning optics 2254 can include a
variety of spectral optical elements, such as optical filters,
gratings, prisms, and chromatic beam splitters.
[1407] In certain embodiments, light conditioning optics 2254
modify the spatial properties of the light generated by light
source 2252. Examples of components that can be used for this
purpose include (but are not limited to) apertures, phase masks,
apodizing elements, and diffusers.
[1408] After modification by light conditioning optics 2254, the
light is received by light delivery optics 2256 and directed onto
sample 2208 or feature array 2214, either of which is positioned on
a mount 2258. Light conditioning optics 2254 generally function to
collect and direct light onto the surface of the sample or array. A
variety of different optical elements can be used for this purpose,
and examples of such elements include, but are not limited to,
lenses, mirrors, beam splitters, and various other elements having
non-zero optical power.
[1409] Light emerging from sample 2208 or feature array 2214 is
collected by light collection optics 2260. In general, light
collection optics 2260 can include elements similar to any of those
described above in connection with light delivery optics 2256. The
collected light can then optionally be modified by light adjusting
optics 2262, which can generally include any of the elements
described above in connection with light conditioning optics
2254.
[1410] The light is then detected by detection sub-system 2264.
Generally, detection sub-system 2264 functions to generate one or
more images of sample 2208 or feature array 2214 by detecting light
from the sample or feature array. A variety of different imaging
elements can be used in detection sub-system 2264, including CCD
detectors and other image capture devices.
[1411] Each of the foregoing components can optionally be connected
to control unit 2228 as shown in FIG. 22B, so that control unit
2228 can adjust various properties of the imaging apparatus. For
example, control unit 2228 can adjust the position of sample 2208
or feature array 2214 relative to the position of the incident
light, and also with respect to the focal plane of the incident
light (if the incident light is focused). Control unit 2228 can
also selectively filter both the incident light and the light
emerging from the sample.
[1412] Imaging apparatus 2250 can typically obtain images in a
variety of different imaging modalities. In some embodiments, for
example, the images are transmitted light images, as shown in FIG.
22B. In certain embodiments, apparatus 2250 is configured to obtain
reflection images. In some embodiments, apparatus 2250 can be
configured to obtain birefringence images, fluorescence images,
phosphorescence images, multiphoton absorption images, and more
generally, any known image type.
[1413] In general, control unit 2228 can perform any of the method
steps described herein that do not expressly require user
intervention by transmitting suitable control signals to the
components of sample handling apparatus 2200 and/or imaging
apparatus 2250. To perform such steps, control unit 2228 generally
includes software instructions that, when executed, cause control
unit 2228 to undertake specific steps. In some embodiments, control
unit 2228 includes an electronic processor and software
instructions that are readable by the electronic processor, and
cause the processor to carry out the steps describe herein. In
certain embodiments, control unit 2228 includes one or more
application-specific integrated circuits having circuit
configurations that effectively function as software
instructions.
[1414] Control unit 2228 can be implemented in a variety of ways.
FIG. 22C is a schematic diagram showing one example of control unit
2228, including an electronic processor 2280, a memory unit 2282, a
storage device 2284, and an input/output interface 2286. Processor
2280 is capable of processing instructions stored in memory unit
2282 or in storage device 2284, and to display information on
input/output interface 2286.
[1415] Memory unit 2282 stores information. In some embodiments,
memory unit 2282 is a computer-readable medium. Memory unit 2282
can include volatile memory and/or non-volatile memory. Storage
device 2284 is capable of providing mass storage, and in some
embodiments, is a computer-readable medium. In certain embodiments,
storage device 2284 may be a floppy disk device, a hard disk
device, an optical disk device, a tape device, a solid state
device, or another type of writeable medium.
[1416] The input/output interface 2286 implements input/output
operations. In some embodiments, the input/output interface 2286
includes a keyboard and/or pointing device. In some embodiments,
the input/output interface 2286 includes a display unit for
displaying graphical user interfaces and/or display
information.
[1417] Instructions that are executed and cause control unit 2228
to perform any of the steps or procedures described herein can be
implemented in digital electronic circuitry, or in computer
hardware, firmware, or in combinations of these. The instructions
can be implemented in a computer program product tangibly embodied
in an information carrier, e.g., in a machine-readable storage
device, for execution by a programmable processor (e.g., processor
2280). The computer program can be written in any form of
programming language, including compiled or interpreted languages,
and it can be deployed in any form, including as a stand-alone
program or as a module, component, subroutine, or other unit
suitable for use in a computing environment. Storage devices
suitable for tangibly embodying computer program instructions and
data include all forms of non-volatile memory, including by way of
example semiconductor memory devices, such as EPROM, EEPROM, and
flash memory devices; magnetic disks such as internal hard disks
and removable disks; magneto-optical disks; and CD-ROM and DVD-ROM
disks. The processor and the memory can be supplemented by, or
incorporated in, ASICs (application-specific integrated
circuits).
[1418] Processor 2280 can include any one or more of a variety of
suitable processors. Suitable processors for the execution of a
program of instructions include, by way of example, both general
and special purpose microprocessors, and the sole processor or one
of multiple processors of any kind of computer or computing
device.
Exemplary Embodiments
[1419] In some non-limiting examples of the workflows described
herein, the sample can be immersed in 100% chilled methanol and
incubated for 30 minutes at -20.degree. C. After 20 minutes, the
sample can be removed and rinsed in ultrapure water. After rinsing
the sample, fresh eosin solution is prepared, and the sample can be
covered in isopropanol. After incubating the sample in isopropanol
for 1 minute, the reagent can be removed by holding the slide at an
angle, where the bottom edge of the slide can be in contact with a
laboratory wipe and air dried. The sample can be uniformly covered
in hematoxylin solution and incubated for 7 minutes at room
temperature. After incubating the sample in hematoxylin for 7
minutes, the reagent can be removed by holding the slide at an
angle, where the bottom edge of the slide can be in contact with a
laboratory wipe. The slide containing the sample can be immersed in
water and the excess liquid can be removed. After that, the sample
can be covered with blueing buffer and can be incubated for 2
minutes at room temperature. The slide containing the sample can
again be immersed in water, and uniformly covered with eosin
solution and incubated for 1 minute at room temperature. The slide
can be air-dried for no more than 30 minutes and incubated for 5
minutes at 37.degree. C. The sample can be imaged using brightfield
imaging setting.
[1420] Further, the biological sample can be processed by the
following exemplary steps for sample permeabilization and cDNA
generation. The sample can be exposed to a permeabilization enzyme
and incubated at 37.degree. C. for the pre-determined
permeabilization time (which is tissue type specific). The
permeabilization enzyme can be removed and the sample prepared for
analyte capture by adding 0.1.times.SSC buffer. The sample can then
subjected to a pre-equilibration thermocycling protocol (e.g., lid
temperature and pre-equilibrate at 53.degree. C., reverse
transcription at 53.degree. C. for 45 minutes, and then hold at
4.degree. C.) and the SSC buffer can be removed. A Master Mix,
containing nuclease-free water, a reverse transcriptase reagent, a
template switch oligo, a reducing agent, and a reverse
transcriptase enzyme can be added to the biological sample and
substrate, and the sample with the Master Mix can be subjected to a
thermocycling protocol (e.g., perform reverse transcription at
53.degree. C. for 45 minutes and hold at 4.degree. C.). Second
strand synthesis can be performed on the substrate by subjecting
the substrate to a thermocycling protocol (e.g., pre-equilibrate at
65.degree. C., second strand synthesis at 65.degree. C. for 15
minutes, then hold at 4.degree. C.). The Master Mix reagents can be
removed from the sample and 0.8M KOH can be applied and incubated
for 5 minutes at room temperature. The KOH can be removed and
elution buffer can be added and removed from the sample. A Second
Strand Mix, including a second strand reagent, a second strand
primer, and a second strand enzyme, can be added to the sample and
the sample can be sealed and incubated. At the end of the
incubation, the reagents can be removed and elution buffer can be
added and removed from the sample, and 0.8M KOH can be added again
to the sample and the sample can be incubated for 10 minutes at
room temperature. Tris-HCl can be added and the reagents can be
mixed. The sample can be transferred to a new tube, vortexed, and
placed on ice.
[1421] Further the biological sample can be processed by the
following exemplary steps for cDNA amplification and quality
control. A qPCR Mix, including nuclease-free water, qPCR Master
Mix, and cDNA primers, can be prepared and pipetted into wells in a
qPCR plate. A small amount of sample can be added to the plated
qPCR Mix, and thermocycled according to a predetermined
thermocycling protocol (e.g., step 1: 98.degree. C. for 3 minutes,
step 2: 98.degree. C. for 5 seconds, step 3: 63.degree. C. for 30
seconds, step 4: record amplification signal, step 5: repeating
98.degree. C. for 5 seconds, 63.degree. C. for 30 seconds for a
total of 25 cycles). After completing the thermocycling, a cDNA
amplification mix, including amplification mix and cDNA primers,
can be prepared and combined with the remaining sample and mixed.
The sample can then be incubated and thermocycled (e.g., lid
temperature at 105.degree. C. for .about.45-60 minutes; step 1:
98.degree. C. for 3 minutes, step 2: 98.degree. C. for 15 seconds,
step 3: 63.degree. C. for 20 seconds, step 4: 72.degree. C. for one
minute, step 5: [the number of cycles determined by qPCR Cq
Values], step 6: 72.degree. C. for 1 minute, and step 7: hold at
4.degree. C.). The sample can then be stored at 4.degree. C. for up
to 72 hours or at -20.degree. C. for up to 1 week, or resuspended
in 0.6.times. SPRIselect Reagent and pipetted to ensure proper
mixing. The sample can then be incubated at 5 minutes at room
temperature, and cleared by placing the sample on a magnet (e.g.,
the magnet is in the high position). The supernatant can be removed
and 80% ethanol can be added to the pellet, and incubated for 30
seconds. The ethanol can be removed and the pellet can be washed
again. The sample can then be centrifuged and placed on a magnet
(e.g., the magnet is on the low position). Any remaining ethanol
can be removed and the sample can be air dried for up to 2 minutes.
The magnet can be removed and elution buffer can be added to the
sample, mixed, and incubated for 2 minutes at room temperature. The
sample can then be placed on the magnet (e.g., on low position)
until the solution clears. The sample can be transferred to a new
tube strip and stored at 4.degree. C. for up to 72 hours or at
-20.degree. C. for up to 4 weeks. A portion of the sample can be
run on an Agilent Bioanalyzer High Sensitivity chip, where a region
can be selected and the cDNA concentration can be measured to
calculate the total cDNA yield. Alternatively, the quantification
can be determined by Agilent Bioanalyzer or Agilent
TapeStation.
[1422] Further, the biological sample can be processed by the
following exemplary steps for spatial gene expression library
construction. A Fragmentation Mix, including a fragmentation buffer
and fragmentation enzyme, can be prepared on ice. Elution buffer
and fragmentation mix can be added to each sample, mixed, and
centrifuged. The sample mix can then be placed in a thermocycler
and cycled according to a predetermined protocol (e.g., lid
temperature at 65.degree. C. for .about.35 minutes, pre-cool block
down to 4.degree. C. before fragmentation at 32.degree. C. for 5
minutes, End-repair and A-tailing at 65.degree. C. for 30 minutes,
and holding at 4.degree. C.). The 0.6.times. SPRIselect Reagent can
be added to the sample and incubated at 5 minutes at room
temperature. The sample can be placed on a magnet (e.g., in the
high position) until the solution clears, and the supernatant can
be transferred to a new tube strip. 0.8.times. SPRIselect Reagent
can be added to the sample, mixed, and incubated for 5 minutes at
room temperature. The sample can be placed on a magnet (e.g., in
the high position) until the solution clears. The supernatant can
be removed and 80% ethanol can be added to the pellet, the pellet
can be incubated for 30 seconds, and the ethanol can be removed.
The ethanol wash can be repeated and the sample placed on a magnet
(e.g., in the low position) until the solution clears. The
remaining ethanol can be removed and elution buffer can be added to
the sample, mixed, and incubated for 2 minutes at room temperature.
The sample can be placed on a magnet (e.g., in the high position)
until the solution clears, and a portion of the sample can be moved
to a new tube strip. An Adaptor Ligation Mix, including ligation
buffer, DNA ligase, and adaptor oligos, can be prepared and
centrifuged. The Adaptor Ligation Mix can be added to the sample,
pipette-mixed, and centrifuged briefly. The sample can then be
thermocycled according to a predetermined protocol (e.g., lid
temperature at 30.degree. C. for .about.15 minutes, step 1:
20.degree. C. for 15 minutes, step 2: 4.degree. C. hold). The
sample can be vortexed to re-suspend SPRIselect Reagent, additional
0.8.times. SPRIselect Reagent can be added to the sample and
incubated for 5 minutes at room temperature, and placed on a magnet
(e.g., in the high position) until the solution clears. The
supernatant can be removed and the pellet can be washed with 80%
ethanol, incubated for 30 seconds, and the ethanol can be removed.
The ethanol wash can be repeated, and the sample can be centrifuged
briefly before placing the sample on a magnet (e.g., in the low
position). Any remaining ethanol can be removed and the sample can
be air dried for a maximum of 2 minutes. The magnet can be removed,
and elution buffer can be added to the sample, and the sample can
be pipette-mixed, incubated for 2 minutes at room temperature, and
placed on a magnet (e.g., in the low position) until the solution
clears. A portion of the sample can be transferred to a new tube
strip. Amplification mix, can be prepared and combined with the
sample. An individual Dual Index TT Set A can be added to the
sample, pipette-mixed and subjected to a pre-determined
thermocycling protocol (e.g., lid temperature at 105.degree. C. for
.about.25-40 minutes, step 1: 98.degree. C. for 45 seconds, step 2:
98.degree. C. for 20 seconds, step 3: 54.degree. C. for 30 seconds;
step 4: 72.degree. C. for 20 seconds, step 5: reverting to step 2
for a predetermined number of cycles, step 6: 72.degree. C. for 1
minute, and 4.degree. C. on hold). Vortex to re-suspend the
SPRIselect Reagent, additional 0.6.times. SPRIselect Reagent can be
added to each sample, mixed, and incubated for 5 minutes at room
temperature. The sample can be placed on a magnet (e.g., in the
high position) until the solution clears, and the supernatant can
be transferred to a new tube strip. The 0.8.times. SPRIselect
Reagent can be added to each sample, pipette-mixed, and incubated
for 5 minutes at room temperature. The sample can then be placed on
a magnet (e.g., in the high position) until the solution clears.
The supernatant can be removed, and the pellet can be washed with
80% ethanol, incubated for 30 seconds, and then the ethanol can be
removed. The ethanol wash can be repeated, the sample centrifuged,
and placed on a magnet (e.g., in the low position) to remove any
remaining ethanol. The sample can be removed from the magnet and
Elution Buffer can be added to the sample, pipette-mixed, and
incubated at 2 minutes at room temperature. The sample can be
placed on a magnet (e.g., in the low position) until the solution
clears and a portion of the sample can be transferred to a new tube
strip. The sample can be stored at 4.degree. C. for up to 72 hours,
or at -20.degree. C. for long-term storage. The average fragment
size can be determined using a Bioanalyzer trace or an Agilent
TapeStation.
[1423] The library can be sequenced using available sequencing
platforms, including, MiSeq, NextSeq 500/550, HiSeq 2500, HiSeq
3000/4000, NovaSeq, and iSeq.
[1424] In alternate embodiments of the above described workflows, a
biological sample can be permeabilized by exposing the sample to
greater than about 1.0 w/v % (e.g., greater than about 2.0 w/v %,
greater than about 3.0 w/v %, greater than about 4.0 w/v %, greater
than about 5.0 w/v %, greater than about 6.0 w/v %, greater than
about 7.0 w/v %, greater than about 8.0 w/v %, greater than about
9.0 w/v %, greater than about 10.0 w/v %, greater than about 11.0
w/v %, greater than about 12.0 w/v %, or greater than about 13.0
w/v %) sodium dodecyl sulfate (SDS). In some embodiments, a
biological sample can be permeabilized by exposing the sample
(e.g., for about 5 minutes to about 1 hour, about 5 minutes to
about 40 minutes, about 5 minutes to about 30 minutes, about 5
minutes to about 20 minutes, or about 5 minutes to about 10
minutes) to about 1.0 w/v % to about 14.0 w/v % (e.g., about 2.0
w/v % to about 14.0 w/v %, about 2.0 w/v % to about 12.0 w/v %,
about 2.0 w/v % to about 10.0 w/v %, about 4.0 w/v % to about 14.0
w/v %, about 4.0 w/v % to about 12.0 w/v %, about 4.0 w/v % to
about 10.0 w/v %, about 6.0 w/v % to about 14.0 w/v %, about 6.0
w/v % to about 12.0 w/v %, about 6.0 w/v % to about 10.0 w/v %,
about 8.0 w/v % to about 14.0 w/v %, about 8.0 w/v % to about 12.0
w/v %, about 8.0 w/v % to about 10.0 w/v %, about 10.0% w/v % to
about 14.0 w/v %, about 10.0 w/v % to about 12.0 w/v %, or about
12.0 w/v % to about 14.0 w/v %) SDS and/or proteinase K (e.g., at a
temperature of about 35.degree. C. to about 50.degree. C., about
35.degree. C. to about 45.degree. C., about 35.degree. C. to about
40.degree. C., about 40.degree. C. to about 50.degree. C., about
40.degree. C. to about 45.degree. C., or about 45.degree. C. to
about 50.degree. C.).
[1425] In non-limiting examples of any of the workflows described
herein, a nucleic acid molecule is produced that includes a
contiguous nucleotide sequence comprising: (a) a first primer
sequence (e.g., Read 1); (b) a spatial barcode; (c) a unique
molecular sequence (UMI); (d) a capture domain; (e) a sequence
complementary to a sequence present in a nucleic acid from a
biological sample; (f) a second primer sequence (e.g., Read 2) that
is substantially complementary to a sequence of a template
switching oligonucleotide (TSO). In some embodiments of these
nucleic acid molecules, the nucleic acid molecule is a
single-stranded nucleic acid molecule. In some embodiments of these
nucleic acid molecules, the nucleic acid molecule is a
double-stranded nucleic acid molecule. In some embodiments of these
nucleic acid molecules, (a) through (f) are positioned in a 5' to
3' direction in the contiguous nucleotide sequence. In some
embodiments of any of these nucleic acid molecules, the nucleic
acid molecule is attached to a substrate (e.g., a slide). In some
embodiments of any of these nucleic acid molecules, the 5' end of
the contiguous nucleic acid sequence is attached to the substrate
(e.g., a slide). In some embodiments of any of these nucleic acid
molecules, the contiguous nucleotide sequence is a chimeric RNA and
DNA sequence. In some embodiments of any of these nucleic acid
molecules, the contiguous nucleotide sequence is a DNA
sequence.
[1426] In non-limiting examples of any of the workflows described
herein, a nucleic acid molecule is produced that includes a
contiguous nucleotide sequence comprising: (a) a sequence
complementary to a first primer sequence (e.g., a sequence
complementary to Read 1); (b) a sequence complementary to a spatial
barcode; (c) a sequence complementary to a unique molecular
sequence; (d) a sequence complementary to a capture domain; (e) a
sequence present in a nucleic acid from a biological sample; and
(f) a sequence of a template switching oligonucleotide (TSO). In
some embodiments of any of these nucleic acid molecules, the
nucleic acid molecule is single-stranded. In some embodiments of
any of these nucleic acid molecules, the nucleic acid molecule is
double-stranded. In some embodiments of any of these nucleic acid
molecules, the contiguous nucleotide sequence is a DNA sequence. In
some embodiments of any of these nucleic acid molecules, (a)
through (f) are positioned in a 3' to 5' direction in the
contiguous nucleotide sequence.
[1427] In non-limiting examples of any of the workflows described
herein, a nucleic acid molecule is produced that includes a
contiguous nucleotide sequence comprising: (a) a first primer
sequence (e.g., Read 1); (b) a spatial barcode; (c) a unique
molecular sequence (UMI); (d) a capture domain; (e) a sequence
complementary to a sequence present in a nucleic acid from a
biological sample; and (f) a second primer sequence (Read 2). In
some embodiments of any of these nucleic acid molecules, the
nucleic acid molecule is a single-stranded nucleic acid molecule.
In some embodiments of any of these nucleic acid molecules, the
nucleic acid molecule is a double-stranded nucleic acid molecule.
In some embodiments of any of these nucleic acid molecules, (a)
through (f) are positioned in a 5' to 3' direction in the
contiguous nucleotide sequence. In some embodiments of any of these
nucleic acid molecules, the contiguous nucleotide sequence is a DNA
sequence. In some embodiments of any of these nucleic acid
molecules, the contiguous nucleotide sequence further comprises 3'
to (0: (g) a sequence complementary to a first adaptor sequence;
and (h) a sequence complementary to a third primer sequence. In
some embodiments of any of the nucleic acid molecules, the first
adaptor sequence is an i7 sample index sequence. In some
embodiments of any of these nucleic acid molecules, the third
primer sequence is a P7 primer sequence. In some embodiments of any
of these nucleic acid molecules, (h) is 3' positioned relative to
(g) in the contiguous nucleotide sequence. In some embodiments of
any of these nucleic acid molecules, the contiguous nucleotide
sequence further comprises 5' to (a): (i) a second adaptor
sequence; and (ii) a fourth primer sequence. In some embodiments of
any of these nucleic acid molecules, the second adaptor sequence is
an i5 sample index sequence. In some embodiments of any of these
nucleic acid molecules, the fourth primer sequence is a P5 primer
sequence. In some embodiments of any of these nucleic acid
molecules, (ii) is 5' positioned relative to (i) in the contiguous
nucleotide sequence.
[1428] In non-limiting examples of any of the workflows described
herein, a nucleic acid molecule is produced that includes a
contiguous nucleotide sequence comprising: (a) a sequence
complementary to a first primer sequence; (b) a sequence
complementary to a spatial barcode; (c) a sequence complementary to
a unique molecular sequence; (d) a sequence complementary to a
capture domain; (e) a sequence present in a nucleic acid from a
biological sample; and (f) a sequence complementary to a second
primer sequence. In some embodiments of these nucleic acid
molecules, a sequence complementary to a first primer sequence is a
sequence complementary to Read 1. In some embodiments of these
nucleic acid molecules, a sequence complementary to a second primer
sequence is a sequence complementary to Read 2. In some embodiments
of any of these nucleic acid molecules, the nucleic acid molecule
is a single-stranded nucleic acid molecule. In some embodiments of
any of these nucleic acid molecules, the nucleic acid molecule is a
double-stranded nucleic acid molecule. In some embodiments of any
of these nucleic acid molecules, (a) through (0 are positioned in a
3' to 5' direction in the contiguous nucleotide sequence. In some
embodiments of any of these nucleic acid molecules, the contiguous
nucleotide sequence is a DNA sequence. In some embodiments of any
of these nucleic acid molecules, the contiguous nucleotide sequence
further comprises 5' to (0: (g) a first adaptor sequence; and (h) a
third primer sequence. In some embodiments of any of these nucleic
acid molecules, the first adaptor sequence is an i7 sample index
sequence. In some embodiments of any of these nucleic acid
molecules, the third primer sequence is a P7 primer sequence. In
some embodiments of any of these nucleic acid molecules, (h) is 5'
positioned relative to (g) in the contiguous nucleotide sequence.
In some embodiments of any of these nucleic acid molecules, the
contiguous nucleotide sequence further comprises 3' to (a): (i) a
sequence complementary to a second adaptor sequence; and (ii) a
sequence complementary to a fourth primer sequence. In some
embodiments of any of these nucleic acid molecules, the second
adaptor sequence is an i5 sample index sequence. In some
embodiments of any of these nucleic acid molecules, the fourth
primer sequence is a P5 primer sequence. In some embodiments of any
of these nucleic acid molecules, (ii) is 3' positioned relative to
(i) in the contiguous nucleotide sequence.
Spatial RIN
[1429] Provided herein is a non-limiting example of a protocol for
determining the spatial RIN in a tissue that can include collecting
breast cancer tissue and snap freezing in liquid nitrogen. Tissue
can be embedded in OCT and sectioned at 10 or 12 .mu.M thickness at
-20.degree. C. and mounted directly on a spatial array including
capture probes having an 18S rRNA capture domain. Tissue can be
fixed and stained using a Hematoxylin and Eosin (H&E) staining
protocol. Briefly, Mayer's Hematoxylin can be added, washed in
water, incubated in Bluing buffer, washed in water, stained with
Eosin, then washed in water, and finally dried. For visualizing
H&E staining, sections can be mounted with 85% glycerol and
covered with a coverslip. Bright field imaging can be performed
using the Metafer Slide Scanning Platform (Metasystems) where raw
images are stitched together with the VSlide software
(Metasystems). Glycerol can be removed by holding the spatial array
or glass slide in water until the coverslip falls off and then was
air dry until the remaining liquid evaporates. Hematoxylin, from
the H&E stain, can be optionally removed from the tissue
section, for example, by washing in dilute HCl (0.01M) prior to
further processing. The tissue sections are then ready for further
processing.
[1430] Following staining, the 18S rRNA present in the tissue
sections are captured by the 18S rRNA specific capture domains on
the spatial array. The 18S rRNA is then converted to cDNA in situ.
Specifically, reverse transcription is performed on the spatial
array in a sealed hybridization cassette by adding 70 .mu.l
reaction mixture including 1.times. First-strand buffer, 5 mM DTT,
1 M Betaine, 6 mM MgCl2, 1 mM dNTPs, 0.2 mg/ml BSA, 50 ng/.mu.l
Actinomycin D, 10% DMSO, 20 U/.mu.l SuperScript III Reverse
Transcriptase, 2 U/.mu.l RNaseOUT Recombinant Ribonuclease
Inhibitor. The reaction is performed overnight at 42.degree. C.
overnight. After incubation cDNA synthesis mixture is removed and
the tissue was washed with 0.1.times.SSC buffer.
[1431] In order to prepare the spatial array for oligonucleotide
probe labeling and imaging, the breast cancer tissue and rRNA is
removed. Tissue removal can be performed first by incubation with
.beta.-mercaptoethanol in RLT lysis buffer at a 3:100 ratio at
56.degree. C. for 1 hour with continuous shaking at 300 rpm. All
tissues can be incubated with a 1:7 ratio of Proteinase K and PKD
buffer for 1 hour at 56.degree. C. using short intervals with
gentle shaking at 300 rpm. The spatial array is then washed with
continuous shaking at 300 rpm as follows: first in 2.times.SSC with
0.1% SDS at 50.degree. C. for 10 min, then in 0.2.times.SSC at RT
for 1 min and finally in 0.1.times.SSC at RT for 1 min. The spatial
array is then spin-dried and put back into the hybridization
cassette. rRNA removal can be performed using a reaction mixture
containing the following final concentrations: 1.times.
First-strand buffer, 0.4 mg/ml BSA and 16.3 mU/.mu.l RNase H. The
reaction can be performed for 1 hour at 37.degree. C. with gentle
shaking at 300 rpm using short intervals. Spatial arrays are then
washed with 0.1.times.SSC buffer and treated with 60% DMSO at room
temperature for 5 minutes and then washed three times with
0.1.times.SSC buffer.
[1432] In order to detect the cDNA produced from the 18S rRNA,
labeled oligonucleotide probes are generated that had sequence
complementarity to the 18S cDNA sequence. Oligonucleotides are
identified that have a length between 18-23 nucleotides with an
optimum at 20 nucleotides, a melting temperature (Tm) between
38-50.degree. C. with the optimal temperature at 42.degree. C., and
a content of guanine and cytosine of 30-60% with an optimum at 50%.
The first five bases of the 3'-ends are set to include two of
either guanine or cytosine or one of each. Oligonucleotides are
checked for quality using Mfold (determination of secondary
structure), Oligo Calc: Oligonucleotide Properties Calculator
(determination of self-dimerization and hairpin formation), and
BLAST (determination of off-target binding). The sequence locations
are picked for compatibility with both human (NR_003286.2) and
mouse (NR_003278.3) 18S rRNA. Four of the oligonucleotide probes
selected include: probe 1 (P1; SEQ ID NO: 4) GAGGAATTCCCAGTAAGT,
probe 2 (P2; SEQ ID NO: 5) GAGATTGAGCAATAACAG, probe 3 (P3; SEQ ID
NO: 6) GTAGTTCCGACCATAAAC, and probe 4 (P4; SEQ ID NO: 7)
GGTGACTCTAGATAACCT. Control oligonucleotide probes can be designed
to include complementary sequences of three detection probes at a
time with a 20 bases spacer sequence between each probe. The
selected oligonucleotide probes can be then designed to incorporate
a Cy3 fluorophore.
[1433] Next, labeled oligonucleotide probes are hybridized to the
spatial array containing cDNA produced from the 18S rRNA or
containing control capture probes. This step can include at least 4
successive rounds of hybridization and imaging, with at least one
round for each of the four labeled oligonucleotide probes.
Following each round of hybridization and imaging, the spatial
array can be washed to remove the hybridized probe before
continuing with a subsequent round of hybridization and
imaging.
[1434] Hybridization of labeled oligonucleotide probes includes
adding to the spatial arrays a pre-heated (e.g., heated to
50.degree. C.) hybridization mixture (10 mM Tris-HCl, 1 mM EDTA, 50
mM
[1435] NaCl, and 0.5 .mu.M of fluorescently labelled probe)
containing at least 0.5 .mu.M of one of the fluorescently labeled
oligonucleotide probes (e.g., one of P1, P2, P3 or P4). The spatial
array can be then imaged using a DNA microarray scanner with the
following settings: excitation wavelength 532 nm set to gain 70 and
635 nm set to 1. Following imaging, the spatial array is incubated
with 60% DMSO at room temperature for 5 minutes and washed three
times with 0.1.times.SSC buffer to remove the hybridized probe.
Subsequent rounds of hybridization and imaging are performed with a
different labeled oligonucleotide probe used in each round (e.g.,
round 2 used P2, round 3 used P3, and round 4 used P4). An initial,
pre-hybridization round (P0) of imaging are performed in order to
assess background fluorescence.
[1436] One image is generated per labeled oligonucleotide probe
(P1-P4) and also one where no fluorescently labeled probes were
hybridized (P0). Normalization of Fluorescence Units (FU) data is
done by subtraction of the auto-fluorescence recorded with P0 and
division with P1. After aligning the five images for a particular
location in the tissue (one image from each probe, P1-P4, and one
image from the location without labeling), the images are loaded
into a script and run in RStudio. The Script can generate two
different plots, one heat-map of spatial RIN values and one image
alignment error plot.
[1437] High quality RNA is defined as full-length (or close to
full-length) transcripts, whereas low quality RNA is defined as
fragmented transcripts. Spatial RIN values are similar to
traditional RIN values in that the RIN values range from 1 to 10,
with higher numbers indicating higher quality (e.g., less degraded,
less fragmented) RNA samples.
Spatial ATAC Compositions
[1438] Provided herein are compositions for identifying the
location of an analyte in a biological sample. In some embodiments,
a nucleotide molecule composition including a) a spatial barcode b)
a unique molecule c) capture domain; d) a functional domain; and e)
a splint oligonucleotide. In some embodiments, a partially
double-stranded nucleotide molecule composition including: a) a
spatial barcode; b) a unique molecular identifier; c) a capture
domain; d) a functional domain; and e) a splint oligonucleotide. In
some embodiments, a composition including a) a capture probe
including i) a spatial barcode; ii) a unique molecular identifier;
iii) a capture domain; iv) a functional domain; and v) a splint
oligonucleotide and b) fragmented genomic DNA including i) a first
adapter sequence comprising a transposon end sequence and a
sequence complementary to the capture domain; and ii) a second
adapter sequence comprising the transposon end sequence and a
second adapter sequence. In some embodiments, a composition
including a) a transposase enzyme monomer complexed with a first
adapter including i) a transposon end sequence; and ii) a sequence
complementary to the capture domain; and b) a transposase enzyme
second monomer complexed with a second adapter including i) a
transposon end sequence; and ii) a second adapter sequence; c)
genomic DNA; and d) a capture probe, including i) a spatial
barcode; ii) a unique molecular identifier; iii) a capture domain;
iv) a functional domain; and v) a splint oligonucleotide. In some
embodiments, a composition including a) a transposase enzyme
monomer complexed with a first adapter including i) a transposon
end sequence; ii) a sequence complementary to the capture domain;
wherein the 5' end of the first adapter is phosphorylated and b) a
transposase enzyme second monomer complexed with a second adapter
including i) the transposon end sequence; ii) a second adapter
sequence; wherein the 5' end of the second adapter is
phosphorylated; c) genomic DNA; and d) a capture probe including i)
a spatial barcode; ii) a unique molecular identifier; iii) a
capture domain; iv) a functional domain; and v) a splint
oligonucleotide. In some embodiments, a composition including a) a
transposase enzyme dimer including i) a transposase enzyme monomer
complexed with a first adapter including 1) a transposon end
sequence, 2) a sequence complementary to a capture domain; ii) a
transposase enzyme second monomer complexed with a second adapter
including 1) the transposon end sequence; 2) a second adapter
sequence; b) genomic DNA; c) a capture probe, including i) a
spatial barcode; ii) a unique molecular identifier; iii) a capture
domain; iv) a functional domain; and v) a splint oligonucleotide.
In some embodiments, a composition including a) a transposase
enzyme dimer including i) a transposase enzyme monomer complexed
with a first adapter including 1) a transposon end sequence, 2) a
sequence complementary to a capture domain wherein the 5' end of
the first adapter is phosphorylated; ii) a transposase enzyme
second monomer complexed with a second adapter including 1) the
transposon end sequence; 2) a second adapter sequence wherein the
5' end of the second adapter is phosphorylated; c) genomic DNA; and
d) a capture probe, including i) a spatial barcode; ii) a unique
molecular identifier; iii) a capture domain; iv) a functional
domain; and v) a splint oligonucleotide.
Spatial Transcriptomics
[1439] In some embodiments, provided herein are methods for
spatially detecting an analyte (e.g., detecting the location of an
analyte, e.g., a biological analyte) from a biological sample
(e.g., present in a biological sample such as a tissue section)
that include: (a) providing a biological sample on a substrate; (b)
staining the biological sample on the substrate, imaging the
stained biological sample, and selecting the biological sample or
subsection of the biological sample to subject to spatial analysis;
(c) providing an array comprising one or more pluralities of
capture probes on a substrate; (d) contacting the biological sample
with the array, thereby allowing a capture probe of the one or more
pluralities of capture probes to capture the biological analyte of
interest; and (e) analyzing the captured biological analyte,
thereby spatially detecting the biological analyte of interest. Any
variety of staining and imaging techniques as described herein or
known in the art can be used in accordance with methods described
herein. In some embodiments, the staining includes optical labels
as described herein, including, but not limited to, fluorescent,
radioactive, chemiluminescent, calorimetric, or colorimetric
detectable labels. In some embodiments, the staining includes a
fluorescent antibody directed to a target analyte (e.g., cell
surface or intracellular proteins) in the biological sample. In
some embodiments, the staining includes an immunohistochemistry
stain directed to a target analyte (e.g., cell surface or
intracellular proteins) in the biological sample. In some
embodiments, the staining includes a chemical stain such as
hematoxylin and eosin (H&E) or periodic acid-schiff (PAS). In
some embodiments, significant time (e.g., days, months, or years)
can elapse between staining and/or imaging the biological sample
and performing spatial transcriptomic analysis. In some
embodiments, reagents for performing spatial analysis are added to
the biological sample before, contemporaneously with, or after the
array is contacted to the biological sample. In some embodiments,
step (d) includes placing the array onto the biological sample. In
some embodiments, the array is a flexible array where the plurality
of spatially-barcoded features (e.g., capture probes) are attached
to a flexible substrate. In some embodiments, measures are taken to
slow down a reaction (e.g., cooling the temperature of the
biological sample or using enzymes that preferentially perform
their primary function at lower or higher temperature as compared
to their optimal functional temperature) before the array is
contacted with the biological sample. In some embodiments, step (e)
is performed without bringing the biological sample out of contact
with the array. In some embodiments, step (e) is performed after
the biological sample is no longer in contact with the array. In
some embodiments, the biological sample is tagged with an analyte
capture agent before, contemporaneously with, or after staining
and/or imaging of the biological sample. In such cases, significant
time (e.g., days, months, or years) can elapse between staining
and/or imaging and performing spatial analysis. In some
embodiments, the array is adapted to facilitate biological analyte
migration from the stained and/or imaged biological sample onto the
array (e.g., using any of the materials or methods described
herein). In some embodiments, a biological sample is permeabilized
before being contacted with an array. In some embodiments, the rate
of permeabilization is slowed prior to contacting a biological
sample with an array (e.g., to limit diffusion of analytes away
from their original locations in the biological sample). In some
embodiments, modulating the rate of permeabilization (e.g.,
modulating the activity of a permeabilization reagent) can occur by
modulating a condition that the biological sample is exposed to
(e.g., modulating temperature, pH, and/or light). In some
embodiments, modulating the rate of permeabilization includes use
of external stimuli (e.g., small molecules, enzymes, and/or
activating reagents) to modulate the rate of permeabilization. For
example, a permeabilization reagent can be provided to a biological
sample prior to contact with an array, which permeabilization
reagent is inactive until a condition (e.g., temperature, pH,
and/or light) is changed or an external stimulus (e.g., a small
molecule, an enzyme, and/or an activating reagent) is provided.
Spatially-Resolved Gene Expression and Clustering in Invasive
Ductal Carcinoma
[1440] The spatial gene expression of invasive ductal carcinoma
tissue from a female patient (ER+, PR-, HER2+) was profiled
(BioIVT: Asterand--Case ID 66320; Specimen ID 116899F). As a
control, the healthy tissue sections adjacent to the tumor were
obtained. 4 replicates were used for each tissue type.
[1441] Spatially-resolved gene expression and clustering in
invasive ductal carcinoma reveal intra-tumor heterogeneity is shown
in FIGS. 23A-H. FIG. 23A shows a histological section of an
invasive ductal carcinoma annotated by a pathologist. The section
contains a large proportion of invasive carcinoma (22.344 mm.sup.2
portion indicated by thick black line (outlined in black in color
figure)), three separate ductal cancer in situ regions (portions
indicated by medium thickness black line 1.329 mm.sup.2, 1.242
mm.sup.2, and 0.192 mm.sup.2 (outlined in green in color figure)),
and fibrous tissue. FIG. 23B shows a tissue plot with spots colored
by unsupervised clustering of transcripts. FIG. 23C shows a t-SNE
plot of spots colored by unsupervised clustering of transcripts.
FIG. 23D shows a gene expression heat map of the most variable
genes between the 9 identified clusters. The region defined as
fibrous tissue mostly corresponds to clusters 1, 7, and 8.
Interestingly, a large region annotated as invasive carcinoma by a
pathologist contained spatial spots that were assigned to DCIS
(cluster 5). In addition, four subtypes of invasive carcinoma with
distinct molecular properties (clusters 2, 3, 4, and 6) were
identified, revealing intra-tumor heterogeneity.
[1442] The expression levels of genes corresponding to human
epidermal growth factor receptor 2 (Her2), estrogen receptor (ER),
and progesterone receptor (PGR) in the tissue section are shown in
FIG. 23E. It is clearly visible that Her2 and ER are highly
expressed in the invasive carcinoma and DCIS regions while the
expression of PR is absent, consistent with the patient's
diagnosis. One of the top differentially expressed genes from each
cluster in the invasive carcinoma region was selected (rectangular
boxes in FIG. 23D), and its expression levels are located in the
tissue as shown in FIG. 23F and overlapped in one plot as shown in
FIG. 23G. With the exception of PGR, all of these genes were highly
up-regulated in the carcinoma tissue compared to the adjacent
normal tissue (FIG. 23H). Analysis revealed that all of these
up-regulated genes have implication in cancer progression.
Interestingly, in the subset of cluster 3, a long non-coding RNA,
of which abnormal expression has recently been implicated in tumor
development (see, e.g., Zhang T, et al. Long Non-Coding RNA and
Breast Cancer. Technol Cancer Res Treat. 2019, 18,
1533033819843889, incorporated herein by reference in its
entirety), is one of the top differentially expressed genes. In
glioblastoma, LINC00645 promotes epithelial-to-mesenchymal
transition by inducing TGF-.beta. (see, e.g., Li, C. et al. Long
non-coding RNA linc00645 promotes TGF-.beta.-induced
epithelial-mesenchymal transition by regulating miR-205-3p-ZEB1
axis in glioma. Cell Death & Dis. 2019, 10, 272, incorporated
herein by reference in its entirety).
[1443] During breast cancer progression, the myoepithelial cells,
which continue to surround preinvasive in situ carcinoma, gradually
disappear (see, e.g., Gudjonsson, T. et al. Myoepithelial Cells:
Their Origin and Function in Breast Morphogenesis and Neoplasia. J.
Mammary Gland Biol. Neoplasia. 2009, 10, 261, incorporated herein
by reference in its entirety). This phenomenon is clearly
visualized in FIGS. 23I and 23J where KRT14 (a gene signature of
myoepithelial cells) was highly expressed around the lining of the
duct in the normal tissue while it was disappearing in the DCIS
region in IDC tissue (FIG. 23I). The extracellular matrix genes
such as COL1A1 and FN1, key genes associated with invasion and
metastasis, were highly upregulated while smooth muscles and basal
keratin were down-regulated in IDC (FIG. 23J).
VI. Generation of Spatial Arrays
[1444] In some aspects, provided herein are methods for generating
arrays for spatial analysis (e.g., spatial arrays). The spatial
arrays can have capture probes that can interact with analytes
present in a biological sample either directly or indirectly. For
example, analytes can be RNA, DNA, protein, lipids, or any other
analytes described herein. In some embodiments, the capture probe
is a nucleic acid or a polypeptide. In some embodiments, the
capture probe is a conjugate (e.g., an oligonucleotide-antibody
conjugate). In some embodiments, the capture probe includes a
barcode (e.g., a spatial barcode and/or a unique molecular
identifier (UMI)) and a capture domain. In some embodiments, a
capture probe includes a cleavage domain.
[1445] In some embodiments, capture probes are attached to an array
feature and an individual array feature can include one or more
capture probes. In some embodiments, an individual array feature
includes hundreds or thousands of capture probes. In some
embodiments, the capture probes are associated with a particular
individual feature, where the individual feature contains a capture
probe including a spatial barcode unique to the individual feature
(e.g., a defined region or location on the array).
[1446] In some embodiments, a particular feature can contain
capture probes including more than one spatial barcode (e.g., one
capture probe at a particular feature can include a non-common
spatial barcode that is different than the non-common spatial
barcode included in another capture probe at the same particular
feature, while both capture probes include a second, common spatial
barcode), where the non-common spatial barcodes of the capture
probes corresponds to a particular defined region or location on
the array. For example, multiple non-common spatial barcode
sequences associated with one particular feature on an array can
provide a stronger address or attribution to a given location by
providing duplicate or independent confirmation of the location. In
some embodiments, the multiple non-common spatial barcodes
represent increasing specificity of the location of the particular
array point
[1447] As used herein, spatial analyte capture can utilize nucleic
acid probes (e.g., capture probes) including spatial barcode
sequences, which are immobilized on a substrate (e.g., directly or
indirectly) to generate an array. The spatial barcode sequences can
correspond to the location of the capture probes on the array. In
some embodiments, biological samples, such as tissue sections, are
placed onto the array and an analyte from the biological sample is
captured. In some embodiments, the captured analyte is a protein.
In some embodiments, the captured analyte is a nucleic acid. In
some embodiments, the captured analyte is RNA (e.g. messenger RNA).
In some embodiments, the captured analyte is DNA (e.g., genomic
DNA). In some embodiments, DNA from the biological sample is
processed, e.g. fragmented and/or provided with a binding domain
that hybridizes to the capture probes on the array. In some
embodiments, DNA polymerase extension or ligation reaction is
performed in the biological sample on the array. In some
embodiments, the probes to which nucleic acids in the biological
sample bind are extended using the bound nucleic acid as a template
to generate complementary nucleic acid strands, which is
immobilized on the surface of the array. In some embodiments,
extended probes (e.g., capture probes that are subjected to
template-directed nucleic acid polymerization using a captured
nucleic acid analyte as a template) carry information about the
position of the "captured" analytes in the biological sample, e.g.
tissue section. In some embodiments, the extended probes can be
released from the surface of the array and analyzed, e.g.
sequenced, which results in a "library" of captured nucleic acid
analytes that are associated with spatial information. In some
embodiments, the sequence data can then be matched to a location in
the biological sample, e.g. tissue section. For instance, the
biological sample can be visualized or imaged, e.g., stained and/or
photographed, before or after analyte capture to facilitate the
correlation of the spatial barcode in the capture probe (e.g., an
extended capture probe that is associated with a given analyte)
with a position within the biological sample and the sequence data
can be overlaid on an image of the biological sample, e.g., using a
computer, to display the distribution pattern of any analyte of
interest across the biological sample. The visualization step is
not essential as the tagged analytes can be correlated with a
location in the biological sample using other means, e.g., the
unique profile of the analytes captured with the same spatial
barcode, e.g., at the same position on the array, can enable the
analyte to be correlated to a location within the biological sample
based on the characteristics of cells or areas of cells within the
biological sample (e.g., a tissue section). Any of a variety of
analytes can be captured and analyzed in accordance with methods
provided herein, including but not limited to RNA, specific
sub-types of RNA, proteins, antibodies, genomic DNA, T-cell
receptors, small molecules, or other analytes.
Capture Probes Via Splint Oligonucleotides
[1448] Capture probes for spatial analysis can be generated by
different methods. In some embodiments, the capture probe is
generated from two or more oligonucleotide sequences. In some
embodiments, two or more oligonucleotide sequences are ligated
together to generate a capture probe. In some embodiments, capture
probe generation can be facilitated by a splint oligonucleotide. In
some embodiments, a plurality of oligonucleotides are attached to a
feature on the substrate. In some embodiments, the oligonucleotides
of the array can be about 20 to about 80 nucleotides long. In some
embodiments, the oligonucleotides of the array can be about 30 to
about 70 nucleotides long. In some embodiments, the
oligonucleotides of the array can be about 40 to about 60
nucleotides long. In some embodiments, the oligonucleotides of the
array can be about 45 to about 55 nucleotides long. In some
embodiments, the capture probe includes one or more constant
sequences (e.g., any of the variety of constant sequences described
herein). For example, the oligonucleotide can have a functional
domain (e.g. any of the variety of functional domains described
herein) and/or a cleavage domain (e.g. any of the variety of
cleavage domains described herein).
[1449] In some embodiments, a primer can hybridize to an
oligonucleotide attached, directly or indirectly to the substrate
(e.g., attached to a feature attached to a substrate, e.g.,
attached via a linker). In some embodiments, the oligonucleotides
can be attached, directly or indirectly, to the substrate at the 3'
end, resulting in the attaching oligonucleotides having free 5'
ends. For example, an array including a plurality of
oligonucleotides attached to a substrate at their 3' ends can be
contacted with a plurality of primers. In some embodiments, the
plurality of primers are complementary or substantially
complementary to the oligonucleotides attached to the substrate at
their 3' ends, or a portion thereof. In some embodiments the primer
can be extended using the oligonucleotide attached to the substrate
as a template to generate a first oligonucleotide. In some
embodiments, the primer is extended by a reverse transcriptase
enzyme. In some embodiments, the primer is extended by a DNA
polymerase enzyme. In some embodiments, additional nucleotides can
be added (e.g., added by an enzyme) to the end of the first
oligonucleotide to generate a 3' overhang. In some embodiments, the
enzyme is a template independent polymerase (e.g., any of the
variety of template independent polymerases described herein or
otherwise known in the art). In some embodiments, the template
independent polymerase is terminal nucleotidyl transferase. In some
embodiments, the template independent polymerase is a poly(A)
polymerase.
[1450] In some embodiments, oligonucleotides attached directly or
indirectly to the substrate at the 3' end and having free 5' ends
can include a sequence that indicates the position of a given
oligonucleotide on the substrate. In some embodiments, such a
sequence is or can function as a spatial barcode. In some
embodiments, different oligonucleotides attached directly or
indirectly to the substrate at the 3' end and having free 5' ends
can include different sequences that can indicate the position of
the different oligonucleotide on the substrate (e.g., the different
oligonucleotides can have different spatial barcodes or sequences
that act as different spatial barcodes).
[1451] In some embodiments, the first oligonucleotide including the
3' overhang generated by the template independent polymerase can be
contacted with a splint oligonucleotide. The term "splint
oligonucleotide", as used herein, refers to an oligonucleotide
that, when hybridized to other polynucleotides, acts as a "splint"
to position the polynucleotides (e.g., oligonucleotides) next to
one another (e.g., abutting one another) so that they can be
ligated together. In some embodiments, the splint oligonucleotide
is DNA. In some embodiments, the splint oligonucleotide is RNA. In
some embodiments, the splint oligonucleotide includes a nucleotide
sequence that is partially complementary to nucleotide sequences
from two or more different oligonucleotides. In some embodiments,
the splint oligonucleotide is approximately 40 nucleotides in
length. In some embodiments, the splint oligonucleotide is between
about 10 and about 50 oligonucleotides in length. In some
embodiments, the splint oligonucleotide is between about 10 and
about 45, about 10 and about 40, about 10 and about 35, about 10
and about 30, about 10 and about 25, or about 10 and about 20
oligonucleotides in length. In some embodiments, the splint
oligonucleotide is between about 15 and about 50, about 15 and
about 45, about 15 and about 40, about 15 and about 35, about 15
and about 30, about 15 and about 30, or about 15 and about 25
nucleotides in length.
[1452] In some embodiments, the splint oligonucleotide provided to
the first oligonucleotides can have a sequence that is
complementary to the nucleotides of a generated 3' overhang of the
first oligonucleotide. A second oligonucleotide can be provided to
the array. In some embodiments, the splint oligonucleotide can have
another sequence that is complementary or substantially
complementary to the second oligonucleotide, or portion thereof. In
some embodiments, the splint oligonucleotide can hybridize to both
the 3' overhang of the first oligonucleotide and the second
oligonucleotide at the same time. In some embodiments, the first
oligonucleotide and the second oligonucleotide are ligated together
to generate a capture probe. In some embodiments, an RNA ligase is
used to ligate the first and second oligonucleotide together. In
some embodiments, a DNA ligase is used to ligate the first and
second oligonucleotides sequences together. In some embodiments,
the DNA ligase is a T4 DNA ligase. In some embodiments the ligase
is App ligase.
[1453] In some embodiments, the splint oligonucleotide includes a
sequence that is complementary to the nucleotides of a generated 3'
overhang of the first oligonucleotide, or a portion thereof, and a
sequence that is complementary to the second oligonucleotide, or a
portion thereof. In some embodiments, the splint oligonucleotide
includes a sequence that is perfectly complementary (e.g., is 100%
complementary) to the nucleotides of a generated 3' overhang of the
first oligonucleotide, or a portion thereof, and/or a sequence that
is perfectly complementary to the second oligonucleotide, or a
portion thereof. In some embodiments, the splint oligonucleotide
includes a sequence that is not perfectly complementary (e.g., is
not 100% complementary) to the nucleotides of a generated 3'
overhang of the first oligonucleotide, or a portion thereof, and/or
a sequence that is not perfectly complementary to the second
oligonucleotide, or a portion thereof. In some embodiments, the
splint oligonucleotide includes a sequence that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the
nucleotides of a generated 3' overhang of the first
oligonucleotide, or a portion thereof, and/or a sequence that is
complementary to the second oligonucleotide, or a portion thereof.
In some embodiments, the splint oligonucleotide includes a sequence
that is perfectly complementary (e.g., is 100% complementary) to
the nucleotides of a generated 3' overhang of the first
oligonucleotide, or a portion thereof, but is not perfectly
complementary to a sequence that is complementary to the second
oligonucleotide, or a portion thereof. In some embodiments, the
splint oligonucleotide includes a sequence that is not perfectly
complementary (e.g., is not 100% complementary) to the nucleotides
of a generated 3' overhang of the first oligonucleotide, or a
portion thereof, but is perfectly complementary to a sequence that
is complementary to the second oligonucleotide, or a portion
thereof. So long as the splint oligonucleotide is capable of
hybridizing to the nucleotides of a generated 3' overhang of the
first oligonucleotide, or a portion thereof, and to a sequence that
is complementary to the second oligonucleotide, or a portion
thereof, the splint oligonucleotide need not have a sequence that
is perfectly complementary to either the generated 3' overhang of
the first oligonucleotide or to the second oligonucleotide. The
second oligonucleotide can be about 20 to about 80 nucleotides
long. In some embodiments, the second oligonucleotide can be about
30 to about 70 nucleotides long. In some embodiments, the second
oligonucleotide can be about 40 to about 60 nucleotides long. In
some embodiments, the second oligonucleotide can be about 45 to
about 55 nucleotides long. The second oligonucleotide can include
various domains. For example, the second oligonucleotide can
include one or more of a sequence complementary to the splint
oligonucleotide, a functional sequence, a unique molecular
identifier, and a capture domain (e.g., any of the variety of
functional sequences, a unique molecular identifiers, and/or a
capture domains described herein). In some embodiments of any of
the methods described herein, the capture probes can include a
unique molecular identifier. In some embodiments of any of the
methods described herein, a unique molecular identifier can be
about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides.
[1454] In some embodiments, the capture domain can include a
poly(dT) sequence. In some embodiments, the poly(dT) sequence can
interact (e.g., hybridize) with poly(A) sequences of analytes
(e.g., mRNA). In some embodiments, the capture domain can include a
gene specific sequence.
Capture Probes from Hydrogel Arrays
[1455] In some aspects, provided herein are methods for preparing a
high-density spatially barcoded array, e.g., a high-diversity and
high-density spatially barcoded array. For example, methods for
preparing a high-density spatially barcoded array can include
contacting an array including one or more pluralities of first
oligonucleotides with a hydrogel including one or more pluralities
of second oligonucleotides. In some embodiments, the first
oligonucleotide, the second oligonucleotide, or both, can have
include a capture domain. The first oligonucleotide and the second
oligonucleotide can be attached to generate capture probes. The
hydrogel containing the capture probes can be reduced in size
(e.g., shrunk). In some embodiments, methods for preparing a
high-density spatially barcoded array can include contacting an
array including one or more pluralities of first oligonucleotides
with a hydrogel including one or more pluralities of second
oligonucleotides; attaching a first oligonucleotide to a second
oligonucleotide to generate a capture probe including a capture
domain; reducing the size of the hydrogel including the capture
probes by dehydration (e.g., removing water), forming a shrunken
hydrogel array including the one or more pluralities of capture
probes and thus generating a high-density spatially barcoded array
(e.g., spatial array). In some embodiments, a capture probe is any
of the variety of capture probes described herein. In some
embodiments, the one or more pluralities of capture probes are
immobilized in the hydrogel.
[1456] The term "partition," as used herein, can be a verb or a
noun. When used as a verb (e.g., "to partition," or
"partitioning"), the term generally refers to the fractionation
(subdivision) of a species or sample (e.g., a polynucleotide) such
that one fraction (or subdivision) is sequestered from another.
Each sequestered section is referred to using the noun "partition."
In some embodiments, a partition maintains separation of its own
contents from the contents of other partitions. A partition may be
any suitable area in which one fraction of a sample is separated or
sequestered from another. In some embodiments, a hydrogel can
include one or more partitions of a species or sample (e.g., a
polynucleotide), wherein the hydrogel matrix maintains the
separation of the contents of one partition from the contents of
other partitions. In some embodiments, a partition has defined
dimensions.
[1457] In some embodiments, the one or more pluralities of
immobilized first oligonucleotides are partitioned. In some
embodiments, the one or more pluralities of immobilized first
oligonucleotides are partitioned into a plurality of first
oligonucleotide partitions. In some embodiments, a partitioned
plurality of immobilized first oligonucleotides is different from
another partitioned plurality of immobilized first
oligonucleotides. In some embodiments, a partitioned plurality of
immobilized first oligonucleotides are partitioned into features on
a substrate.
[1458] In some embodiments, one or more first pluralities of first
oligonucleotides include a barcode sequence (e.g., spatial
barcode). In some embodiments, one or more pluralities of first
oligonucleotides include a spatial barcode sequence different from
the spatial barcode sequence of one or more other pluralities of
first oligonucleotides. In some embodiments, one or more first
oligonucleotide partitions (e.g., a population) include a plurality
of immobilized first oligonucleotides including a spatial barcode
sequence different from the spatial barcode sequence of the
plurality of immobilized first oligonucleotides of one or more
other first oligonucleotide partitions. In some embodiments, a
first oligonucleotide of a population of first oligonucleotides in
one or more first oligonucleotide partitions includes a spatial
barcode. In some embodiments, the first oligonucleotide of a
population of first oligonucleotides in one or more first
oligonucleotide partitions includes a spatial barcode that is
different from the spatial barcode of a first oligonucleotide of a
population of first oligonucleotides in another first
oligonucleotide partition. In some embodiments, a spatial barcode
of the first oligonucleotide of a population of first
oligonucleotides in one or more first oligonucleotide partitions is
different from a spatial barcode of a second oligonucleotide of a
population of second oligonucleotides in one or more second
oligonucleotide partitions. In some embodiments, a first
oligonucleotide partition comprises a first population of first
oligonucleotides comprising a spatial barcode that is different
from a spatial barcode of a second population of first
oligonucleotides in another first oligonucleotide partition.
[1459] In some embodiments, the one or more pluralities of second
oligonucleotides are immobilized in the hydrogel. In some
embodiments, the immobilized second oligonucleotides are
partitioned. In some embodiments, the immobilized second
oligonucleotides are partitioned into a first population of
immobilized second oligonucleotides (e.g., in a partition). In some
embodiments, one or more immobilized second oligonucleotide
partitions include another population of immobilized second
oligonucleotides different from the first population of immobilized
second oligonucleotide partitions. In some embodiments, the first
immobilized second oligonucleotide of a partitioned population
(e.g., a first population) of immobilized second oligonucleotides
is different from a second immobilized second oligonucleotide of
another partitioned population of immobilized second
oligonucleotides. In some embodiments, the first immobilized second
oligonucleotide, the second immobilized second oligonucleotide, or
both include a spatial barcode. In some embodiments, the first
immobilized second oligonucleotide of a partitioned population of
immobilized second oligonucleotides includes a spatial barcode that
is different from a spatial barcode of the second immobilized
second oligonucleotide of another partitioned population of
immobilized second oligonucleotides.
[1460] In some embodiments, one or more pluralities of second
oligonucleotides include a spatial barcode sequence. In some
embodiments, one or more pluralities of second oligonucleotides
include a spatial barcode sequence different from the spatial
barcode sequence of one or more pluralities of second
oligonucleotides. In some embodiments, the spatial barcode sequence
of one or more pluralities of first oligonucleotides is different
from the spatial barcode sequence of one or more pluralities of
second oligonucleotides. In some embodiments, one or more second
oligonucleotide partitions include a plurality of second
oligonucleotides including a spatial barcode sequence different
from the spatial barcode sequence of the plurality of second
oligonucleotides of one or more second oligonucleotide
partitions.
[1461] In some embodiments, each first oligonucleotide from the
plurality of first oligonucleotides includes a spatial barcode. In
some embodiments, each first oligonucleotide from the plurality of
first oligonucleotides includes a unique molecular identifier. In
some embodiments, each first oligonucleotide from the plurality of
first oligonucleotides includes a cleavage domain, a spatial
barcode, a unique molecular identifier, and a capture domain. In
some embodiments, at least one first oligonucleotide from the
plurality of first oligonucleotides includes a spatial barcode. In
some embodiments, at least one first oligonucleotide from the
plurality of first oligonucleotides includes a unique molecular
identifier. In some embodiments, at least one first oligonucleotide
from the plurality of first oligonucleotides includes a cleavage
domain, a spatial barcode, a unique molecular identifier, and a
capture domain.
[1462] A "capture probe" as used herein can refer to a capture
probe that is a product of attaching a first oligonucleotide to
second oligonucleotide is a hydrogel. For example, one or more
domains of the first oligonucleotide can be attached to one or more
domains of a second oligonucleotide (e.g., a second oligonucleotide
in a hydrogel). "Attaching" the first and second oligonucleotide in
a hydrogel can be accomplished by any suitable means described
herein. For example, a first oligonucleotide can be ligated to an
end of a second oligonucleotide. As another example, PCR can be
used to attach one or more domains of a first oligonucleotide to
one or more domains of a second oligonucleotide (e.g., a second
oligonucleotide in a hydrogel).
[1463] In some embodiments, methods provided herein that include
attaching a first oligonucleotide to second oligonucleotide is a
hydrogel can be used to generate a spatial array of relatively high
complexity. In some embodiments, a plurality of first
oligonucleotides (e.g., partitioned first oligonucleotides) is
attached to a plurality of second oligonucleotides (e.g.,
partitioned second oligonucleotides) to generate a plurality of
capture probes such that the plurality of capture probes exhibits a
high complexity (e.g., a high sequence complexity) relative to the
complexity of the plurality of first and second oligonucleotides.
For example, a plurality of first oligonucleotides can have a
plurality of first partial spatial barcodes, and a plurality of
second oligonucleotides can have a plurality of second partial
spatial barcodes. By attaching the plurality of first and second
oligonucleotides to each other in a combinatorial fashion, a
plurality of spatial barcodes can be generated, which plurality of
generated spatial barcodes can have a higher sequence complexity
than the sequence complexity of either of the first or second
partial spatial barcodes. As a non-limiting example, if a plurality
of first oligonucleotides includes a plurality of M first partial
spatial barcodes (e.g., the plurality of first oligonucleotides can
be partitioned such that each of the M first partial spatial
barcodes is in a different partition) and a plurality of second
oligonucleotides includes a plurality of N second partial spatial
barcodes (e.g., the plurality of second oligonucleotides can be
partitioned such that each of the N second partial spatial barcodes
is in a different partition), the plurality of first and second
oligonucleotides can be attached in a combinatorial fashion to
generate a plurality of spatial barcodes that includes M.times.N
spatial barcodes.
[1464] In some embodiments, attaching members of the one or more
pluralities of first oligonucleotides to members of the one or more
pluralities of second oligonucleotides to form one or more
pluralities of capture probes includes using a polymerase chain
reaction (PCR). In some embodiments, the hydrogel includes PCR
reagents such as a DNA polymerase, a plurality of primers, and a
plurality of nucleotides. In some embodiments, members of the
plurality of features are amplified (see, e.g., Mitra and Church,
Nucleic Acids Res. 15; 27(24):e34 (1999) which is herein
incorporated by reference in its entirety).
[1465] In some embodiments, chemical ligation can be used to ligate
two or more oligonucleotides (e.g., a first oligonucleotide and a
second oligonucleotide from a hydrogel as described herein). In
some embodiments, chemical ligation involves the use of condensing
reagents. In some embodiments, condensing reagents are utilized to
activate a phosphate group. In some embodiments, condensing
reagents may be one or more of 1-25 ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCI), cyanogen bromide, imidazole derivatives, and
1-hydroxybenzotriazole (HOAt). In some embodiments, functional
group pairs selected from one or more of a nucleophilic group and
an electrophilic group, or an alkyne and an azide group are used
for chemical ligation. In some embodiments, chemical ligation of
two or more oligonucleotides requires a template strand that is
complementary to the oligonucleotides to be ligated (e.g., a splint
oligonucleotide). In some embodiments, the chemical ligation
process is similar to oligonucleotide synthesis.
[1466] In some embodiments, not all of the domains of a first
oligonucleotide are attached to a second oligonucleotide to form a
capture probe. For example, a capture probe can include 1, 2, 3, 4,
or 5 domains of the original first oligonucleotide. In some
embodiments, all the domains of the first oligonucleotide are
attached to the second oligonucleotide. For example, the capture
probe includes all the domains of the first oligonucleotide.
[1467] In some embodiments, members of the one or more pluralities
of the first oligonucleotide are cross-linked to a hydrogel.
[1468] As used herein "shrinking" or "reducing the size of" a
hydrogel refers to any process causing the hydrogel to physically
contract and/or the size of the hydrogel to decrease in volume. For
example, the scaffold of the gel may shrink or "implode" upon
solvent removal (see, e.g., Long and Williams. Science. 2018;
362(6420):1244-1245, and Oran et al. Science. 2018;
362(6420):1281-1285; each of which is incorporated herein by
reference in its entirety). As another example, the process to
shrink or reduce the size of a hydrogel may be one that removes
water (e.g., a dehydrating process) from the hydrogel. There are
many methods known to one of skill in the art for shrinking or
reducing the size of a hydrogel. Non-limiting examples of a method
to shrink or reduce the size of a hydrogel include exposing the
hydrogel to one or more of: a dehydrating solvent, a salt, heat, a
vacuum, lyophilization, desiccation, filtration, and
air-drying.
[1469] In some embodiments, the removal of water includes a
dehydrating solvent. Non-limiting examples of a dehydrating solvent
include a ketone (such as methyl ethyl ketone (MEK)), isopropanol
(IPA), acetone, 1-butanol, methanol (MeOH), dimethyl sulfoxide
(DMSO), glycerol, propylene glycol, ethylene glycol, ethanol, (k)
1,4-dioxane, propylene carbonate, furfuryl alcohol,
N,N-dimethylformamide (DMF), acetonitrile, and an aldehyde (such as
formaldehyde or glutaraldehyde). In some embodiments, the solvent
includes at least one of an alcohol, a ketone, an aldehyde,
acetonitrile, and a combination thereof. In some embodiments, the
solvent includes a solvent selected from the group consisting of:
glycerol, ethanol, acetonitrile, acetone, 2-propanol, and a
combination thereof.
[1470] In some embodiments, the removal of water includes the use
of a salt. In some embodiments, the salt includes an inorganic
salt. Non-limiting examples of salts that may be used to shrink a
hydrogel include inorganic salts such as aluminum, ammonium,
barium, beryllium, calcium, cesium, lithium, magnesium, potassium,
rubidium, sodium, and strontium salts. Further non-limiting
examples of inorganic salts include sodium chloride, potassium
chloride, lithium chloride, cesium chloride, sodium fluoride,
sodium bromide, sodium iodide, sodium nitrite, potassium sulfate,
potassium nitrate, potassium carbonate, potassium bicarbonate,
sodium sulfate, sodium nitrate, sodium carbonate, sodium
bicarbonate, calcium sulfate, copper oxychloride, calcium chloride,
calcium carbonate, calcium bicarbonate, magnesium sulfate,
magnesium nitrate, magnesium chloride, magnesium carbonate,
magnesium bicarbonate, ammonium sulfate, ammonium chloride,
ammonium nitrate, ammonium carbonate, ammonium bicarbonate,
trisodium phosphate, tripotassium phosphate, calcium phosphate,
copper(II) sulfate, sodium sulfide, potassium sulfide, calcium
sulfide, potassium permanganate, iron(II) chloride, iron(III)
chloride, iron (2+) sulfate, iron(III) sulfate, iron(II) nitrate,
iron(III) nitrate, manganese(II) chloride, manganese(III) chloride,
manganese(II) sulfate, manganese(II) nitrate, zinc chloride, zinc
nitrate, zinc sulfate, ammonium orthomolybdate, monopotassium
phosphate, nickel(II) sulfate, nickel(II) nitrate, sodium
metavanadate, sodium paravanadate, potassium dichromate, ammonium
dichromate, antipyonin, ammonium nitrite, potassium fluoride,
sodium fluoride, ammonium fluoride, calcium fluoride, chrome alum,
potassium alum, potassium iodide, sodium hypochlorite, tin(II)
sulfate, tin(II) nitrate, gold selenite, dicesium chromate,
potassium perchlorate, calcium perchlorate, aluminium sulphate,
lead(II) bisulfate, barium phosphate, barium hydrogen
orthophosphate, barium dihydrogen phosphate, silver dichromate,
potassium bromate, sodium bromate, sodium iodate, sodium silicate,
diammonium phosphate, ammonium phosphate, ammonium dihydrogen
phosphate, chromium orthophosphate, copper(II) chloride, copper(I)
chloride, sodium tetrametaphosphate, potassium heptafluoroniobate,
zinc phosphate, sodium sulfite, copper(I) nitrate, copper(II)
nitrate, potassium silicate, copper(II) carbonate basic, copper(II)
carbonate salts of acrylic acid and sulfopropyl acrylate.
[1471] In some embodiments, the removal of water includes an acid.
In some embodiments, the hydrogel is exposed to a different pH
environment. For example, the hydrogel can be exposed to an acidic
pH or a basic pH. In some embodiments, the hydrogel is exposed to a
pH of less than about 6.5, e.g., a pH of about 6, about 5.5, about
5, about 4.5, about 4, about 3.5, about 3, about 2.5, about 2,
about 1.5, or about 1. In some embodiments, the hydrogel is exposed
to a pH of greater than about 7.5, e.g., a pH of about 8, about
8.5, about 9, about 9.5, about 10, about 10.5, about 11, about
11.5, 10 about 12, about 12.5, about 13, about 13.5, or about
14.
[1472] In some embodiments, the removal of water includes a
dehydrating process such as heat, a vacuum, lyophilization,
desiccation, filtration, and air-drying. In some embodiments, the
hydrogel is exposed to an increased temperature, e.g., a higher
temperature than about room temperature. For example, a temperature
of about 37.degree. C., 38 15.degree. C., 39.degree. C., 40.degree.
C., 41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C. 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., or 70.degree. C., or higher. As another example, the
hydrogel is exposed to an increase in temperature from about
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C., or
49.degree. C. to about 50.degree. C., 51 20.degree. C., 52.degree.
C., 53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C. 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., or 70.degree. C., or higher).
[1473] In some embodiments, more than one method to shrink or
reduce the size of a hydrogel can be used. For example, a hydrogel
can be exposed to an acid and subsequently dehydrated by another
method such as heat, a vacuum, lyophilization, desiccation,
filtration, or a combination thereof.
[1474] In some embodiments, the hydrogel can further include a
cross-linking agent. The cross linking agent may help stabilize the
hydrogel and/or modulate the extent the hydrogel shrinks.
Non-limiting examples of a cross-linking agent include
disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS),
formalin, dimethyladipimidate (DMA), dithio-bis(-succinimidyl
propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene
glycol bis(succinimidyl succinate) (EGS). In some embodiments, the
shrunken hydrogel is at least about 2-fold smaller in a linear
dimension (e.g., along one axis) than the pre-shrunk hydrogel. For
example, at least about 2.5, about 3, about 4, about 5, about 6,
about 7, about 8, about 9, about 10, or more fold smaller in a
linear dimension than the pre-shrunk hydrogel. In some embodiments,
a shrunken hydrogel is about 2 to about 10 fold smaller in a linear
dimension than the pre-shrunk hydrogel. For example, about 2 to
about 3, about 2 to about 4, about 2 to about 5, about 2 to about
6, about 2 to about 7, about 2 to about 8, about 2 to about 9,
about 9 to about 10, about 8 to about 10, about 7 to about 10,
about 6 to about 10, about 5 to about 10, about 4 to about 10, or
about 3 to about 10. In some embodiments, a shrunken hydrogel is
about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5
to about 7, about 6 to about 8 fold smaller in a linear dimension
than the pre-shrunk hydrogel. In some embodiments, a shrunken
hydrogel is about 2, about 3, about 4, about 5, about 6, about 7,
about 8, about 9, or about 10 fold smaller in a linear dimension
than the pre-shrunk hydrogel. In some embodiments, the shrunken
hydrogel is about 2 to about 10 fold smaller in a linear dimension
than the size of the hydrogel prior to reducing the size of the
pre-shrunk hydrogel.
[1475] In some embodiments, the size of the hydrogel is reduced
along more than one axes, e.g., along 2 or 3 axes. In some
embodiments, each axis intersects each other axis at 90 degrees. In
some embodiments, the size of the hydrogel along the first axis is
about 2 to about 10 or more fold smaller than the pre-shrunk
hydrogel, e.g., about 2, about 3, about 4, about 5, about 6, about
7, about 8, about 9, about 10 or more fold smaller than the
pre-shrunk hydrogel. In some embodiments, the size of the hydrogel
along the second axis is about 2 to about 10 or more fold smaller
than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10 or more fold
smaller than the pre-shrunk hydrogel. In some embodiments, the size
of the hydrogel along the third axis is about 2 to about 10 or more
fold smaller than the preshrunk hydrogel, e.g., about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10 or
more fold smaller than the pre-shrunk hydrogel. In some
embodiments, the reduction in the size of the hydrogel is
isometric.
[1476] In some embodiments, a hydrogel is prepared from hydrogel
subunits including at least one of: acrylamide, bis-acrylamide, a
poly(ethylene glycol), a polyaliphatic polyurethane, a polyether
polyurethane, a polyester polyurethane, a polyethylene copolymer, a
polyamide, a polyvinyl alcohol, polypropylene glycol,
polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,
poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate),
collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin,
alginate, a protein polymer, methylcellulose, and any combination
thereof.
[1477] In some embodiments, the size of a hydrogel including
capture probes is reduced, thus increasing the density of the
capture probes. In some embodiments, the volume of a partition of
members of the plurality of capture probes in a hydrogel is reduced
when the size of the hydrogel is reduced.
[1478] In some embodiments, a high-density spatially barcoded array
(e.g., a high density spatially barcoded array as described herein)
can be used for spatial analysis of analytes present in the
biological sample. For example, methods for spatially determining
the location of an analyte or a plurality of analytes in a
biological sample can include providing a high-density spatially
barcoded array as described herein; contacting the biological
sample with the high-density spatially barcoded array under
conditions such that one or more analytes present in the biological
sample are captured by one or more capture probes of the array; and
determining the spatial location of the captured analytes by any of
the methods described herein.
Methods for Spatial Analysis of Analytes
[1479] In some embodiments, a high-density spatially barcoded array
(e.g., a high density spatially barcoded array as described herein)
can be used for spatial analysis of analytes present in a
biological sample. For example, methods for spatially determining a
location of an analyte or a plurality of analytes in a biological
sample can include providing a high-density spatially barcoded
array as described herein; contacting the biological sample with
the high-density spatially barcoded array under conditions such
that one or more analytes present in the biological sample are
captured by one or more capture probes of the array; and
determining the spatial location of the captured analyte(s).
[1480] In some aspects, provided herein methods for building an
array (e.g., a spatial array, e.g. a high-density spatial array).
In some embodiments, methods of generating an array comprise: (a)
providing an substrate including a plurality of features
immobilized on the substrate, wherein the features (e.g., each
feature or a substantial portion thereof) comprises a plurality of
first oligonucleotides, and wherein the first oligonucleotides
(e.g., each first nucleotide or a substantial portion thereof)
include a first spatial barcode (e.g., a first partial spatial
barcode); (b) selecting one or more first oligonucleotides from the
plurality of first oligonucleotides; (c) delivering a second
oligonucleotide to the one or more selected first oligonucleotides,
wherein the second oligonucleotide includes a second spatial
barcode (e.g., a second partial spatial barcode) and a capture
domain; and (d) attaching the second oligonucleotide to the one or
more selected first oligonucleotides, thereby generating one or
more capture probes including the first spatial barcode and the
second spatial barcode, thereby generating an array.
[1481] In some embodiments, each feature of the plurality of
features is associated with one of a number of different first
spatial barcodes. In some embodiments, each second oligonucleotide
is associated with one of a number of different second
barcodes.
[1482] In some embodiments, step (d) includes ligating the second
oligonucleotide to the one or more selected first oligonucleotides.
In some embodiments, the method includes ligating the second
oligonucleotide to the one or more selected first oligonucleotides
using a ligase. In some embodiments, the second oligonucleotide is
attached to the one or more selected first oligonucleotides by a
chemical coupling reaction. In some embodiments, one or more of
(e.g., each of) the first oligonucleotides includes a 3' end that
is masked by a protective group (e.g., a photocleavable protective
group). In some embodiments, the method includes removing the
photocleavable protective group from the one or more selected first
oligonucleotides prior to delivering a second oligonucleotide to
the one or more selected first oligonucleotides. In some
embodiments, the protective group is a photocleavable protective
group (e.g., any of the variety of a photocleavable protective
groups described herein or otherwise known in the art). In some
embodiments, the protective group is removed by exposing the one or
more selected first oligonucleotides to light. In some embodiments,
the protective group coupled to the one or more selected first
oligonucleotides is a photomask (e.g., any of the variety of a
photomasks described herein or otherwise known in the art). In some
embodiments, the photomask is removed by exposing the one or more
selected first oligonucleotides to light.
[1483] In some embodiments, the selected first oligonucleotide(s)
are separated from the other first oligonucleotides of the
plurality by one or more physical barriers, wherein the one or more
physical barriers form one or more chambers, and wherein the second
oligonucleotide is delivered to the one or more chambers including
the one or more selected first oligonucleotides. In some
embodiments, the one or more physical barriers include a
hydrophobic surface. In some embodiments, the one or more physical
barriers include one or more chamber walls having a tapered end. In
some embodiments, the selected first oligonucleotide(s) are
separated from the other first oligonucleotides of the plurality,
but are not separated by one or more physical barriers. For
example, the selected first oligonucleotide(s) can be separated
from the other first oligonucleotides of the plurality simply by
virtue of their spatial locations. In some embodiments, the second
oligonucleotide includes a unique molecular identifier. In some
embodiments, the second oligonucleotide includes a 3' end that is
masked. In some embodiments, a free 3'-OH group on the capture
probes is created.
[1484] In some embodiments, methods of generating an array
comprise: (a) providing an array including two or more partitions,
wherein the partitions (e.g., each partition or a substantial
portion thereof) includes a plurality of features immobilized on a
substrate, wherein the features (e.g., each feature or a
substantial portion thereof) includes a plurality of first
oligonucleotides, and wherein the first oligonucleotides (e.g.,
each first oligonucleotide or a substantial portion thereof)
include a first spatial barcode (e.g., a first partial spatial
barcode); (b) delivering a second oligonucleotide to a first
partition, wherein the second oligonucleotide includes a second
barcode (e.g., a second partial spatial barcode) and a capture
domain; and (c) attaching the second oligonucleotide to one or more
first oligonucleotides in the first partition, thereby generating
one or more capture probes including the first spatial barcode and
the second spatial barcode in the first partition, thereby
generating an array.
[1485] In some embodiments, methods provided herein further
include: (d) delivering a third oligonucleotide to a second
partition of the two or more partitions, wherein the third
oligonucleotide includes a third spatial barcode (e.g., a third
partial spatial barcode); and (e) attaching the third
oligonucleotide to a fourth oligonucleotide in the second
partition, wherein the fourth oligonucleotide includes the first
spatial barcode (e.g., the first partial spatial barcode), thereby
generating capture probes including the first spatial barcode and
the third spatial barcode in the second partition. In some
embodiments, methods provided herein further include: (d)
delivering a third oligonucleotide to a second partition, wherein
the third oligonucleotide includes a third spatial barcode (e.g., a
third partial spatial barcode); and (e) attaching the third
oligonucleotide to a fourth oligonucleotide in the second
partition, wherein the fourth oligonucleotide includes the fourth
spatial barcode (e.g., a fourth partial spatial barcode) thereby
generating capture probes including the fourth spatial barcode and
the third spatial barcode in the second partition.
[1486] In some embodiments, the two or more partitions are
separated by one or more physical barriers. In some embodiments,
the one or more physical barriers are printed on the substrate. In
some embodiments, the one or more physical barriers form one or
more chambers including one or more chamber walls. In some
embodiments, the one or more chamber walls include a hydrophobic
surface. In some embodiments, the one or more chamber walls include
a tapered end in contact with the substrate. In some embodiments,
the second oligonucleotide is delivered using a microfluidic
device.
Chemical Ligation of Oligonucleotides
[1487] In some embodiments, chemical ligation can be used to ligate
two or more oligonucleotides. In some embodiments, chemical
ligation involves the use of condensing reagents. In some
embodiments, condensing reagents are utilized to activate a
phosphate group. In some embodiments, condensing reagents may be
one or more of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDCI), cyanogen bromide, imidazole derivatives, and
1-hydroxybenzotriazole (HOAt). In some embodiments, functional
group pairs selected from one or more of a nucleophilic group and
an electrophilic group, or an alkyne and an azide group are used
for chemical ligation. In some embodiments, chemical ligation of
two or more oligonucleotides requires a template strand that is
complementary to the oligonucleotides to be ligated (e.g., a splint
oligonucleotide). In some embodiments, the chemical ligation
process is similar to oligonucleotide synthesis.
Photocleavable Protective Groups
[1488] In some embodiments, a photocleavable protective group (e.g.
any photocleavable, photoremovable, photoreleasable,
photoactivatable, or photosensitive protective group described
herein or known in the art) can be coupled to one or both ends of
an oligonucleotide on a substrate, a surface probe (e.g. any of the
surface probes described herein), or a capture probe (e.g., any of
the capture probes described herein). In some embodiments, a
photocleavable protective group is a chemical modification to a
molecule that can be removed with light. In some embodiments, the
photocleavable protective group (PPG) is at least one of a
nitrobenzyl-based PPG, a carbonyl-based PPG, or a benzyl-based PPG.
In some embodiments, the photocleavable protective group is removed
from one or more surface probes by exposing the one or more surface
probes to a light.
Shallow Sequencing
[1489] As used herein, the coverage or depth of sequencing is the
number of unique reads that include a given nucleotide in a
reconstructed sequence. As used herein, shallow sequencing is a
small or low number of unique reads of an identified analyte. In
some embodiments, shallow sequencing can be performed to identify a
spatial region of interest. In some embodiments, shallow sequencing
can be performed on an entire biological sample. In some
embodiments, shallow sequencing can be performed on an identified
region of a biological sample (e.g., a region of interest). In some
embodiments, shallow sequencing can be performed after identifying
and/or selecting a region of a biological sample by some other
method (e.g., immunostaining).
Target Spatial Barcodes
[1490] In some embodiments, a target spatial barcode (e.g.,
sometimes referred to herein as a target barcode sequence) is a
barcode sequence corresponding to a spatial region of interest. In
some embodiments, a target spatial barcode is or includes a spatial
barcode. In some embodiments, a target spatial barcode is
identified by identifying a spatial region of interest and
identifying the spatial barcode associated with the spatial region
of interest on the array. In some embodiments, a target spatial
barcode is identified by performing shallow sequencing, and
selecting a spatial barcode based on the captured analytes. As used
herein, the coverage or depth of sequencing is the number of unique
reads that include a given nucleotide in a reconstructed sequence.
As used herein, shallow sequencing is a small or low number of
unique reads of an identified analyte. In some embodiments, shallow
sequencing can be performed to identify a spatial region of
interest. In some embodiments, shallow sequencing can be performed
on an entire biological sample. In some embodiments, shallow
sequencing can be performed on an identified region of a biological
sample (e.g., a region of interest). In some embodiments, shallow
sequencing can be performed after identifying and/or selecting a
region of a biological sample by some other method (e.g.,
immunostaining). In some embodiments, a target spatial barcode is
identified by a co-localization of a target spatial barcode with
one or more additional spatial barcode sequences. In some
embodiments, a target spatial barcode is identified by imaging. In
some embodiments, imaging includes immunohistochemistry and/or
other staining or microscopy methods. In some embodiments, a target
spatial barcode is identified manually. In some embodiments, a
target spatial barcode is identified using a computer. In some
embodiments, a target spatial barcode is identified
algorithmically. In some embodiments, the target spatial barcode
corresponds to a plurality of spatial barcode sequences that
identify a region, a sub-region, a sub-sub-region, or further
spatial demarcation. In some embodiments, a target spatial barcode
is used to identify and characterize captured analytes at
single-cell resolution. In some embodiments, the identifying can
include sequencing a plurality of capture probes to generate a
plurality of sequencing reads, and analyzing the plurality of
sequencing reads to identify the at least one target spatial
barcode. In some embodiments, each of the plurality of capture
probes can further include one or more functional sequences
selected from the group consisting of a sequencing primer sequence
and a sequence configured to attach to a flow cell of a sequencer.
In some embodiments, the plurality of capture probes can include,
from 5' to 3', a first functional domain (e.g., adapter sequence),
a sequence derived from a template nucleic acid, and a second
adapter sequence. The first adapter sequence can include a first
sequence configured to attach to a flow cell of a sequencer, a
first sequencing primer sequence, and a spatial barcode. The second
adapter sequence can include a second sequencing primer sequence
and a second sequence configured to attach to a flow cell of a
sequencer. The second adapter sequence can further include an index
sequence.
Enriching Barcoded Nucleic Acids
[1491] In an aspect, the present disclosure provides methods of
enriching a sample for target spatial barcoded nucleic acids. The
methods can include providing a plurality of capture probes
including a plurality of different spatial barcodes and a target
capture probe of the population of captures, identifying at least
one target spatial barcode from the plurality of different target
spatial barcodes, and enriching the population of target capture
probes including the spatial barcode from the plurality of capture
probes.
[1492] In an aspect, the present disclosure provides methods of
enriching a sample for target spatial barcoded nucleic acids. The
methods can include providing a plurality of capture probes
including a plurality of different spatial barcodes, identifying at
least one target spatial barcode from the plurality of different
target spatial barcodes, and enriching the analyte detected by
(e.g., specifically bound to the capture probe) the capture probes
including the at least one target spatial barcode (e.g., sometimes
referred to herein as a target barcode sequence). Enriching can be
performed by purifying capture probes including the at least one
target spatial barcode from a plurality of capture probes.
Additionally or alternatively, enriching can be performed by
amplifying (e.g., increasing the numbers of) capture probes
including the at least one target spatial barcode from a plurality
of capture probes. The amplification can include any of the variety
of amplification methods described herein, including isothermal
amplification and/or PCR. Enriching can be performed by both
purifying and amplifying the capture probe including the at least
one target spatial barcode from a plurality of capture probes.
[1493] Spatial barcodes can be included in capture probes. For
example, the capture probes can be co-partitioned into a partition
with spatial barcodes to facilitate the barcoding reaction for
spatial analysis. The spatial barcodes can be attached to a bead
(e.g., in the context of a capture probe). The bead can be a gel
bead. In some embodiments, the spatial barcodes are releasably
attached to the bead (e.g., gel bead) as described elsewhere herein
(e.g., via a cleavage domain). The plurality of capture probes can
be spatially barcoded as described elsewhere herein.
[1494] In some embodiments, methods provided herein can include
enriching nucleic acids including the at least one target spatial
barcode (e.g., capture probes that have been extended using a
captured analytes as a template). The enriching can include
performing a nucleic acid amplification reaction on at least one
capture probe including the at least one target spatial barcode to
generate an enriched plurality of capture probes including the at
least one target spatial barcode. The nucleic acid amplification
reaction can be a polymerase chain reaction (PCR). The nucleic acid
extension amplification reaction can be an isothermal amplification
reaction. The nucleic acid amplification reaction can include a
polymerase. The nucleic acid extension reaction can result in
amplification of the nucleic acid.
[1495] In some embodiments, methods provided herein can further
include performing an additional amplification reaction (e.g., PCR)
on the enriched plurality of capture probes including the at least
one target spatial barcode. The amplification reaction PCR can
include use of an additional primer including a sequence specific
for the enriched plurality of capture probes including the at least
one target spatial barcode sequence. In some embodiments, the
additional primer can include a sequence specific for the spatial
barcode of the enriched plurality of capture probes including the
at least one target spatial barcode. In some embodiments, the
additional primer can include a sequence substantially specific for
the spatial barcode sequence of the enriched plurality of capture
probes including the at least one target spatial barcode. For
example, the additional primer can include a sequence having at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more sequence
complementarity or sequence identity to the spatial barcode
sequence of the enriched plurality of capture probes including the
at least one target spatial barcode. In some embodiments, the
additional primer can include a sequence specific for a 5' region
of a spatial barcode sequence of the enriched plurality of capture
probes including the at least one target spatial barcode. In some
cases, the additional primer can include a sequence that has no (or
low) sequence complementarity or identity to the spatial barcode
sequence of the enriched plurality of capture probes including the
at least one target spatial barcode.
[1496] In some embodiments, the additional primer can include one
or more functional sequences that facilitate sequencing of the
enriched plurality of capture probes. The one or more functional
sequences can include a sequencing primer sequence. The one or more
functional sequences can include a sequence configured to attach to
a flow cell of a sequencer.
[1497] In some embodiments, methods provided herein can further
include, subsequent to the nucleic acid amplification reaction,
performing a size selection process to remove unincorporated
primers. The size selection process can include using a gel or
gel-like polymer. The size selection process can use gel
electrophoresis. In some cases, the size selection process can
include using a substrate. The substrate can be an array.
[1498] In some embodiments, the enriched plurality of nucleic acids
can include an affinity group. The affinity group can be
incorporated to the enriched plurality of the nucleic acid via a
polymerization reaction with a primer including the affinity group.
The affinity group can be incorporated via enzymatic reaction with
a nucleotide and the affinity group. The affinity group may be
attached covalently or non-covalently. The method can further
include coupling the enriched plurality of capture probes including
the affinity group to a substrate specific for the affinity group.
The substrate can be an array. The substrate can be a polymer
matrix. The substrate can be a glass slide. The substrate can be a
resin. In some embodiments, the substrate can include an antibody
or antibody fragment. In some embodiments, the substrate includes
avidin or streptavidin and the affinity group includes (or is)
biotin. In some embodiments, the substrate includes maltose and the
affinity group includes (or is) maltose binding protein. In some
embodiments, the substrate includes maltose-binding protein and the
affinity group includes (or is) maltose.
[1499] In some embodiments, prior to the nucleic acid amplification
reaction, a first capture probe complementary to a first portion of
the at least one target spatial barcode and a second capture probe
complementary to a second portion of the at least one target
spatial barcode can be hybridized. The first capture probe can be
ligated to the second capture probe to generate the primer
including the sequence specific for the at least one target spatial
barcode. The ligating can include using a DNA ligase.
[1500] In some embodiments, the enriching can result in at least
10-fold enrichment of capture probes including the at least one
target spatial barcode. In some embodiments, the enriching can
result in at least 20-fold enrichment of capture probes including
the at least one target spatial barcode. The enrichment of nucleic
acids can be observed by an increase in the percentage of nucleic
acids including the at least one target spatial barcode in the
enriched plurality of capture probes compared to the percent amount
of nucleic acids including the at least one target spatial barcode
in the plurality of capture probes. The enriching can result in at
least 50-fold enrichment of capture probes including the at least
one target spatial barcode. The enriching can result in at least
100-fold enrichment of capture probes including the at least one
target spatial barcode. The enriching can result in at least
200-fold enrichment of capture probes including the at least one
target spatial barcode. The enriching can result in at least
500-fold enrichment of capture probes including the at least one
target spatial barcode. The enriching can result in at least
1000-fold enrichment of capture probes including the at least one
target spatial barcode.
[1501] Primers used for extension or amplification reactions
described herein can be a specific length or include a specific
number of nucleotides. For example, the primers may be at least
about 5, about 6, about 7, about 8, about 9, about 10, about 12,
about 14, about 16, about 18, about 20, about 22, about 24, about
25, about 26, about 28, about 30, about 40, about 50 or more
nucleotides long. Alternatively or in addition, the primers can be
no more than about 50, about 40, about 30, about 28, about 26,
about 25, about 24, about 22, about 20, about 18, about 16, about
14, about 12, about 10, about 9, about 8, about 7, about 6, about
5, or fewer nucleotides long.
[1502] The primers may have a specific melting temperature. The
melting temperature of the primer can be at least about 35 degrees
Celsius (.degree. C.), 40.degree. C., 41.degree. C., 42.degree. C.,
43.degree. C., 44.degree. C., 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C., 50.degree. C.,
51.degree. C., 52.degree. C., 53.degree. C., 54.degree. C.,
55.degree. C., 56.degree. C., 57.degree. C., 58.degree. C.,
59.degree. C. 60.degree. C., 61.degree. C., 62.degree. C.,
63.degree. C., 64.degree. C., 65.degree. C., or higher. The melting
temperature of the primer can be no more than about 35.degree. C.,
40.degree. C., 41.degree. C., 42.degree. C., 43.degree. C.,
44.degree. C., 45.degree. C., 46.degree. C., 47.degree. C.,
48.degree. C., 49.degree. C., 50.degree. C., 51.degree. C.,
52.degree. C., 53.degree. C., 54.degree. C., 55.degree. C.,
56.degree. C., 57.degree. C., 58.degree. C., 59.degree. C.
60.degree. C., 61.degree. C., 62.degree. C., 63.degree. C.,
64.degree. C., 65.degree. C., or lower.
[1503] In some cases, primers are ligated together to form a longer
primer. The longer primer can be subsequently used for any PCR
reactions, as described elsewhere herein. Ligating together shorter
primers into a longer primer can increase the stringency of binding
such that the targeted spatial barcode is enriched. For example,
the shorter primers can be able to distinguish mismatches better
and thus bind only to sequences with perfect or near perfect
complementarity. The longer primer can be more able to accommodate
mismatches in sequences and bind to sequences that are not the
targeted barcode. In some cases, more than 2 primers are ligated
together to form a longer primer. For example, at least about 3, 4,
5, 6, 7, 8, 9, 10, or more primers can be ligated together to form
a longer primer.
[1504] In some embodiments, the primer includes an affinity group
and the enriched plurality of capture probes includes the affinity
group. The affinity group can include biotin. The affinity group
can include streptavidin or avidin. The affinity group can include
dioxigenin. The affinity group can include a protein or
polypeptide. The affinity group can include an antibody or antibody
fragment. The affinity group can include a receptor or receptor
ligand. The affinity group can includes a sugar, a monosaccharide,
a disaccharide, or a polysaccharide. The affinity group can include
maltose.
[1505] Methods of the present disclosure can be used for various
applications, such as, processing a single analyte (e.g., RNA, DNA,
or protein) or multiple analytes (e.g., DNA and RNA, DNA and
protein, RNA and protein, or RNA, DNA, and protein) from a single
cell. For example, multiple analytes from the biological sample can
be processed. The multiple analytes can be from the single cell.
This can enable, for example, simultaneous proteomic,
transcriptomic and genomic analysis of the cell (e.g.,
multiplexing).
Hierarchal Barcode Enrichment
[1506] In some aspects, the present disclosure provides methods of
spatially designing or designating spatial barcodes, such that the
position of the spatial barcodes are specifically identified. In
some embodiments, a particular individual feature on a spatial
array (e.g., a spot or bead) can contain more than one spatial
barcode, wherein each spatial barcode corresponds to a particular
defined region on the spotted array. For example, multiple spatial
barcode sequences associated with one particular feature on an
array can provide a stronger address or attribution to a given
location by providing duplicate or independent confirmation of the
location. In some cases, the multiple spatial barcodes represent
increasing specificity of the location of the particular feature.
In a non-limiting example, a particular feature can be coded with
two different spatial barcodes, wherein each spatial barcode
identifies a particular defined region within the array, and a
feature possessing both spatial barcodes identifies the sub-region
where two defined regions overlap, e.g., such as the overlapping
portion of a Venn diagram. In another non-limiting example, a
particular feature can be coded with three or more different
spatial barcodes, wherein the first spatial barcode identifies a
first region within the array, the second spatial barcode (e.g.,
sub region spatial barcode, second common spatial barcode)
identifies a second region, wherein the second region is a
subregion entirely within the first region (a sub region), and the
third spatial barcode (e.g., sub-sub region spatial barcode, third
common spatial barcode) identifies a third region, wherein the
third region is a subregion entirely within the first and second
subregions (a sub region), and so on. In some embodiments, each
capture probe has a unique molecular identifier (UMI). In some
embodiments, each capture probe has a UMI, as well as one or more
target spatial barcodes corresponding to one or more regions or sub
regions on an array.
[1507] In some embodiments, an array has multiple features (also
sometimes referred to as "regions", "spots", or "locations") and
subregions. In some embodiments, an array has 10 features or more.
In some embodiments, an array has 20, 30, 40, 50, 60, 70, 80, 90,
100 features, or more. In some embodiments, an array has 100, 200,
300, 400, 500, 600, 700, 800, 900, 1000 features or more. In some
embodiments, an array has 1000, 2000, 3000, 4000, 5000, 6000, 7000,
8000, 9000, 1000 features or more. In some embodiments, the
features are identical in size. In some embodiments, the features
are not identical in size. The features may take any shape,
including but not limited to square, rectangle, circle, oval,
pentagon, hexagon, octagon arc, arches, lens, crescent, and any
combinations thereof. In some embodiments, a feature of an array
has multiple subregions. In some embodiments, a feature of an array
has 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,
600, 700, 800, 900, 1000 subregions or more. In some embodiments,
the subregions are identical in size to other subregions. In some
embodiments, the subregions are not identical in size to other
subregions. In some embodiments, the subregions are completely
surrounded by a larger region. In some embodiments, subregions are
partially surrounded by a larger region. In some embodiments,
different subregions of a feature include different regional
spatial barcodes that are characteristic of the given subregions.
In some embodiments, different subregions of a feature include
different regional spatial barcodes that are characteristic of the
given subregions and a common spatial barcode that is
characteristic of the feature. Such embodiments permit enrichment
of capture analytes having the common spatial barcode and/or one or
more regional spatial barcodes (e.g., via any of the variety of
enrichment methods described herein). In some embodiments, an array
has multiple sub-subregions. In some embodiments, an array has 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,
800, 900, 1000 sub-subregions or more. In some embodiments, the
sub-subregions are identical in size to other sub-subregions. In
some embodiments, the sub-subregions are not identical in size to
other sub-subregions. In some embodiments, sub-subregions are
completely surrounded by a larger region. In some embodiments, the
larger region is a subregion. In some embodiments, the larger
region is a region. In some embodiments, sub-subregions are
partially surrounded by a larger subregion. In some embodiments,
sub-subregions are partially surrounded by a larger region. In some
embodiments, different sub-subregions of a subregion include
different sub-regional spatial barcodes that are characteristic of
the given sub-subregions. In some embodiments, different
sub-subregions of a feature include different sub-regional spatial
barcodes that are characteristic of the given subregions and a
common regional spatial barcode that is characteristic of the
region. In some embodiments, different sub-subregions of a feature
include different sub-regional spatial barcodes that are
characteristic of the given subregions, a common regional barcode
that is characteristic of the region, and a common spatial barcode
that is characteristic of the feature. Such embodiments permit
enrichment of capture analytes having the common spatial barcode
and/or one or more regional spatial barcodes (e.g., via any of the
variety of enrichment methods described herein). In some
embodiments, individual features or spots or beads include a
region. In some embodiments, individual features of spots or beads
include a subregion. In some embodiments, individual features or
spots or beads include a sub-subregion. In some embodiments,
individual features or spots or beads include a region
corresponding to single cell resolution of the biological
sample.
[1508] In some embodiments, the present disclosure provides methods
of enriching a sample including more than one analyte specifically
bound to the capture probe. The methods can include providing a
plurality of capture probes including a plurality of different
spatial barcodes, identifying more than one target spatial barcode
(e.g., sometimes referred to as a target barcode sequence) from the
plurality of different spatial barcodes, and enriching capture
probes including the more than one target spatial barcode.
Enriching can be performed by purifying capture probes including
the more than one target spatial barcode from a plurality of
capture probes. Enriching can be performed by amplifying (e.g.,
increasing the numbers of) capture probes including the more than
one target spatial barcode from a plurality of capture probes.
Enriching can be performed by both purifying and amplifying the
capture probe including the more than one target spatial barcode
from a plurality of capture probes. Enriching can be performed
using target spatial barcodes associated with smaller regions.
Enriching can be performed using target spatial barcodes of
increasing specificity to a particular feature or spot or location.
For example, enriching can be performed by amplifying and/or
purifying a first plurality of capture probes including a target
spatial barcode that is associated with a first spatial location
(e.g., a first feature, a first subregion, and/or a first
sub-subregion). The enriching may further include amplifying and/or
purifying a second plurality of capture probes including a target
spatial barcode that is associated with a second spatial location
(e.g., a second feature, a second subregion, and/or a second
sub-subregion) that is partially or completely within the first
spatial location. The enriching can further include amplifying
and/or purifying a second plurality of capture probes including a
target spatial barcode that is associated with a second spatial
location that is partially within the first region. The enriching
can further include amplifying and/or purifying a second plurality
of capture probes including a target spatial barcode that is
associated with a second spatial location that is completely within
the first spatial location. The enriching can further include
amplifying and/or purifying a third plurality of capture probes
including a target spatial barcode that is associated with a third
spatial location that is partially or completely within the first
and second spatial locations. The enriching can further include
amplifying and/or purifying a third plurality of capture probes
including a target spatial barcode that is associated with a third
spatial location that is partially within the first and second
spatial locations. The enriching can further include amplifying
and/or purifying a third plurality of capture probes including a
target spatial barcode that is associated with a third spatial
location that is completely within the first and second spatial
locations. The enriching can further include amplifying and/or
purifying a fourth plurality of capture probes including additional
target spatial barcodes that are associated with four or more
spatial locations that are partially or completely within the
first, second, and third spatial locations. The enriching may
further include amplifying and/or purifying a fifth plurality of
capture probes including additional target spatial barcodes that
are associated with five or more spatial locations that are
partially or completely within the first, second, third, and fourth
spatial locations. The enriching can further include amplifying
and/or purifying additional pluralities of capture probes including
additional target spatial barcodes that are associated with 6, 7,
8, 9, 10, or more spatial locations that are partially or
completely within the previously identified regions and/or sub
regions.
[1509] In some embodiments, the method can include enriching
nucleic acids including more than one target spatial barcode. The
enriching can include performing a nucleic acid extension reaction
using a capture probe including more than one target spatial
barcode and a primer including a sequence specific for more than
one target spatial barcode to generate an enriched plurality of
capture probes including more than one target spatial barcode. The
nucleic acid extension reaction can be a polymerase chain reaction
(PCR). The nucleic acid extension reaction can include a
polymerase. The nucleic acid extension reaction can result in
amplification of the nucleic acid.
[1510] In some embodiments, methods provided herein may further
include performing additional rounds of PCR on the enriched
plurality of capture probes corresponding to the number of spatial
barcode sequences of a given location. The additional rounds of PCR
may include use of additional primers including a sequence specific
for the additional target spatial barcode (e.g., target spatial
barcode associated with a region, a subregion, or sub-subregion of
interest). In some embodiments, the additional primers may include
a sequence specific for the spatial barcode sequence of the
enriched plurality of capture probes including more than one target
spatial barcode. In some embodiments, the additional primers may
include a sequence substantially specific for an additional target
spatial barcode of the enriched plurality of capture probes
including more than one target spatial barcode. For example, the
additional primer may include a sequence having at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.5%, 99.9% or more sequence complementarity or
sequence identity to a target spatial barcode of the enriched
plurality of capture probes including the more than one target
spatial barcode. In some embodiments, the additional primers may
include a sequence specific for a 5' region of a spatial barcode
sequence of the enriched plurality of capture probes including more
than one target spatial barcode. In some cases, the additional
primer can includes a sequence that has no (or low) sequence
complementarity or identity to one or more spatial barcode
sequences of the enriched plurality of capture probes including
more than one target spatial barcode.
[1511] In some embodiments, each spatial barcode sequence of the
plurality of different spatial barcode sequences can identify a
capture probe as derived from a single cell. Alternatively or in
addition, each spatial barcode sequence of the plurality of
different spatial barcode sequences may identify a capture probe as
derived from a single partition.
Identifying a Spatial Region of Interest
[1512] In some embodiments, identifying a spatial region of
interest includes any technique for observing and/or locating an
area within a tissue. In some embodiments, identifying a spatial
region of interest includes immunostaining techniques, including
but not limited to, immunohistochemistry techniques. In some
embodiments, identifying a spatial region of interest includes
microscopy techniques, including but not limited to, optical
microscopy, fluorescence microscopy, and confocal microscopy. In
some embodiments, identifying a spatial region of interest includes
visual inspection. In some embodiments, identifying a spatial
region of interest includes shallow sequencing a spatial region. In
some embodiments, identifying a spatial region of interest includes
shallow sequencing the entire biological sample. Any of the variety
of other methods described herein or otherwise known in the art to
identify a spatial region of interest can be used.
Generating Spatial Arrays with Light
[1513] In at least one embodiment of the compositions and methods
of the present disclosure, a plurality of first oligonucleotide are
attached to a substrate by any suitable means that immobilizes the
first oligonucleotide by either their 3' or 5' end in a region of
the substrate and allows the unattached end to be available for
further synthesis. Further synthesis can include in situ
base-by-base step-wise synthesis (e.g., phosphoramidite chemistry)
or biosynthesis via enzyme reactions (e.g., DNA polymerization or
reverse transcription). In some embodiments, the means of
attachment is also be suitable to allow at least a portion of the
first oligonucleotide to be capable of hybridization.
[1514] In at least one embodiment of the compositions and methods
of the present disclosure, the plurality of first oligonucleotides
are attached to a substrate by a covalent linkage. Accordingly, in
at least one embodiment, the plurality of first oligonucleotides
are attached to the substrate by a chemical reaction that forms one
or more covalent bonds between a functional group on the substrate
(or a linker group attached to the substrate) and a functional
group on the 3'- or 5'-end of the first oligonucleotide (or a
linker group attached to the 3' or 5'-end of the first
oligonucleotide). Such functional groups can include a naturally
occurring nucleotide functional group, e.g. a terminal hydroxyl
group, or a non-natural functional group introduced during
oligonucleotide synthesis, such as a 5'-amine group. Reagents and
techniques for chemically modifying substrate materials and/or
nucleotides with functional groups that are useful in the methods
of the present disclosure are well known in the art. For example,
functional groups that may be introduced on a substrate that allow
for attachment of a first oligonucleotide (e.g., 5'-amine modified
oligonucleotide) to a substrate include linkers with available
carboxy, aldehyde or epoxy groups. Other functionalized linker
compounds are known to the person skilled in the art.
[1515] The first oligonucleotide of the plurality of first
oligonucleotides to be attached to a substrate in preparing a
spatial array of the present disclosure can be understood as having
at least three exemplary oligonucleotide: a first oligonucleotide
of about 2-mer to about 40-mer that is attached to the substrate
through either its 3'-end or its 5'-end; a second oligonucleotide
that is attached to the unattached end of the first, and includes a
spatial barcode sequence of about 8-mer to about 30-mer in length
that indicates the region on the substrate where the second is
located (e.g., a spatial barcode); and a plurality of third
oligonucleotide s of about 5-mer to about 50-mer attached to the
second oligonucleotide, wherein the third oligonucleotide includes
a capture sequence at its 3'-end. In some embodiments, the
exemplary oligonucleotides include one or more additional
sequences. For example, a first oligonucleotide can include a
functional domain (e.g., any of the variety of functional domains
described herein). In some embodiments, two or more domains present
in the exemplary oligonucleotides each include a portion of a
domain, such that when the exemplary oligonucleotides are joined, a
full domain is generated. For example, a first oligonucleotide can
include a first portion of a domain (e.g., a spatial barcode) and a
second oligonucleotide can include a second portion of a domain
(e.g., a spatial barcode) such that the spatial barcode is
generated upon joining the first and second oligonucleotides.
[1516] FIG. 31 depicts at least one embodiment of a method for
preparing an exemplary spatial array composition of the present
disclosure. First, as shown in FIG. 31A, a substrate is provided
that includes a first oligonucleotide attached through its 5'-end
to the substrate. In at least one embodiment, the first
oligonucleotide includes a sequence that is identical across all of
the plurality of the different oligonucleotides attached to the
substrate. In some embodiments, the first oligonucleotide can be
understood as a common oligonucleotide "lawn" across the surface of
the substrate that does not differ in sequence in the various
regions of the substrate. The free, unattached end of the first
oligonucleotide, thus, effectively acts as the starting point on
the spatial array for the spatially-directed in situ synthesis of
the second oligonucleotide that includes the spatial barcode.
[1517] In at least one embodiment, the length of the first
oligonucleotide is the same across the surface of the substrate.
While typically not critical to the function of the spatial array,
in at least one embodiment, the length of the first oligonucleotide
sequence should be at least about 2 nucleotides in length.
Furthermore, in some embodiments the length of the first
oligonucleotide is at least about 5 nucleotides in length, at least
about 10 nucleotides in length, at least about 15 nucleotides in
length, at least about 20 nucleotides in length, at least about 25
nucleotides in length, at least about 27 nucleotides in length, at
least about 30 nucleotides in length, at least about 33 nucleotides
in length, or at least about 35 nucleotides in length. Similarly,
in some embodiments the first oligonucleotide has a length of about
2 nucleotides in length to about 40 nucleotides in length, about 5
nucleotides in length to about 40 nucleotides in length, about 10
nucleotides in length to about 30 nucleotides in length, about 25
nucleotides in length to about 35 nucleotides in length, about 20
nucleotides in length to about 40 nucleotides in length, about 25
nucleotides in length to about 40 nucleotides in length, about 25
nucleotides in length to about 35 nucleotides in length, or about
27 nucleotides in length to about 33 nucleotides in length.
[1518] As further depicted in FIG. 31A, in at least one embodiment,
the first oligonucleotide can include phosphodiester and exocyclic
amine moieties that are protected. Protection of the phosphodiester
and exocyclic amine moieties of the first oligonucleotide allows
for optimal in situ synthesis of the second oligonucleotides that
includes the spatial barcode. Suitable protecting groups for
phosphodiester and exocyclic amine moieties are described herein
and are well-known in the art and include the protecting groups
employed in standard oligonucleotide synthesis using
phosphoramidite chemistry.
[1519] The present disclosure describes a variety of embodiments
for preparing a substrate having attached thereto a first
oligonucleotide, wherein the first oligonucleotide includes
protected phosphodiester and exocyclic amine moieties (see e.g.,
embodiments of FIGS. 32-36).
[1520] In at least one exemplary embodiment, depicted in FIG. 32,
the method further includes preparing the first oligonucleotide ex
situ (e.g., on a separate substrate or feature using solid-phase
automated synthesis), cleaving it from the separate substrate or
feature with the protecting groups intact (see FIG. 32A), and
subsequently attaching the ex situ synthesized oligonucleotide with
protecting groups it to the substrate (see FIG. 32B). A free
hydroxyl at the unattached end is available for subsequent
spatially-directed in situ synthesis of a second oligonucleotide,
e.g., a second oligonucleotide including a spatial barcode (see
FIG. 32C).
[1521] In at least another exemplary embodiment, depicted in FIG.
33, the method further includes preparing the first oligonucleotide
in situ directly on the substrate using base-by-base
phosphoramidite synthesis chemistry with phosphodiester and
exocyclic amine protecting groups. Such a method can be use
standard solid-phase oligonucleotide synthesis chemistry to provide
a substrate with first oligonucleotide attached thereto by the
3'-end and having protecting groups. In at least one embodiment, as
depicted in FIG. 33A, oligonucleotide synthesis chemistry in a
5'-to-3' direction can be carried out to provide the protected
first oligonucleotides attached to the substrate through the
5'-end. In both cases, the free hydroxyl at the unattached end is
available for subsequent spatially-directed in situ synthesis of a
second oligonucleotide, e.g., a second oligonucleotide including a
spatial barcode (see FIG. 32C).
[1522] In some embodiments, as depicted in FIG. 31B, a plurality of
second oligonucleotides of about 8-mer to about 30-mer are prepared
by spatially-directed in situ synthesis extending from the free end
of the first oligonucleotide. As shown in FIG. 31B, the
spatially-directed in situ synthesis can be a light-directed
spatial synthesis which is carried out in the 5'-to-3' direction.
This in situ synthesis method results in a plurality of second
oligonucleotide each attached through its 5'-end to a 3'-end of a
first oligonucleotide. FIG. 31B is a schematic representation of
only two distinct regions each having four individual
oligonucleotide molecules. Each region can have a different second
oligonucleotide sequence, and all of the second oligonucleotide
molecules in a region can have the same sequence, although as
discussed further below, less than 100% yield in the in situ
synthesis process results in less than perfect purity (or
homogeneity) of oligonucleotide sequence (e.g., a second
oligonucleotide) in a region.
[1523] In at least one embodiment of the present disclosure,
spatially-directed synthesis of a plurality of oligonucleotides on
a substrate in spatially distinct regions is carried out using
light-directed synthesis or by mechanically-directed synthesis
methods. A spatially-directed in situ synthesis method inherently
correlates each sequence synthesized with the location of
synthesis. In the schematic illustration of FIG. 31B, a
light-directed spatial synthesis is used to synthesize a second
oligonucleotide in a base-by-base step-wise fashion in each region.
The region where each nucleotide base is added can be controlled by
the precise location (and duration) of spatially-directed
illumination of the substrate. The direction and duration of
illumination can be precisely controlled, for example with maskless
array synthesis (MAS) technology using computer-controlled digital
micromirror devices (DMD), to provide distinct regions of 1
.mu.m.sup.2, or even smaller. It is only at these precisely
illuminated regions on the substrate where deprotection of a
photo-labile protecting group occurs, thereby allowing coupling of
a nucleoside phosphoramidite that provides the next base in the
oligonucleotide sequence. In some embodiments, the synthesis of the
first oligonucleotide is not light-directed synthesis.
[1524] In some embodiments, light-directed spatial in situ
synthesis provides optimal control in the synthesis of
high-fidelity second oligonucleotide spatial barcode sequences at
precise region locations on a substrate. Light-directed spatial
synthesis of oligonucleotides refers to synthetic methods using
photolabile nucleoside protecting groups and spatially-directed
illumination to effect selective deprotection and base-by-base
synthesis at specific locations on a substrate. Light-directed
spatial synthesis of oligonucleotides uses an iterative cycle of
phosphoramidite-based coupling chemistry with photo-labile
protecting groups rather than dimethoxytrityl (DMT) protecting
groups. The synthesis cycle, thus, is very similar to that used in
standard solid-phase phosphoramidite synthesis of oligonucleotides
with the following general steps: (1) photo-deprotection; (2)
activation and coupling; (3) capping; and (4) drying. The drying
step (4) can involve blowing helium to dry the substrate surface
after coupling and acetonitrile wash, but before the next
photodeprotection step. Techniques, protocols, reagents, materials,
and other instrumentation useful for light-directed spatial
synthesis of high-density oligonucleotide microarrays are described
herein, are well-known in the art, and are commercially available.
For example, U.S. Pat. Nos. 7,844,940, 8,030,477, PCT Publ. No. WO
99/63385A1, US Pat. Publ. No. 20150246336A1, Agbavwe et al.,
Efficiency, error and yield in light-directed maskless synthesis of
DNA microarrays, Journal of Nanobiotechnology 9.1 (2011): 57; and
Sack et al., Express photolithographic DNA microarray synthesis
with optimized chemistry and high-efficiency photolabile groups,
Journal of Nanobiotechnology 14.1 (2016): 14, describe materials
and methods that can be used in the light-directed spatial
synthesis methods of the present disclosure. The aforementioned
patents and publications are each incorporated herein by reference
in their entireties.
[1525] In some embodiments, the photo-deprotection step involves
spatially-directed illumination of the substrate and is the step
that ultimately defines the size and location of a region.
Efficiency and accuracy of the photo-deprotection step in defining
the regions on the substrate where specific oligonucleotides are
synthesized depends largely on the illumination techniques and the
photo-labile groups used. Photo-labile groups and illumination
techniques useful for light-directed spatial synthesis methods are
described herein, are well-known, and are commercially available
(e.g., from Sigma-Aldrich, St. Louis, Mo., USA). For example,
photo-labile protecting groups that can be used to prepare spatial
array compositions of the present disclosure are described in US
Pat. Publ. No. 20150246336A1; Agbavwe et al., Efficiency, error and
yield in light-directed maskless synthesis of DNA microarrays
Journal of Nanobiotechnology 9.1 (2011): 57; Kretschy et al.,
Next-Generation o-Nitrobenzyl Photolabile Groups for Light-Directed
Chemistry and Microarray Synthesis, Angewandte Chemie International
Edition 54.29 (2015): 8555-8559; Sack et al., Express
photolithographic DNA microarray synthesis with optimized chemistry
and high-efficiency photolabile groups, Journal of
Nanobiotechnology 14.1 (2016): 14; Lietard et al., High-Density RNA
Microarrays Synthesized In Situ by Photolithography, Angew. Chem.
Int. Ed. 57 (2016): 15257-15261; and Holz et al., High-Efficiency
Reverse (5'.fwdarw.3') Synthesis of Complex DNA Microarrays,
Scientific reports 8.1 (2018): 15099, each of which are
incorporated herein by reference in their entireties.
[1526] In at least one embodiment, the photo-labile protecting
groups useful in the light-direct spatial synthesis methods of the
present disclosure include NPPOC, Bz-NPPOC, and SPh-NPPOC which are
shown in Table 2.
TABLE-US-00002 TABLE 2 Exemplary Photo-labile Protecting Groups
Structure Name (e.g., protecting 3'-OH of nucleoside) NPPOC
##STR00042## Bz--NPPOC ##STR00043## SPh--NPPOC ##STR00044##
[1527] The schematic illustration of FIG. 31B depicts
light-directed spatial synthesis of second oligonucleotide in the
5'-to-3' direction. The resulting plurality of the second
oligonucleotide are thus attached through their 5'-ends to the free
3'-ends of the first oligonucleotide on the substrate. Accordingly,
in at least one embodiment the light-directed spatial synthesis of
the second oligonucleotides is carried out in the 5'-to-3'
direction. Techniques, reagents, and photo-labile protecting groups
useful for synthesis in the 5'-to-3' direction include those
described in e.g., Holz et al., High-Efficiency Reverse
(5'.fwdarw.3') Synthesis of Complex DNA Microarrays, Scientific
reports 8.1 (2018): 15099, which is incorporated herein by
reference. In at least one embodiment, light-directed spatial
synthesis of a plurality of second oligonucleotides can be carried
out using one or more of the photo-labile protecting groups of
Table 1 to protect the 3'-OH. In at least one embodiment, the
photo-labile protecting group used in light-directed spatial
synthesis of the second oligonucleotide is Bz-NPPOC.
[1528] In at least one embodiment, the spatially-directed in situ
synthesis can be carried out using mechanically-directed spatial
synthesis. Mechanically-directed spatial synthesis of
oligonucleotides refers to methods that use computer-controlled
liquid dispensing and/or placement technology to mechanically
direct nucleoside phosphoramidite reagents at distinct locations on
a substrate, and thereby synthesizing an oligonucleotide (e.g., a
second oligonucleotide) using standard solid-phase phosphoramidite
chemistry at a distinct region on a substrate. A range of
technologies for mechanical spatially-directed in situ synthesis of
oligonucleotides are described herein and are well-known in the
art.
[1529] The schematic illustration of FIG. 31B depicts only two
regions, however it is contemplated that a spatial array
composition made by the methods of the present disclosure can
include millions of different regions on a substrate, each
including a different second oligonucleotide spatial barcode. In at
least one embodiment, the number of second oligonucleotides
including different sequences is at least about 100,000, at least
about 250,000, at least about 500,000, at least about 750,000, at
least about 1,000,000, at least about 1,500,000, at least about
3,000,000, at least about 5,000,000, at least about 10,000,000, at
least about 100,000,000, at least about 500,000,000, or at least
about 1,000,000,000. In some embodiments, the number of second
oligonucleotide including different sequences is about 100,000 to
about 1,000,000,000, about 750,000 to about 1,500,000, about
1,000,000 to about 1,000,000,000, about 1,000,000 to about
10,000,000, or about 1,500,000 to about 5,000,000.
[1530] In order for a spatial array to accommodate these large
numbers of different oligonucleotides on a substrate, it also is
contemplated that the areas of the regions can be sufficiently
small. Accordingly, in at least one embodiment of the method of the
present disclosure the regions of the substrate have an area of
less than about 2500 .mu.m.sup.2, less than about 1600 .mu.m.sup.2,
less than about 400 .mu.m.sup.2, less than about 100 .mu.m.sup.2,
less than about 50 .mu.m.sup.2, less than about 25 .mu.m.sup.2,
less than about 15 .mu.m.sup.2, less than about 10 .mu.m.sup.2,
less than about 5 .mu.m.sup.2, less than about 2 .mu.m.sup.2, less
than about 1 .mu.m.sup.2, or smaller.
[1531] In at least one embodiment, methods of the present
disclosure contemplate the spatially-directed in situ synthesis of
second oligonucleotides including millions of different spatial
barcodes each in a different region. In order to accommodate
millions of distinct regions each with a distinct spatial barcode,
the present disclosure contemplates that the second
oligonucleotides have a length range of about 8 nucleotides to
about 30 nucleotides. A length range of about 8 nucleotides to
about 30 nucleotides allows for the plurality of second
oligonucleotide to have extremely large number of distinct
oligonucleotides that are indexed for their location on the
substrate. For example, in the case of an 8 nucleotides sequence,
there are 48 or 65,536 different possible barcode sequences that
can be used to identify distinct locations on a substrate. In the
case of a 12 nucleotides sequence, there are 412 or 16,777,216
different possible spatial barcodes that can be used to identify
distinct locations on a substrate. Accordingly, in at least one
embodiment of the method of the present disclosure the second
oligonucleotide have a length selected from 8 nucleotides, 9
nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13
nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17
nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21
nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25
nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29
nucleotides, or 30 nucleotides.
[1532] In order to provide for optimal functionality of a spatial
array, the correlation of the spatial barcodes with the locations
should be high. Accordingly, the spatially-directed in situ
synthesis of the spatial barcode should result in high fidelity or
purity of spatial barcode in each of the regions. It is an
advantage of the spatially-directed synthesis methods of the
present disclosure that the second oligonucleotide are prepared on
a substrate with high fidelity. Generally, the fidelity (or
sequence purity) of oligonucleotide sequences prepared by step-wise
solid-phase synthesis decreases as the length of the
oligonucleotide being synthesized increases. For example, assuming
a spatially-directed in situ synthesis having a coupling efficiency
of 99.5%, the fidelity of 12 nucleotide barcode sequences in a
region is .about.95%. In some embodiments (e.g., as in FIG. 32),
the use of spatially-directed in situ synthesis to prepare only the
8 to 30 nucleotide second oligonucleotides results in spatial
barcodes of high-fidelity. Accordingly, in at least one embodiment
of the methods of the present disclosure the resulting second
oligonucleotide synthesized in each of the different regions of the
substrate has a purity of at least 75%, at least 80%, at least 85%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or more.
[1533] In at least one embodiment, the second oligonucleotide is
unique for each region of the substrate. Additionally or
alternatively, in some embodiments, the second oligonucleotide
spatial barcode includes a sequence that uniquely identifies it
from the plurality of other spatial barcode sequences on the
spatial array but does not interfere with the capture sequences
(e.g., capture domains) of the oligonucleotides (e.g., capture
probes) that target binding to nucleic acids in a sample. Unique
spatial barcodes can be generated using random sequence generation,
along with stringent filtering of sequences by mapping to the
genomes of all common reference species using desired Tm intervals,
and GC content, to minimize interference with capture sequence
(e.g., capture domain) binding to analytes, and to optimize the
difference from the other spatial barcodes. Thus, in at least one
embodiment, the spatial barcodes of the spatial array are selected
such that desired analytes of the biological sample applied to the
array do not hybridize specifically to the spatial barcode of the
second oligonucleotide. For example, in some embodiments, the
spatial barcode of the spatial array have less than 80% sequence
complementarity to the targeted nucleic acid sequences (e.g.,
analytes) in the sample applied to the spatial array. In some
embodiments, spatial barcode has less than 70% sequence id
complementarity entity, less than 60% sequence complementarity,
less than 50% sequence complementarity, or less than 40% sequence
complementarity to the nucleic acids in the biological sample
targeted for capture by the plurality of oligonucleotide
sequences.
[1534] In some embodiments, as depicted in FIG. 31C, a plurality of
third oligonucleotides are attached to the unattached end of the
second oligonucleotide. In at least one embodiment, the third
oligonucleotide is in the same orientation as the second
oligonucleotide. Thus, if the second oligonucleotide is attached to
the first oligonucleotide through its 5'-end (in a 5'-to-3'
orientation relative to the substrate) then the third
oligonucleotide is attached through its 5'-end to the 3'-end of the
second oligonucleotide. As exemplified in FIG. 31C, in at least one
embodiment, the third oligonucleotides are attached through their
5'-ends to the 3'-ends of the plurality of second oligonucleotides.
In some embodiments, the third oligonucleotide has a length of
about 5 to about 40 nucleotides and includes a capture sequence
(e.g., capture domain) capable of hybridizing to an analyte (e.g.,
nucleic acid) in a biological sample. As described further herein,
in some embodiments, the third oligonucleotide includes a free
3'-OH and is capable of hybridizing to a complementary nucleic acid
(e.g., an analyte) in a biological sample and then undergoing
biosynthetic (e.g., polymerase catalyzed) extension from it
3'-OH.
[1535] In at least one embodiment, the plurality of third
oligonucleotides have a length of at least about 5 nucleotides, at
least about 10 nucleotides, at least about 15 nucleotides, at least
about 20 nucleotides, at least about 27 nucleotides, at least about
30 nucleotides, at least about 33 nucleotides, or at least about 35
nucleotides. In some embodiments, the plurality of third
oligonucleotide have a length of about 5 nucleotides to about 40
nucleotides, about 10 nucleotides to about 40 nucleotides, about 20
nucleotides to about 40 nucleotides, about 25 nucleotides to about
35 nucleotides, or about 27 nucleotides to about 33
nucleotides.
[1536] In at least one embodiment of the methods of the present
disclosure, the third oligonucleotides are prepared off the
substrate and attached as a whole segment by chemical coupling or
ligation to the plurality of second oligonucleotides. In at least
one embodiment, the plurality of third oligonucleotides are
attached by ligation. In some embodiments, protected moieties of
the first and second oligonucleotides are deprotected prior to
ligating the plurality of third oligonucleotides.
[1537] In at least one embodiment where the method of the present
disclosure further includes attaching a plurality of third
oligonucleotides, the first oligonucleotide is attached to the
substrate through its 5'-end, the plurality of second
oligonucleotides are prepared by spatially-directed in situ
synthesis on the 3'-end of the first oligonucleotide, and the
plurality of third oligonucleotides are attached through their
5'-end to the 3'-end of the plurality of second
oligonucleotides.
[1538] Alternatively, in at least one embodiment where the method
of the present disclosure further includes attaching a plurality of
third oligonucleotides, the first oligonucleotide can be attached
to the substrate through its 3'-end, the plurality of second
oligonucleotides are prepared by spatially-directed in situ
synthesis on the 5'-end of the first oligonucleotide, and the
plurality of third oligonucleotides are attached through their
3'-end to the 5'-end of the plurality of second
oligonucleotides.
[1539] In at least one embodiment, the third oligonucleotide
includes a capture sequence (e.g., a capture domain) capable of
hybridizing to a nucleic acid sequence (e.g., an analyte) in a
biological sample. In at least one embodiment, the capture sequence
is located at the free 3'-end of the third oligonucleotide which is
oriented with its 5'-end attached to the second oligonucleotide. In
this orientation the free 3'-end can be extended (e.g., by template
dependent polymerization). It is contemplated that in at least one
embodiment, the capture sequence (e.g., capture domain) of the
third oligonucleotide includes a sequence capable of hybridizing to
a specific type of target nucleic acid that is present in a
biological sample. For example, the capture sequence of the third
oligonucleotide can be designed to selectively capture mRNA by
hybridization to the poly-A tail. Accordingly, in at least one
embodiment, the capture sequence of the third oligonucleotide
includes a poly-T oligonucleotide. In some embodiments, the capture
sequence can include nucleotides which are functionally analogous
to poly-T and capable of binding (e.g., hybridizing) selectively to
poly-A, such as a poly-U oligonucleotide. In at least one
embodiment, the capture sequence of the third oligonucleotide
includes a poly-T or poly-U sequence of at least about 5
nucleotides to about 40 nucleotides in length. In some embodiments,
the capture sequence (e.g., capture domain) includes a poly-T or
poly-U sequence of at least 5 nucleotides, 10 nucleotides, at least
15 nucleotides, at least 20 nucleotides, at least 25 nucleotides,
at least 30 nucleotides, at least 35 nucleotides, or at least 40
nucleotides in length.
[1540] In some embodiments, it is contemplated that the capture
sequence can be designed to selectively bind to a desired sub-type
or subset of nucleic acid targets suspected of being present in a
biological sample. For example, a particular type of RNA, such as
mRNA, microRNA or rRNA, or a particular mRNA species that
corresponds to a particular group of genes (e.g., gene specific
capture domain). Thus, the capture sequence of the third
oligonucleotide may include a sequence that specifically binds to a
particular target gene or set of genes, and can be based on a
particular sequence motif sequence or conserved sequence according
to methods well known in the art.
[1541] In some embodiments, the third oligonucleotides of the
plurality of the third oligonucleotides can include a random
sequence. As is well-known in the art, such random sequences (e.g.
random hexamers or nonamers) are effective for the capture nucleic
acid targets (e.g., analytes) by selective binding. Accordingly, it
is contemplated that the capture sequence (e.g., capture domain) of
the third oligonucleotides of the plurality of third
oligonucleotides can include a random sequence, where the random
sequences are at least 6-mers, at least 8-mers, at least 10-mers,
at least 12-mers, at least 14-mers, at least 16-mers, or longer. In
some embodiments, the plurality of third oligonucleotides can
include random sequences together with a poly-T or poly-U capture
sequence. Accordingly, where a third oligonucleotide includes a
poly-T or poly-U capture sequence, it can also include a random
sequence. In some embodiments, where the third oligonucleotide is
in a 5'-to-3' orientation, the random sequences are located at the
5'-end of the third oligonucleotide adjacent to the 3-end of the
second oligonucleotide including the spatial barcode.
[1542] In at least one embodiment, the first, second, and third
oligonucleotides are linked via phosphodiester bonds. Additionally,
in some embodiments, it is contemplated that the oligonucleotides
can be attached via non-natural oligonucleotide linkages, such as
methylphosphonate or phosphorothioate bonds, or via other
non-natural biocompatible linkages such as click-chemistry
linkages. The bonds linking the oligonucleotides can be formed by
synthetic phosphoramidite coupling chemistry, by a synthetic
click-chemistry described herein, by a ligation reaction, by
enzymatic biosynthesis of nucleic acid polymers (e.g., using a
polymerase or transcriptase), or a combination of bond forming
reactions. For example, in at least one embodiment, the second
oligonucleotide is attached to the first oligonucleotide by
standard phosphoramidite chemistry forming a phosphodiester
linkage, and the third oligonucleotide is attached to the
unattached end of the second oligonucleotide via a ligation
reaction.
[1543] The plurality of first oligonucleotides on a spatial array
can be attached (e.g., covalently bound or immobilized) to the
substrate via either the 3'-end or 5'-end of the oligonucleotide.
The attachment of an oligonucleotide to a substrate of a spatial
array through a covalent bond can be either direct (e.g., substrate
to 3'-end of oligonucleotide) or indirect (e.g., substrate to
linker molecule to 3'-end of oligonucleotide). Typically, for
commercially available DNA microarrays, the oligonucleotides are
attached via a 3' linkage, thereby leaving a free 5'-end. The free
5'-end can be used for further synthesis on the oligonucleotide
strand. However, oligonucleotides attached to the substrate via the
5'-end, provide an advantage in having their 3'-ends available for
further biosynthetic extension (e.g., using polymerase or
transcriptase) as described elsewhere herein.
[1544] In at least one embodiment, the first oligonucleotide is
attached to the substrate through its 5'-end, the second
oligonucleotide is attached to the 3'-end of the first
oligonucleotide through its 5'-end, and the third oligonucleotide
is attached to the 3'-end of the second oligonucleotide through its
5'-end.
[1545] In at least one embodiment, the first oligonucleotide is
attached to the substrate through its 3'-end; the second
oligonucleotide that is attached to the 5'-end of the first
oligonucleotide through its 3'-end, and the third oligonucleotide
is attached to the 5'-end of the second oligonucleotide through its
3'-end.
[1546] In at least one embodiment of the composition of the present
disclosure the length of the oligonucleotide (e.g., a first
oligonucleotide, a second oligonucleotide, and/or a third
oligonucleotide) in each region is at least 10 nucleotides, at
least 20 nucleotides, at least 30 nucleotides, at least 40
nucleotides, at least 50 nucleotides, or at least 60 nucleotides.
In some embodiments the length of the oligonucleotide in each
region is from 5 nucleotides to 100 nucleotides, from 10
nucleotides to 100 nucleotides, from 20 nucleotides to 100
nucleotides, from 40 nucleotides to 100 nucleotides, from 50
nucleotides to 90 nucleotides, or from 60 nucleotides to 80
nucleotides.
[1547] As described elsewhere herein, in some embodiments of the
spatial arrays of the present disclosure, the first oligonucleotide
of the spatial array includes a 2-mer to 40-mer having the common
sequence attached across all, or a substantial contiguous portion,
of the surface of the substrate. Thus, the step of attaching the
common first oligonucleotide to the substrate does not need to be
spatially directed. Accordingly, a variety of different embodiments
can be used to attach prepare a substrate having a common first
oligonucleotide of 2 to 40 nucleotides in length attached thereto.
In some embodiments, the first oligonucleotide of the spatial array
can include various domains described herein. For example, the
first oligonucleotide can include a cleavage domain, a functional
domain (e.g., sequencing handle), a unique molecular identifier, or
combinations thereof.
[1548] As illustrated by FIG. 32, in at least one embodiment, a
first oligonucleotide can be prepared ex situ by standard automated
solid-phase oligonucleotide synthesis in a 3'-to-5' direction using
phosphoramidite chemistry. However, as described elsewhere herein,
protection of the phosphodiester and exocyclic amine moieties of
the first oligonucleotide facilitates the spatially-directed in
situ synthesis of the second oligonucleotide on the unattached end
of the first oligonucleotide. Thus, as shown in FIG. 32A, in at
least one embodiment, the first oligonucleotide is synthesized ex
situ using standard techniques, however, the synthesized
oligonucleotide is cleaved from the substrate without deprotecting
the phosphodiester and exocyclic amine moieties that are protected
during synthesis. The protected common oligonucleotide used for the
first oligonucleotide is attached to the substrate with the
protecting groups intact as shown in FIG. 32B.
[1549] The standard oligonucleotide synthesis method using
phosphoramidite chemistry was developed in the 1980s and later
enhanced with solid-phase supports and automation. It the method
used by commercial synthesis of DNA and RNA oligonucleotides.
Generally, solid-phase oligonucleotide synthesis proceeds in the
3'.fwdarw.5' direction rather than 5'.fwdarw.3' direction of
natural biosynthesis. The solid-phase phosphoramidite synthesis
cycle generally includes four steps: (1) detritylation; (2)
coupling; (3) oxidation; and (4) capping. Briefly, the four steps
in the synthesis cycle are as follows. In step (1),
"detritylation," a 5'-DMT protecting group is removed from the
first, solid-support-linked nucleoside. In step (2), "coupling,"
the de-protected 5'-OH of the first, solid-support-linked
nucleoside attacks the phosphorus of the incoming second nucleoside
to form a phosphite triester. In step (3), "oxidation," the
relatively unstable phosphite triester is converted to a stable
phosphate triester. In step (4), prior to moving to step (1) of the
next cycle, "capping," is carried out wherein unreacted 5'-OH
groups are acetylated, thereby preventing elongation of sequences
with deletion mutations.
[1550] In at least one embodiment of the ex situ synthesis method,
the final monomer unit added to the 5'-end during ex situ synthesis
of the first oligonucleotide can include a non-natural nucleoside
that facilitates attachment or immobilization of the first
oligonucleotide to the substrate or linkers attached to the
substrate via its 5'-end. Alternatively, in some embodiments of the
ex situ synthesis method, the first monomer unit added to the
3'-end during ex situ synthesis of the first oligonucleotide can
include a non-natural nucleoside that upon cleavage from the
synthesis substrate or feature can facilitate attachment or
immobilization of the first oligonucleotide to the substrate or
linkers attached to the substrate via the 3'-end.
[1551] In at least one embodiment, the first oligonucleotide
attached to the substrate is prepared by in situ synthesis on the
substrate. Generally, because the sequence of the first
oligonucleotide is the same across all regions of the substrate, it
is not necessary to use spatially-directed in situ synthesis in
such an embodiment. For example, in situ synthesis of the first
oligonucleotide can be carried out on the substrate using standard
solid-phase phosphoramidite chemistry and reagents. As noted above,
maintaining protection of the phosphodiester and exocyclic amine
moieties of the first oligonucleotide facilitates the
spatially-directed in situ synthesis of the second oligonucleotide
on the unattached end of the first oligonucleotide. Accordingly, in
at least one embodiment, it is contemplated that the in situ
synthesis first oligonucleotide on the substrate is carried out
without the final step of deprotecting the phosphodiester and
exocyclic amine moieties before the second oligonucleotide is
synthesized.
[1552] FIG. 33 illustrates an exemplary embodiment of a method for
in situ synthesis of a common first oligonucleotide directly on the
substrate. The synthesis can be carried out using step-wise
solid-phase phosphoramidite chemistry but without final cleavage or
deprotection steps. As shown in FIG. 33A, in this embodiment, the
synthesis would result in a first oligonucleotide attached across
the surface of the substrate with protected phosphodiester and
exocyclic amine moieties. Following this in situ synthesis of the
first oligonucleotide with protective groups intact, light-directed
spatial synthesis of the second oligonucleotide to the 3'-ends of
the first oligonucleotides can be carried out as shown in FIG.
33B.
[1553] In at least one embodiment, the first oligonucleotide
attached to the substrate is prepared by spatially-directed in situ
synthesis on the substrate. The spatially-directed oligonucleotide
synthesis method used can include light-directed spatial synthesis
and/or mechanically-directed spatial synthesis described elsewhere
herein. In this embodiment, the spatially-directed synthesis can be
used in essentially the same way as the in situ standard
oligonucleotide synthesis for the preparation of the first
oligonucleotide having a single sequence across all regions of the
substrate. It is contemplated, however, that in some embodiments
spatially-directed synthesis can be used to prepare a substrate
having the first oligonucleotide attached in selected regions of
the substrate. For example, in some embodiments, a spatial array
could be prepared having more than one first oligonucleotide
sequence in different locations of the substrate.
[1554] Accordingly, as shown in FIG. 34 that in at least one
embodiment the common first oligonucleotide can be in situ
synthesized directly on the substrate however using light-directed
synthesis with photolabile groups, rather than standard protective
groups, such as dimethoxytrityl groups. In some embodiments, the
synthesized first oligonucleotides would be prepared using
light-directed deprotection but would not be cleaved from the
substrate or deprotected as shown in FIG. 34A. Following this
light-directed in situ synthesis of the first oligonucleotide with
protective groups intact, light-directed spatial synthesis of the
second oligonucleotide to the 3'-ends of the first oligonucleotides
can be carried out as shown in FIG. 34B.
[1555] Generally, during standard automated oligonucleotide
synthesis, following cleavage of the oligonucleotide from the
synthesis substrate or feature, the protecting groups on the
phosphates and bases are removed (or deprotected) by treatment in
concentrated aqueous ammonia with heat. Accordingly, it is also
contemplated in at least one embodiment that a first
oligonucleotide is synthesized ex situ using standard automated
synthesis, including cleavage and deprotection of the
phosphodiesters and/or exocyclic amine moieties. As illustrated by
the schematic of FIG. 35A, the cleaved and deprotected first
oligonucleotides synthesized ex situ can be attached to the
substrate surface through a linkage to the 5'-end as described
elsewhere herein. Then, as depicted by FIG. 35B, the phosphodiester
and/or exocyclic amine moieties can be protected in situ on the
substrate. Following this in situ protection step, light-directed
spatial synthesis of the second oligonucleotide to the 3'-ends of
the first oligonucleotides can be carried out as shown in FIG.
35C.
[1556] In an embodiment illustrated by the schematic of FIG. 36A,
ex situ synthesized first oligonucleotides can be cleaved from
their synthesis substrate or feature, deprotected, and then
attached to a substrate surface through a linkage to the 5'-end as
described elsewhere herein. Then, as depicted by FIG. 36B, a
complementary oligonucleotide can be hybridized to the common first
oligonucleotides located across the substrate. The formation of a
double-stranded structure by hybridization can effectively protect
the phosphodiester and/or exocyclic amine moieties of the first
oligonucleotide on the substrate. Following this in situ
protection-by-hybridization step, light-directed spatial synthesis
of the second oligonucleotide to the 3'-ends of the first
oligonucleotides can be carried out as shown in FIG. 36C.
[1557] In at least one embodiment, the spatial array compositions
prepared using the methods of the present disclosure can be useful
for spatial transcriptomics applications as described in e.g., U.S.
Patent App. No. US2014/0066318A1, incorporated by reference herein
in its entirety. Briefly, in spatial transcriptomics a complex
multi-cellular sample, such as a biological sample (e.g., tissue
section), is immobilized on a spatial array of oligonucleotides.
Typically, the oligonucleotide sequences are attached to the array
substrate through their 5'-end and include an mRNA capture sequence
at their 3'-end. Each of the oligonucleotides also includes
5'-adjacent to the capture sequence, a random primer sequence of at
least 16-mer that allows for amplification of any captured
sequences. The detection process results in amplification of the
captured mRNA sequence as well as the spatial barcode. Sequencing
of the amplicons thereby indicates not only the identity of the
transcribed gene but also the location in the multi-cellular sample
where the gene was transcribed.
[1558] Furthermore, in order to survey the genes undergoing
transcription in a complex multi-cellular biological sample, the
spatial array can include an extremely high density of different
oligonucleotides specifically located on the substrate. For
example, it is contemplated that spatial arrays useful in various
applications, such as spatial transcriptomics, can include 1.5
million, 10 million, 100 million, and even up to 1.0 billion
different oligonucleotides, each of which has a different spatial
barcode sequence and is located in a different region on a
substrate, wherein the regions are less than 5 .mu.m.sup.2, even as
small as 1.0 .mu.m.sup.2.
[1559] As noted elsewhere herein, oligonucleotides of a spatial
array can include a different spatial barcode that correlates with
its location. In some embodiments, it is contemplated that
additional oligonucleotides can be present on the same substrate as
the spatial array that include a sequence identical to a barcode
sequence of the spatial array. Such additional oligonucleotides can
arise due to errors in synthesis or can be prepared intentionally
on the substrate (e.g., for calibration or quality control
purposes). Generally, these sequences located in a different
location on the same substrate are not considered part of the
spatial array and are kept to a level and/or location that avoids
interference with the intended function of the spatial array.
[1560] In some embodiments, methods provided herein can be used to
generate an array that allows resolution equal to or less than 10
.mu.m, allowing single-cell resolution. In some embodiments, a
spatial array is generated by providing a plurality of sets of
primers on a substrate. In some embodiments, a set of the sets of
primers includes a first primer and a second primer present at a
1:1 ratio. In some embodiments, the second primer has a sequence
that is complementary to the first primer or to a template
oligonucleotide.
[1561] In some embodiments, methods of preparing a spatial array
provided herein include providing a substrate, immobilizing a
plurality of sets of primers on the substrate, providing a template
oligonucleotide having a reverse complement of a spatial barcode
domain, a reverse complement of a capture domain, a first
hybridization domain capable of hybridizing to the first primer,
and a second hybridization domain, wherein a reverse complement of
the second hybridization domain is capable of hybridizing to the
second primer; hybridizing the template oligonucleotide to the
first primer; extending the first primer using the template
oligonucleotide as a template to generate a capture probe
comprising the first primer, the spatial barcode, and the capture
domain, which capture probe is immobilized on the substrate; and
releasing the template oligonucleotide from the spatial array.
[1562] In some embodiments, the spatial array comprises a plurality
of sets of primers affixed to the substrate. In some embodiments, a
first primer and a second primer that are at a 1:1 ratio or about
at a 1:1 ratio (e.g., at or about a 2:1, 1.9:1, 1.8:1, 1.7:1,
1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3,
1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2 ratio) and that
are less than 10 .mu.m apart on the substrate. In some embodiments,
the first primer and the second primer are about 0.1 .mu.m, 0.2
.mu.m, 0.3 .mu.m, 0.4 .mu.m, 0.5 .mu.m, 0.6 .mu.m, 0.7 .mu.m, 0.8
.mu.m, 0.9 .mu.m, 1.0 .mu.m, 2.0 .mu.m, 3.0 .mu.m, 4.0 .mu.m, 5.0
.mu.m, 6.0 .mu.m, 7.0 .mu.m, 8.0 .mu.m, 9.0 .mu.m, 10.0 .mu.m, 15
.mu.m, 20 .mu.m, 25 .mu.m, 30 .mu.m, 35 .mu.m, 40 .mu.m, 45 .mu.m,
50 .mu.m, 55 .mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80
.mu.m, 85 .mu.m, 90 .mu.m, 95 .mu.m, or 100 .mu.m apart on the
substrate.
[1563] In some embodiments, the first primer and the second primer
are about 0.1 .mu.m to about 100 .mu.m apart on the substrate. In
some embodiments, the first primer and the second primer are about
0.5 .mu.m to about 50 .mu.m apart on the substrate. In some
embodiments, the first primer and the second primer are about 0.7
.mu.m to about 25 .mu.m apart on the substrate. In some
embodiments, the first primer and the second primer are about 1.0
.mu.m to about 20 .mu.m apart on the substrate. In some
embodiments, the first primer and the second primer are about 1.0
.mu.m to about 15 .mu.m apart on the substrate. In some
embodiments, the first primer and the second primer are about 1.0
.mu.m to about 10 .mu.m apart on the substrate. In some
embodiments, the first primer and the second primer are about 1.0
.mu.m to about 5 .mu.m apart on the substrate. In some embodiments,
the first primer and the second primer are about 1.0 .mu.m to about
2 .mu.m apart on the substrate.
[1564] In some embodiments, the first primer is affixed to the
substrate at the 5' end of the first primer. In some embodiments,
the second primer is affixed to the substrate at the 5' end of the
second primer.
[1565] In some aspects, the first primer is single stranded DNA. In
some aspects, the first primer is RNA. In some aspects, the second
primer is single stranded DNA. In some aspects, the second primer
is RNA.
[1566] In some embodiments, the first primer on the substrate
comprises a sequence that is complementary to the template
oligonucleotide and allows the template oligonucleotide to bind to
the first primer. In some embodiments, the first primer can also
comprise a poly (A) or poly (T) domain that is complementary to a
poly(T) or poly(A) domain of the second primer. In some
embodiments, the first primer can also comprise a cleavage domain.
In some embodiments, the second primer can also comprise a cleavage
domain.
[1567] In some embodiments, the first primer and the second primer
are used to amplify a template oligonucleotide to create a cluster
of one or more capture probes. In some aspects, the template
oligonucleotide includes a reverse complement of a unique molecular
identifier (UMI).
[1568] In some embodiments, the template oligonucleotide is
attached to a bead (e.g., any of the variety of beads described
herein, e.g., a gel bead). In some embodiments, the template
oligonucleotide is attached to the bead at the 3' end. In some
embodiments, the template oligonucleotide is attached to the bead
at the 5' end. In some embodiments, template oligonucleotides that
have a bead at the 3' end also have 5' modification, which allows
the template oligonucleotide to attach to a support (e.g., any of
the variety of functionalized groups for attaching nucleic acids to
substrates, e.g., slides, described herein or otherwise known in
the art). In some embodiments, template oligonucleotides that have
a bead at the 5' end also have 3' modification, which allows the
template oligonucleotide to attach to a support (e.g., any of the
variety of functionalized groups for attaching nucleic acids to
substrates, e.g., slides, described herein or otherwise known in
the art). In some aspects, the template oligonucleotide comprises a
cleavage sequence that allows the template oligonucleotide to
detach from the bead (e.g., after the oligonucleotide has been
attached to the support).
[1569] In another embodiment, the primer on the support has a
non-barcoded universal primer that hybridizes to the template
oligonucleotide on beads. In some embodiments, after one round of
synthesis, the template oligonucleotide (e.g., a barcode on the
template oligonucleotide) is incorporated.
[1570] In some aspects, the bead is a gel bead. In some aspects,
the bead is less than 10 .mu.m in diameter. In some aspects, the
bead is less than 5 .mu.m in diameter. In some aspects, the bead is
2 .mu.m in diameter. In some aspects, the bead is less than 2 .mu.m
in diameter. In some aspects, the bead is 1 .mu.m in diameter. In
some aspects, the bead is less than 1 .mu.m in diameter.
[1571] In some aspects, after the template oligonucleotide binds to
a primer, the bead is removed. In some aspects, the bead is removed
prior to amplification of the template oligo. In some aspects, the
bead is removed after amplification of the template oligo. In some
embodiments, the bead is removed by a washing step. Removal of
beads can be performed using techniques known in the art.
[1572] In some aspects, after binding of the template
oligonucleotide to the first primer, the sequence of the first
primer is extended. In some aspects, one or more beads (e.g., all
of the beads or substantially all of the beads) that are attached
to the template oligonucleotide(s) is removed after extension of
the first primer. In some aspects, the one or more beads (e.g., all
of the beads or substantially all of the beads) that are attached
to the oligonucleotide(s) is removed before extension of the
primer.
[1573] In some aspects, the one or more beads is non-spherical. In
some aspects, the non-spherical bead or one or more non-spherical
beads is primer loaded. In some aspects, the non-spherical bead or
one or more non-spherical beads is hexagonal. In some aspects,
hexagonal beads can form in a honeycomb pattern when placed on a
support. In some aspects, the non-spherical bead or one or more
non-spherical beads is cuboidal. In some aspects, one or more
hexagonal or cuboidal beads allows for more compact packing of
beads on a substrate than would occur with spherical beads. In some
embodiments, after initial binding of the template oligonucleotide
and a first primer, a capture probe is generated. In some aspects,
the capture probe is generated using cluster expansion.
[1574] In some aspects, the capture probe is generated using bridge
amplification. The term "bridge amplification" refers to a method
of amplifying nucleic acid in which primers (e.g., a first primer
and a second primer) that are extended in the reaction are tethered
to a substrate by their 5' ends. In some embodiments, the process
is repeated to provide amplified, immobilized nucleic acid
molecules.
[1575] In some embodiments, bridge amplification comprises a first
step comprising hybridization of the target nucleic acid to the
first primer attached to the support. A first amplification
product, which is complementary to the target nucleic acid, is then
formed by extension of the first primer sequence. On subjecting the
support to denaturation conditions the target nucleic acid is
released and can then participate in further hybridization
reactions with other first primer sequences which may be attached
to the support. The first amplification product which is attached
to the support, may then hybridize with the second primer attached
to the support, forming a "bridge." In some embodiments, a second
amplification product comprising a nucleic acid sequence
complementary to the first amplification product can be formed by
extension of the second primer sequence and is also attached to the
support. Thus, the target nucleic acid and the first and second
amplification products are capable of participating in a plurality
of hybridization and extension processes, limited only by the
initial presence of the target nucleic acid and the number of first
primer and second primer sequences initially present and the result
is a number of copies of the target sequence attached to the
surface. Bridge amplification is further disclosed U.S. Appls. Nos.
US-2008-0160580 and US-2008-0286795, each of which is incorporated
by reference in its entirety. In some aspects, the capture probe
includes a UMI (e.g., any of the variety of UMIs described herein).
In some aspects, the capture probe includes the sequence of the
first primer, a spatial barcode (e.g., any of the variety of
spatial barcodes described herein), and/or a capture domain (e.g.,
any of the variety of capture domains described herein). In some
aspects, the capture probe is immobilized on the substrate. In some
aspects, the capture domain comprises a sequence complementary to
an analyte of interest. In some aspects, the capture domain
comprises a sequence designed to capture messenger RNA. In some
aspects, the capture domain comprises a poly(T) domain. In some
aspects, the capture domain comprises a target-specific sequence
(e.g., a sequence complementary to the sequence of a target of
interest, e.g., a target nucleic acid molecule, e.g., a specific
messenger RNA or DNA). In some aspects, the capture domain
comprises a random sequence (e.g. a hexamer).
[1576] In some embodiments, after a copy of the capture probe is
generated, the copy of the template oligonucleotide is released by
cleaving the cleavage domain after the copy of the capture probe is
generated.
[1577] In some embodiments, after cluster amplification, all or a
portion of the sequence (e.g., identity) of the generated capture
probe is determined. In some aspects, the all or a portion of the
sequence (e.g., identity) of the generated capture probe is
determined after the template oligonucleotide is released. In some
aspects, the all or a portion of the sequence (e.g., identity) of
the generated capture probe is determined in situ. In some aspects,
the all or a portion of the sequence (e.g., identity) of the
generated capture probe is determined by in situ sequencing. As
non-limiting examples, in situ sequencing can be performed via
sequencing-by-synthesis (SBS), sequential fluorescence
hybridization, sequencing by ligation, nucleic acid hybridization,
or high-throughput digital sequencing techniques.
[1578] In some embodiments, after the probe is clonally expanded,
spatial analysis of a biological analyte in a biological sample can
be performed. For example, spatial analysis can include determining
a location of an analyte in a biological sample. In some aspects,
methods of performing spatial analysis of a biological analyte in a
biological sample include preparing the spatial array (e.g., via
any method discussed herein). In some aspects, the methods further
include decoding the one or more capture probes by determining the
identity of the spatial barcode of the one or more capture probes,
thereby associating the spatial barcode of the one or more capture
probe with a location on the spatial array; contacting the
biological sample to the spatial array under conditions wherein the
biological analyte binds a capture probe present in a cluster;
extending the capture probe via a polymerization reaction using the
biological analyte as a template to generate an extended capture
probe that include the sequence of the capture probe and the
reverse complement of the sequence of the biological analyte;
determining the sequence of the extended capture probe; and
associating the spatial barcode of the capture probe with the
captured biological analyte, thereby determining spatial location
of the analyte in the biological sample.
Generating Capture Probes
[1579] Capture probes described herein can contain various
functional sequences and a capture domain. In some embodiments, the
capture domain captures analytes in an unbiased manner. For
example, if the capture domain is a poly(T) sequence, the capture
domain can interact (e.g., hybridize) to the poly(A) tail of any
given mRNA present in the biological sample, regardless of the
identity of that mRNA. In some embodiments, the capture domain can
capture analytes in an unbiased manner with via a capture domain
having a random or degenerate sequence. As non-limiting examples, a
capture domain can include a random hexamer or octamer sequence for
unbiased capture. In some embodiments, the capture domain captures
analytes in a biased manner. For example, the capture domain can be
gene specific, such that a specific analyte (e.g., a specific
nucleic acid, e.g., a specific mRNA or DNA sequence) or set of
analytes (e.g., set of nucleic acids, e.g., a set of mRNAs or a set
of DNAs, e.g., a set of mRNAs or a set of DNAs sharing a common or
similar sequence) can be captured for spatial analysis in the
biological sample. In some embodiments, the capture domain can
capture rRNA analytes. For example, the capture domain can contain
a degenerate sequence capable of interacting (e.g., hybridizing)
with 16s rRNA. In some embodiments, the capture domain can interact
with analytes containing V(D)J sequences. In some embodiments, the
capture domain can interact with a capture agent barcode domain
associated with an analyte capture agent (e.g., an analyte capture
sequence). Capture domains for both unbiased analyte capture and
biased capture can be generated from an existing array with a
ligation strategy.
[1580] Capture probes can be generated from existing arrays with a
ligation strategy. In some embodiments, an array containing a
plurality of oligonucleotides (e.g., in situ synthesized
oligonucleotides) can be modified to generate a variety of capture
probes. The oligonucleotides can include various domains such as,
spatial barcodes, UMIs, functional domains (e.g., sequencing
handle), cleavage domains, and/or ligation handles. As used herein,
the term "ligation handle" refers to a portion of an
oligonucleotide that is complementary to a portion of a different
oligonucleotide. In some embodiments, the different oligonucleotide
is a splint oligonucleotide having a sequence complementary to a
donor oligonucleotide, and the donor oligonucleotide is ligated to
the ligation handle. In some embodiments, the donor oligonucleotide
includes a capture domain, such that upon ligation to
oligonucleotide having a ligation handle, a capture probe is
generated. For example, FIG. 41 shows an acceptor oligonucleotide
on a immobilized substrate (e.g., an array), that contains from 5'
to 3' an amino modifier C6, followed by 12 uracils that can
function as a cleavage domain, a sequencing handle (e.g., R1), a 16
base pair spatial barcode, a 12 base pair UMI, and a ligation
handle. In some embodiments, a splint oligonucleotide with a
sequence complementary to the ligation handle of the acceptor
oligonucleotide and an additional sequence complementary to an
oligonucleotide (e.g., a donor oligonucleotide) containing a
capture domain (e.g., a capture domain of interest) facilitates the
ligation of the oligonucleotide and the oligonucleotide containing
the capture domain. In some embodiments, the additional sequence
complementary to the capture domain is a poly(A) sequence. In some
embodiments, the additional sequence complementary to the capture
domain is a sequence specific to a nucleic acid (e.g., an mRNA or
DNA sequence). In some embodiments, the additional sequence
complementary to the capture domain is a sequence specific to a
subset of nucleic acids (e.g., a subset of mRNA or DNA sequences
sharing a common or similar sequence). In embodiments where the
splint oligonucleotide sequence complementary to the capture domain
is a poly(A) sequence, the splint oligonucleotide can facilitate
the ligation of a poly(T) capture domain to the oligonucleotide. In
embodiments where the splint oligonucleotide sequence complementary
to the capture domain is a sequence specific to a nucleic acid, the
splint oligonucleotide can facilitate the ligation of that capture
domain to the oligonucleotide. In some embodiments, the gene
specific capture domain is for Penk mRNA. In embodiments where the
splint oligonucleotide sequence complementary to the capture domain
is a sequence specific to a subset of nucleic acids, the splint
oligonucleotide can facilitate the ligation of that capture domain
to the oligonucleotide.
[1581] Capture probes can be generated from existing arrays with a
ligation strategy. In some embodiments, an array containing a
plurality of oligonucleotides (e.g., in situ synthesized
oligonucleotides) can be modified to generate a variety of capture
probes. The oligonucleotides can include various domains such as,
spatial barcodes, UMIs, functional domains (e.g., sequencing
handle), cleavage domains, and/or ligation handles. For example,
FIG. 44 shows an exemplary strategy where an acceptor
oligonucleotide having a ligation handle is immobilized on a
substrate (e.g., an array). In some embodiments, the acceptor
oligonucleotide shown in FIG. 44 contains from 5' to 3' one or more
of (e.g., each of) an amino modifier C6, 5 thymines, a sequencing
handle (e.g., R1), a 16 base pair barcode, a 12 base pair, UMI, and
a 7 base pair ligation handle (SEQ ID NO: 8). FIG. 44 shows the
ligation handle at the 3' end of the acceptor oligonucleotide
(e.g., acceptor oligonucleotide 2). In some embodiments, the
ligation handle can be complementary to a sequence in the splint
oligonucleotide. In some embodiments, the splint oligonucleotide
can have a sequence complementary to a second ligation handle that
is present in an oligonucleotide that also includes the capture
domain. In some embodiments, the capture domain can be any of the
capture domains described herein.
[1582] In some embodiments, the splint oligonucleotide includes a
sequence that is complementary to an oligonucleotide (e.g., an
acceptor oligonucleotide), or a portion thereof, and a sequence
that is complementary to an oligonucleotide (e.g., a donor
oligonucleotide) containing a capture domain, or a portion thereof
(e.g., complementary to the capture domain itself or to a second
ligation handle). In some embodiments, the splint oligonucleotide
includes a sequence that is perfectly complementary (e.g., is 100%
complementary) to an oligonucleotide (e.g., an acceptor
oligonucleotide), or a portion thereof, and/or a sequence that is
perfectly complementary to an oligonucleotide containing a capture
domain, or a portion thereof (e.g., perfectly complementary to the
capture domain itself or to a second ligation handle). In some
embodiments, the splint oligonucleotide includes a sequence that is
not perfectly complementary (e.g., is not 100% complementary) to an
oligonucleotide (e.g., an acceptor oligonucleotide), or a portion
thereof, and/or a sequence that is not perfectly complementary to
an oligonucleotide containing a capture domain, or a portion
thereof (e.g., not perfectly complementary to the capture domain
itself or to a second ligation handle). In some embodiments, the
splint oligonucleotide includes a sequence that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to an
oligonucleotide (e.g., an acceptor oligonucleotide), or a portion
thereof, and/or a sequence that is complementary to an
oligonucleotide containing a capture domain, or a portion thereof
(e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
complementary to the capture domain itself or to a second ligation
handle). In some embodiments, the splint oligonucleotide includes a
sequence that is perfectly complementary (e.g., is 100%
complementary) to an oligonucleotide (e.g., an acceptor
oligonucleotide), or a portion thereof, but is not perfectly
complementary to a sequence that is complementary to an
oligonucleotide containing a capture domain, or a portion thereof
(e.g., not perfectly complementary to the capture domain itself or
to a second ligation handle). In some embodiments, the splint
oligonucleotide includes a sequence that is not perfectly
complementary (e.g., is not 100% complementary) to an
oligonucleotide (e.g., an acceptor oligonucleotide), or a portion
thereof, but is perfectly complementary to a sequence that is
complementary to an oligonucleotide containing a capture domain, or
a portion thereof (e.g., perfectly complementary to the capture
domain itself or to a second ligation handle). So long as the
splint oligonucleotide is capable of hybridizing to an
oligonucleotide (e.g., an acceptor oligonucleotide), or a portion
thereof, and to a sequence that is complementary to an
oligonucleotide containing a capture domain, or a portion thereof
(e.g., to the capture domain itself or to a second ligation
handle), the splint oligonucleotide need not have a sequence that
is perfectly complementary to either the oligonucleotide (e.g., the
acceptor oligonucleotide) or to the an oligonucleotide containing a
capture domain.
[1583] The splint oligonucleotide can be about 20 to about 80
nucleotides long. In some embodiments, the splint oligonucleotide
can be about 30 to about 70 nucleotides long. In some embodiments,
the splint oligonucleotide can be about 40 to about 60 nucleotides
long. In some embodiments, the splint oligonucleotide can be about
45 to about 55 nucleotides long.
[1584] In some embodiments, the spatial array is generated after
ligating capture domains (e.g., poly(T) or gene specific capture
domains) to the oligonucleotides (e.g., generating capture probes).
The spatial array can be used with any of the spatial analysis
methods described herein. For example, a biological sample can be
provided to the generated spatial array generated. In some
embodiments, the biological sample is permeabilized. In some
embodiments, the biological sample is permeabilized under
conditions sufficient to allow one or more analytes present in the
biological sample to interact with the capture probes of the
spatial array. After capture of analytes in the biological sample
the analytes can be analyzed (e.g., reverse transcribed, amplified,
sequenced) by any of the variety of methods described herein.
Exemplary Embodiments
[1585] This disclosure relates to methods for building an array. In
one aspect, provided herein are methods of generating an array for
spatial analysis that includes: (a) providing an array that
includes a plurality of in situ synthesized oligonucleotides,
wherein each of the in situ synthesized oligonucleotides comprise a
spatial barcode, and wherein the 3' end of each of the in situ
synthesized oligonucleotides are attached to a solid support; (b)
providing a plurality of primers complementary to a portion of one
or more in situ synthesized oligonucleotides; (c) extending the
primers using the one or more in situ synthesized oligonucleotides
as a template, thereby generating a first oligonucleotide; (d)
extending the first oligonucleotide to produce a 3' end overhang;
(e) ligating a splint oligonucleotide to the 5' end of the one or
more in situ synthesized oligonucleotides; and (f) ligating a
second oligonucleotide to the 3' end of the first oligonucleotide,
where the second oligonucleotide includes a sequence that is
complementary to a portion of the splint oligonucleotide and a
capture domain, thereby generating a plurality of capture probes to
be used in an array for spatial analysis. In some embodiments, the
splint oligonucleotide includes a sequence that is complementary to
the 3' end overhang of the first oligonucleotide. In some
embodiments, the plurality of in situ synthesized oligonucleotides
include a first constant sequence. In some embodiments, the
plurality of in situ synthesized oligonucleotides further comprise
a second constant sequence. In some embodiments, the plurality of
in situ synthesized oligonucleotides have a length of between 1 and
30 nucleotides. In some embodiments, the second oligonucleotide
further includes a constant sequence, and where the constant
sequence is complementary to a portion of the splint
oligonucleotide. In some embodiments, the second oligonucleotide
further includes a degenerate sequence. In some embodiments, the
capture domain includes a poly-T sequence. In some embodiments, the
3' end overhang of step (d) is produced by an enzyme. In some
embodiments, the enzyme is a template-independent polymerase. In
some embodiments, the template-independent polymerase is at least
one of terminal deoxynucleotidyl transferase (TdT) and poly A
polymerase.
[1586] In one aspect, provided herein are methods for spatial
analysis of a biological sample that includes: (a) providing an
array produced by the method of claim 1; (b) contacting a
biological sample with the array; (c) permeabilizing the biological
sample under conditions sufficient to allow a plurality of
biological analytes in the biological sample to interact with a
plurality of capture probes on the array to generate a plurality of
spatially barcoded biological analytes; and analyzing the plurality
of spatially barcoded biological analytes, thereby determining the
spatial location of the plurality of biological analytes.
[1587] Capture Probes
[1588] In some embodiments, the capture probe is a product of two
or more oligonucleotide sequences. In some embodiments, two or more
oligonucleotide sequences are ligated together. In some
embodiments, the capture probe includes a splint oligonucleotide.
In some embodiments, the capture probe includes one or more
constant sequences. In some embodiments, the capture probe is
generated by having an enzyme add polynucleotides at the end of an
oligonucleotide sequence. In some embodiments, the capture probe
includes a degenerate sequence. In some embodiments, the degenerate
sequence functions as a unique molecular identifier.
[1589] In Situ Synthesized Oligonucleotides
[1590] In some embodiments, the capture probe includes an in situ
synthesized oligonucleotide. In some embodiments, the in situ
synthesized oligonucleotide includes one or more constant
sequences. In some embodiments, a constant sequence may be a
cleavable sequence. In some embodiments, the in situ synthesized
oligonucleotide includes a barcode sequence. In some embodiments,
the in situ synthesized oligonucleotide includes a variable barcode
sequence. In some embodiments, the in situ synthesized oligo have a
length of between 1 and 50 nucleotides (e.g. between 1 and 40,
between 1 and 30, between 1 and 20, or between 1 and 10
nucleotides). In some embodiments, the in situ synthesized
oligonucleotide is attached to an array. In some embodiments, the
in situ synthesized oligonucleotide is attached to a feature. In
some embodiments, the attachment is direct attachment. In some
embodiments, the attachment is an indirect attachment.
[1591] Splint Oligonucleotides
[1592] The term "splint oligonucleotide", as used herein, refers to
an oligonucleotide that, when hybridized to other polynucleotides,
acts as a "splint" to position the polynucleotides next to one
another so that they can be ligated together. In some embodiments,
the splint oligonucleotide is DNA. In some embodiments, the splint
oligonucleotide is RNA. In some embodiments, the splint
oligonucleotide comprises a nucleotide sequence that is partially
complimentary to nucleotide sequences from two or more different
oligonucleotides. In some embodiments, the splint oligonucleotide
assists in ligating a "donor" oligonucleotide and an "acceptor"
oligonucleotide. In some embodiments, the splint oligonucleotide is
approximately 40 nucleotides in length. In some embodiments, the
splint oligonucleotide is between 10 and 50 oligonucleotides in
length. In some embodiments, the splint oligonucleotide is between
10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20
oligonucleotides in length. In some embodiments, the splint
oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and
35, 15 and 30, 15 and 30, or 15 and 25 nucleotides in length. In
some embodiments, an RNA ligase is used to ligate two nucleotide
sequences together. In some embodiments, a DNA ligase is used to
ligate two oligonucleotide sequences together. In some embodiments,
the DNA ligase is a T4 DNA ligase.
[1593] Degenerate Sequence
[1594] In some embodiments of any of the methods described herein,
the capture probes may include a degenerate sequence. In some
embodiments, a degenerate sequence is a sequence in which some
positions of a nucleotide sequence contain a number of possible
bases. In some embodiments of any of the methods described herein,
a degenerate sequence can be a degenerate nucleotide sequence
including about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In some
embodiments, a nucleotide sequence contains 1, 2, 3, 4, 5, 6, 7, 8,
9, 0, 10, 15, 20, 25, or more degenerate positions within the
nucleotide sequence. In some embodiments of any of the methods
described herein, the degenerate sequence is used as a unique
molecular identifier.
[1595] This disclosure relates to methods for preparing
high-density spatially barcoded arrays and methods for spatial
analysis (e.g., analysis of the spatial location(s) of one or more
species of analytes in a biological sample) of analytes in a
biological sample using a high-density spatially barcoded
arrays.
[1596] In one aspect, provided herein are methods for preparing a
high-density spatially barcoded array, comprising: (a) contacting
an array comprising one or more pluralities of capture probes with
a hydrogel including one or more pluralities of oligonucleotides;
(b) attaching members of the one or more pluralities of capture
probes to members of the one or more pluralities of
oligonucleotides to form one or more pluralities of coupled capture
probes; (c) reducing the size of the hydrogel including the one or
more pluralities of coupled capture probes by removing water,
forming a shrunken hydrogel including the one or more pluralities
of coupled capture probes; thus generating a high-density spatially
barcoded array.
[1597] In some embodiments, the one or more pluralities of capture
probes are immobilized. In some embodiments, the one or more
pluralities of immobilized capture probes are partitioned. In some
embodiments, the one or more pluralities of immobilized capture
probes are partitioned into a plurality of capture probe
partitions. In some embodiments, a partitioned plurality of
immobilized capture probes is different from another partitioned
plurality of immobilized capture probes.
[1598] In some embodiments, one or more first pluralities of
capture probes comprise a barcode sequence. In some embodiments,
one or more pluralities of capture probes comprise a barcode
sequence different from the barcode sequence of one or more other
pluralities of capture probes. In some embodiments, one or more
capture probe partitions comprise a plurality of immobilized
capture probes including a barcode sequence different from the
barcode sequence of the plurality of immobilized capture probes of
one or more other capture probe partitions.
[1599] In some embodiments, the one or more pluralities of
oligonucleotides are partitioned. In some embodiments, the one or
more pluralities of oligonucleotides are partitioned into a
plurality of oligonucleotide partitions. In some embodiments, one
or more oligonucleotide partitions comprise a plurality of
oligonucleotides different from one or more other oligonucleotide
partitions. In some embodiments, one or more pluralities of
oligonucleotides comprise a barcode sequence. In some embodiments,
one or more pluralities of oligonucleotides comprise a barcode
sequence different from the barcode sequence of one or more
pluralities of oligonucleotides. In some embodiments, the barcode
sequence of one or more pluralities of capture probes is different
from the barcode sequence of one or more pluralities of
oligonucleotides. In some embodiments, one or more oligonucleotide
partitions comprise a plurality of oligonucleotides including a
barcode sequence different from the barcode sequence of the
plurality of oligonucleotides of one or more oligonucleotide
partitions.
[1600] In some embodiments, the reduction in the size of the
hydrogel is isometric.
[1601] In some embodiments, the removal of water comprises a
dehydrating solvent. In some embodiments, the solvent comprises at
least one of an alcohol, a ketone, an aldehyde, acetonitrile, or a
combination thereof. In some embodiments, the solvent comprises at
least one of glycerol, ethanol, acetonitrile, acetone, 2-propanol,
and a combination thereof.
[1602] In some embodiments, the removal of water comprises a salt.
In some embodiments, the salt comprises an inorganic salt.
[1603] In some embodiments, the removal of water comprises an
acid.
[1604] In some embodiments, the removal of water comprises a
dehydrating process.
[1605] In some embodiments, the shrunken hydrogel is about 2 to
about 10 fold smaller in a linear dimension than the pre-shrunk
hydrogel. In some embodiments, the shrunken hydrogel is at least
2-fold smaller in a linear dimension than the pre-shrunk hydrogel.
In some embodiments, the shrunken hydrogel is at least 4-fold
smaller in a linear dimension than the pre-shrunk hydrogel. In some
embodiments, the shrunken hydrogel is at least 8-fold smaller in a
linear dimension than the pre-shrunk hydrogel.
[1606] In some embodiments, the attaching comprises polymerase
chain reaction.
[1607] In some embodiments, the hydrogel comprises a DNA
polymerase, a plurality of primers, and a plurality of
nucleotides.
[1608] In some embodiments, members of the plurality of capture
probes are cross-linked to the hydrogel.
[1609] In some embodiments, members of the one or more pluralities
of coupled capture probes are amplified.
[1610] In some embodiments, the coupled capture probe is amplified
prior to the reduction of the size of the hydrogel.
[1611] In some embodiments, the hydrogel is prepared from hydrogel
subunits including at least one of: acrylamide, bis-acrylamide, a
poly(ethylene glycol), a polyaliphatic polyurethane, a polyether
polyurethane, a polyester polyurethane, a polyethylene copolymer, a
polyamide, a polyvinyl alcohol, polypropylene glycol,
polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,
poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate),
collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin,
alginate, a protein polymer, methylcellulose, and a combination
thereof.
[1612] In some embodiments, one or more capture probes comprise a
spatial barcode. In some embodiments, one or more capture probes
comprise a unique molecular identifier. In some embodiments, one or
more pluralities of capture probes comprise a spatial barcode, and
a capture domain.
[1613] Also provided herein are methods for spatially profiling a
plurality of analytes in a biological sample, including: (a)
providing a high-density spatially barcoded array as described
herein; (b) contacting the biological sample with the high-density
spatially barcoded array under conditions such that one or more
analytes present in the biological sample are captured by one or
more features of the array; and (c) determining the spatial profile
of the captured analytes.
[1614] In some embodiments, the biological sample is a tissue
sample. In some embodiments, the tissue sample is a fresh-frozen
tissue sample. In some embodiments, the tissue sample comprises a
tumor cell. In some embodiments, the tissue sample comprises a
tissue section.
[1615] In some embodiments, the analytes in the biological sample
are migrated to the bead array. In some embodiments, the migrating
comprises passive migration. In some embodiments, the migrating
comprises active migration.
[1616] In some embodiments, the one or more analytes comprises at
least one of RNA, DNA, protein, lipid, peptide, metabolite, small
molecule, and a cell labeling agent.
Methods for Preparing High-Density Spatially Barcoded Arrays
[1617] Provided herein are methods for preparing a high-density
spatially barcoded array, e.g., a high-diversity and high-density
spatially barcoded array. For example, methods for preparing a
high-density spatially barcoded array can include attaching capture
probes to oligonucleotides in a hydrogel to form coupled capture
probes and reducing the size of the hydrogel including the coupled
capture probes. In some embodiments, a method for preparing a
high-density spatially barcoded array, can include contacting an
array including one or more pluralities of capture probes with a
hydrogel including one or more pluralities of oligonucleotides;
attaching members of the one or more pluralities of capture probes
to members of the one or more pluralities of oligonucleotides to
form one or more pluralities of coupled capture probes; reducing
the size of the hydrogel including the one or more pluralities of
coupled capture probes by removing water, forming a shrunken
hydrogel including the one or more pluralities of coupled capture
probes; thus generating a high-density spatially barcoded
array.
[1618] In some embodiments, a capture probe is any capture probe as
described herein. In some embodiments, the one or more pluralities
of capture probes are immobilized.
[1619] The term "partition," as used herein, may be a verb or a
noun. When used as a verb (e.g., "to partition," or
"partitioning"), the term generally refers to the fractionation
(subdivision) of a species or sample (e.g., a polynucleotide) such
that one fraction (or subdivision) is sequestered from another.
Each sequestered section is referred to using the noun "partition."
In some embodiments, a partition maintains separation of its own
contents from the contents of other partitions. A partition may be
any suitable area in which one fraction of a sample is separated or
sequestered from another. In some embodiments, a hydrogel can
comprise one or more partitions of a species or sample (e.g., a
polynucleotide), wherein the hydrogel matrix maintains the
separation of the contents of one partition from the contents of
other partitions. In some embodiments, a partition has defined
dimensions.
[1620] In some embodiments, the one or more pluralities of
immobilized capture probes are partitioned. In some embodiments,
the one or more pluralities of immobilized capture probes are
partitioned into a plurality of capture probe partitions. In some
embodiments, a partitioned plurality of immobilized capture probes
is different from another partitioned plurality of immobilized
capture probes.
[1621] In some embodiments, one or more first pluralities of
capture probes comprise a barcode sequence. In some embodiments,
one or more pluralities of capture probes comprise a barcode
sequence different from the barcode sequence of one or more other
pluralities of capture probes. In some embodiments, one or more
capture probe partitions comprise a plurality of immobilized
capture probes including a barcode sequence different from the
barcode sequence of the plurality of immobilized capture probes of
one or more other capture probe partitions.
[1622] In some embodiments, the one or more pluralities of
oligonucleotides are partitioned. In some embodiments, the one or
more pluralities of oligonucleotides are partitioned into a
plurality of oligonucleotide partitions. In some embodiments, one
or more oligonucleotide partitions comprise a plurality of
oligonucleotides different from one or more other oligonucleotide
partitions. In some embodiments, one or more pluralities of
oligonucleotides comprise a barcode sequence. In some embodiments,
one or more pluralities of oligonucleotides comprise a barcode
sequence different from the barcode sequence of one or more
pluralities of oligonucleotides. In some embodiments, the barcode
sequence of one or more pluralities of capture probes is different
from the barcode sequence of one or more pluralities of
oligonucleotides. In some embodiments, one or more oligonucleotide
partitions comprise a plurality of oligonucleotides including a
barcode sequence different from the barcode sequence of the
plurality of oligonucleotides of one or more oligonucleotide
partitions.
[1623] In some embodiments, each capture probe comprises a spatial
barcode. In some embodiments, each capture probe from the plurality
of capture probes comprises a unique molecular identifier. In some
embodiments, each capture probe comprises a cleavage domain, a
spatial barcode, a unique molecular identifier, and a capture
domain.
[1624] A "coupled capture probe" as used herein refers to a capture
probe that is attached to an oligonucleotide (e.g., an
oligonucleotide in a hydrogel). For example, one or more domains of
a capture probe can be attached to one or more domains of an
oligonucleotide (e.g., an oligonucleotide in a hydrogel).
"Attaching" can refer to any method that binds a capture probe to
an oligonucleotide (e.g., an oligonucleotide in a hydrogel). For
example, an oligonucleotide can be ligated to an end of a capture
probe. As another example, PCR can be used to attach one or more
domains of a capture probe to one or more domains of an
oligonucleotide (e.g., an oligonucleotide in a hydrogel).
[1625] In some embodiments, members of the one or more pluralities
of capture probes comprise a nucleic acid, e.g., an
oligonucleotide. In some embodiments, attaching members of the one
or more pluralities of capture probes to members of the one or more
pluralities of oligonucleotides to form one or more pluralities of
coupled capture probes comprises polymerase chain reaction (PCR).
In some embodiments, the hydrogel includes PCR reagents such as a
DNA polymerase, a plurality of primers, and a plurality of
nucleotides. In some embodiments, members of the plurality of
features are amplified (see, e.g., Mitra and Church, Nucleic Acids
Res. 1999 Dec. 15; 27(24):e34, which is incorporated by reference
herein in its entirety).
[1626] In some embodiments, chemical ligation can be used to ligate
two or more oligonucleotides (e.g., a capture probe and an
oligonucleotide from a hydrogel as described herein). In some
embodiments, chemical ligation involves the use of condensing
reagents. In some embodiments, condensing reagents are utilized to
activate a phosphate group. In some embodiments, condensing
reagents may be one or more of 1-25 ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCI), cyanogen bromide, imidazole derivatives, and
1-hydroxybenzotriazole (HOAt). In some embodiments, functional
group pairs selected from one or more of a nucleophilic group and
an electrophilic group, or an alkyne and an azide group are used
for chemical ligation. In some embodiments, chemical ligation of
two or more oligonucleotides requires a template strand that is
complementary to the oligonucleotides to be ligated. In some
embodiments, the chemical ligation process is similar to
oligonucleotide synthesis.
[1627] In some embodiments, not all of the domains of a capture
probe are attached to an oligonucleotide to form a coupled capture
probe. For example, a coupled capture probe may comprise 1, 2, 3,
4, or 5 domains of the original capture probe. In some embodiments,
all the domains of the capture probe are attached to the
oligonucleotide. For example, the coupled capture probe comprises
all the domains of the original capture probe.
[1628] In some embodiments, members of the one or more pluralities
of capture probes are cross-linked to a hydrogel.
[1629] As used herein "shrinking" or "reducing the size of" a
hydrogel refers to any process causing the hydrogel to physically
contract and/or the size of the hydrogel to decrease in volume. For
example, the scaffold of the gel may shrink or "implode" upon
solvent removal (see, e.g., Long and Williams. Science. 2018;
362(6420):1244-1245, and Oran et al. Science. 2018;
362(6420):1281-1285; each of which is incorporated herein by
reference in its entirety). As another example, the process to
shrink or reduce the size of a hydrogel may be one that removes
water (i.e., a dehydrating process) from the hydrogel. There are
many methods known to one of skill in the art for shrinking or
reducing the size of a hydrogel. Non-limiting examples of a method
to shrink or reduce the size of a hydrogel include exposing the
hydrogel to one or more of: a dehydrating solvent, a salt, heat, a
vacuum, lyophilization, desiccation, filtration, and
air-drying.
[1630] In some embodiments, the removal of water comprises a
dehydrating solvent. Non-limiting examples of a dehydrating solvent
include a ketone (such as methyl ethyl ketone (MEK)), isopropanol
(IPA), acetone, 1-butanol, methanol (MeOH), dimethyl sulfoxide
(DMSO), glycerol, propylene glycol, ethylene glycol, ethanol, (k)
1,4-dioxane, propylene carbonate, furfuryl alcohol,
N,N-dimethylformamide (DMF), acetonitrile, and an aldehyde (such as
formaldehyde or glutaraldehyde). In some embodiments, the solvent
comprises at least one of an alcohol, a ketone, an aldehyde,
acetonitrile, and a combination thereof. In some embodiments, the
solvent comprises a solvent selected from the group consisting of:
glycerol, ethanol, acetonitrile, acetone, 2-propanol, and a
combination thereof.
[1631] In some embodiments, the removal of water comprises a salt.
In some embodiments, the salt comprises an inorganic salt.
Non-limiting examples of salts that may be used to shrink a
hydrogel include inorganic salts such as aluminum, ammonium,
barium, beryllium, calcium, cesium, lithium, magnesium, potassium,
rubidium, sodium, and strontium salts. Further non-limiting
examples of inorganic salts include sodium chloride, potassium
chloride, lithium chloride, cesium chloride, sodium fluoride,
sodium bromide, sodium iodide, sodium nitrite, potassium sulfate,
potassium nitrate, potassium carbonate, potassium bicarbonate,
sodium sulfate, sodium nitrate, sodium carbonate, sodium
bicarbonate, calcium sulfate, copper oxychloride, calcium chloride,
calcium carbonate, calcium bicarbonate, magnesium sulfate,
magnesium nitrate, magnesium chloride, magnesium carbonate,
magnesium bicarbonate, ammonium sulfate, ammonium chloride,
ammonium nitrate, ammonium carbonate, ammonium bicarbonate,
trisodium phosphate, tripotassium phosphate, calcium phosphate,
copper(II) sulfate, sodium sulfide, potassium sulfide, calcium
sulfide, potassium permanganate, iron(II) chloride, iron(III)
chloride, iron (2+) sulfate, iron(III) sulfate, iron(II) nitrate,
iron(III) nitrate, manganese(II) chloride, manganese(III) chloride,
manganese(II) sulfate, manganese(II) nitrate, zinc chloride, zinc
nitrate, zinc sulfate, ammonium orthomolybdate, monopotassium
phosphate, nickel(II) sulfate, nickel(II) nitrate, sodium
metavanadate, sodium paravanadate, potassium dichromate, ammonium
dichromate, antipyonin, ammonium nitrite, potassium fluoride,
sodium fluoride, ammonium fluoride, calcium fluoride, chrome alum,
potassium alum, potassium iodide, sodium hypochlorite, tin(II)
sulfate, tin(II) nitrate, gold selenite, dicesium chromate,
potassium perchlorate, calcium perchlorate, aluminium sulphate,
lead(II) bisulfate, barium phosphate, barium hydrogen
orthophosphate, barium dihydrogen phosphate, silver dichromate,
potassium bromate, sodium bromate, sodium iodate, sodium silicate,
diammonium phosphate, ammonium phosphate, ammonium dihydrogen
phosphate, chromium orthophosphate, copper(II) chloride, copper(I)
chloride, sodium tetrametaphosphate, potassium heptafluoroniobate,
zinc phosphate, sodium sulfite, copper(I) nitrate, copper(II)
nitrate, potassium silicate, copper(II) carbonate basic, copper(II)
carbonate salts of acrylic acid and sulfopropyl acrylate.
[1632] In some embodiments, the removal of water comprises an acid.
In some embodiments, the hydrogel is exposed to a different pH
environment. For example, the hydrogel can be exposed to an acidic
pH or a basic pH. In some embodiments, the hydrogel is exposed to a
pH of less than about 6.5, e.g., a pH of about 6, about 5.5, about
5, about 4.5, about 4, about 3.5, about 3, about 2.5, about 2,
about 1.5, or about 1. In some embodiments, the hydrogel is exposed
to a pH of greater than about 7.5, e.g., a pH of about 8, about
8.5, about 9, about 9.5, about 10, about 10.5, about 11, about
11.5, 10 about 12, about 12.5, about 13, about 13.5, or about
14.
[1633] In some embodiments, the removal of water comprises a
dehydrating process such as heat, a vacuum, lyophilization,
desiccation, filtration, and air-drying. In some embodiments, the
hydrogel is exposed to an increased temperature, e.g., a higher
temperature than about room temperature. For example, a temperature
of about 37.degree. C., 38 15.degree. C., 39.degree. C., 40.degree.
C., 41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C. 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., or 70.degree. C., or higher. As another example, the
hydrogel is exposed to an increase in temperature from about
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C., or
49.degree. C. to about 50.degree. C., 51 20.degree. C., 52.degree.
C., 53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C. 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., or 70.degree. C., or higher).
[1634] In some embodiments, more than one method to shrink or
reduce the size of a hydrogel can be used. For example, a hydrogel
can be exposed to an acid and subsequently dehydrated by another
method such as heat, a vacuum, lyophilization, desiccation,
filtration, or a combination thereof.
[1635] In some embodiments, the hydrogel can further comprise a
cross-linking agent. The cross linking agent may help stabilize the
hydrogel and/or modulate the extent the hydrogel shrinks.
Non-limiting examples of a cross-linking agent include
disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS),
formalin, dimethyladipimidate (DMA), dithio-bis(-succinimidyl
propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene
glycol bis(succinimidyl succinate) (EGS). In some embodiments, the
shrunken hydrogel is at least about 2-fold smaller in a linear
dimension (e.g., along one axis) than the pre-shrunk hydrogel. For
example, at least about 2.5, about 3, about 4, about 5, about 6,
about 7, about 8, about 9, about 10, or more fold smaller in a
linear dimension than the pre-shrunk hydrogel. In some embodiments,
a shrunken hydrogel is about 2 to about 10 fold smaller in a linear
dimension than the pre-shrunk hydrogel. For example, about 2 to
about 3, about 2 to about 4, about 2 to about 5, about 2 to about
6, about 2 to about 7, about 2 to about 8, about 2 to about 9,
about 9 to about 10, about 8 to about 10, about 7 to about 10,
about 6 to about 10, about 5 to about 10, about 4 to about 10, or
about 3 to about 10. In some embodiments, a shrunken hydrogel is
about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5
to about 7, about 6 to about 8 fold smaller in a linear dimension
than the pre-shrunk hydrogel. In some embodiments, a shrunken
hydrogel is about 2, about 3, about 4, about 5, about 6, about 7,
about 8, about 9, or about 10 fold smaller in a linear dimension
than the pre-shrunk hydrogel.
[1636] In some embodiments, the size of the hydrogel is reduced
along more than one axes, e.g., along 2 or 3 axes. In some
embodiments, each axis intersects each other axis at 90 degrees. In
some embodiments, the size of the hydrogel along the first axis is
about 20 2 to about 10 or more fold smaller than the pre-shrunk
hydrogel, e.g., about 2, about 3, about 4, about 5, about 6, about
7, about 8, about 9, about 10 or more fold smaller than the
pre-shrunk hydrogel. In some embodiments, the size of the hydrogel
along the second axis is about 2 to about 10 or more fold smaller
than the pre-shrunk hydrogel, e.g., about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10 or more fold
smaller than the pre-shrunk hydrogel. In some embodiments, the size
of the hydrogel along the third axis is about 2 to about 10 or more
fold smaller than the preshrunk hydrogel, e.g., about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10 or
more fold smaller than the pre-shrunk hydrogel. In some
embodiments, the reduction in the size of the hydrogel is
isometric.
[1637] In some embodiments, a hydrogel is prepared from hydrogel
subunits including at least one of: acrylamide, bis-acrylamide, a
poly(ethylene glycol), a polyaliphatic polyurethane, a polyether
polyurethane, a polyester polyurethane, a polyethylene copolymer, a
polyamide, a polyvinyl alcohol, polypropylene glycol,
polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,
poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate),
collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin,
alginate, a protein polymer, methylcellulose, and a combination
thereof.
[1638] In some embodiments, the size of a hydrogel including
coupled capture probes is reduced, thus increasing the density of
the coupled capture probe. In some embodiments, the volume of a
partition of members of the plurality of capture probes in a
hydrogel is reduced when the size of the hydrogel is reduced.
Methods for Spatial Analysis of Analytes Using a Barcoded
Biological Sample
[1639] In some embodiments, a high-density spatially barcoded array
(e.g., a high density spatially barcoded array as described herein)
can be used for spatial analysis of analytes in the biological
sample. For example, methods for spatially profiling a plurality of
analytes in a biological sample can include providing a
high-density spatially barcoded array as described herein;
contacting the biological sample with the high-density spatially
barcoded array under conditions such that one or more analytes
present in the biological sample are captured by one or more
capture probes of the array; and determining the spatial profile of
the captured analytes.
[1640] This disclosure relates to methods for building an array. In
some embodiments, methods of generating an array comprise: (a)
providing an array including a plurality of features immobilized on
a solid substrate, wherein each feature comprises a plurality of in
situ synthesized oligonucleotides, and wherein each in situ
synthesized oligonucleotide comprises a first barcode; (b)
selecting one or more in situ synthesized oligonucleotides from the
plurality of in situ synthesized oligonucleotides; (c) delivering
an oligonucleotide to the one or more selected in situ synthesized
oligonucleotides, wherein the oligonucleotide comprises a second
barcode and a capture domain; and (d) attaching the oligonucleotide
to the one or more selected in situ synthesized oligonucleotides,
thereby generating one or more capture probes including the first
barcode and the second barcode, thereby generating an array.
[1641] In some embodiments, each feature of the plurality of
features is associated with one of a number of different first
barcodes. In some embodiments, each oligonucleotide is associated
with one of a number of different second barcodes.
[1642] In some embodiments, step (d) comprises ligating the
oligonucleotide to the one or more selected in situ synthesized
oligonucleotides. In some embodiments, the method comprises
ligating the oligonucleotide to the one or more selected in situ
synthesized oligonucleotides using a ligase. In some embodiments,
the oligonucleotide is attached to the one or more selected in situ
synthesized oligonucleotides by a chemical coupling reaction. In
some embodiments, each of the in situ synthesized oligonucleotide
comprises a 3' end that is masked by a protective group. In some
embodiments, the method comprises removing the protective group
from the one or more selected in situ synthesized oligonucleotides
prior to delivering an oligonucleotide to the one or more selected
in situ synthesized oligonucleotides. In some embodiments, the
protective group is a photocleavable protective group. In some
embodiments, the protective group is removed by exposing the one or
more selected in situ synthesized oligonucleotides to light. In
some embodiments, the protective group coupled to the one or more
selected in situ synthesized oligonucleotides is removed by
applying a photomask.
[1643] In some embodiments, the selected in situ synthesized
oligonucleotides are separated from the remaining in situ
synthesized oligonucleotides by one or more physical barriers,
wherein the one or more physical barriers form one or more
chambers, and wherein the oligonucleotide is delivered to the one
or more chambers including the one or more selected in situ
synthesized oligonucleotides. In some embodiments, the one or more
physical barriers include a hydrophobic surface. In some
embodiments, the one or more physical barriers include one or more
chamber walls having a tapered end. In some embodiments, the
oligonucleotide comprises a unique molecular identifier. In some
embodiments, the oligonucleotide comprises a 3' end that is masked.
In some embodiments, a free 3'-OH group on the capture probes is
created.
[1644] In some embodiments, methods of generating an array
comprise: (a) providing an array including two or more partitions,
wherein each partition comprises a plurality of features
immobilized on a substrate, wherein each feature comprises a
plurality of in situ synthesized oligonucleotides, and wherein each
in situ synthesized oligonucleotide comprises a first barcode; (b)
delivering an oligonucleotide to a first partition, wherein the
oligonucleotide comprises a second barcode and a capture domain;
and (c) attaching the oligonucleotide to one or more in situ
synthesized oligonucleotides in the first partition, thereby
generating one or more capture probes including the first barcode
and the second barcode in the first partition, thereby generating
an array.
[1645] In some embodiments, methods provided herein further
comprise: (d) delivering a second oligonucleotide to a second
partition, wherein the second oligonucleotide includes a third
barcode; and (e) attaching the second oligonucleotide to one or
more in situ synthesized oligonucleotides in the second partition,
thereby generating capture probes including the first barcode and
the third barcode in the second partition.
[1646] In some embodiments, the two or more partitions are
separated by one or more physical barriers. In some embodiments,
the one or more physical barriers are printed on the solid
substrate. In some embodiments, the one or more physical barriers
form one or more chambers comprising one or more chamber walls. In
some embodiments, the one or more chamber walls comprise a
hydrophobic surface. In some embodiments, the one or more chamber
walls comprise a tapered end in contact with the solid substrate.
In some embodiments, the second nucleotide is delivered using a
microfluidic device.
Chemical Ligation of Oligonucleotides
[1647] In some embodiments, chemical ligation can be used to ligate
two or more oligonucleotides. In some embodiments, chemical
ligation involves the use of condensing reagents. In some
embodiments, condensing reagents are utilized to activate a
phosphate group. In some embodiments, condensing reagents may be
one or more of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDCI), cyanogen bromide, imidazole derivatives, and
1-hydroxybenzotriazole (HOAt). In some embodiments, functional
group pairs selected from one or more of a nucleophilic group and
an electrophilic group, or an alkyne and an azide group are used
for chemical ligation. In some embodiments, chemical ligation of
two or more oligonucleotides requires a template strand that is
complementary to the oligonucleotides to be ligated. In some
embodiments, the chemical ligation process is similar to
oligonucleotide synthesis.
Photocleavable Protective Group
[1648] In some embodiments, a photocleavable protective group (e.g.
any photocleavable, photoremovable, photoreleasable,
photoactivatable, or photosensitive protective group described
herein or known in the art) may be coupled to one or both ends of
an in situ synthesized oligonucleotide, a tagging probe (e.g. any
of the tagging probes described herein), or a capture probe (e.g.
any of the capture probe described herein). In some embodiments, a
photocleavable protective group is a chemical modification to a
molecule that can be removed with light. In some embodiments, the
photocleavable protective group (PPG) is at least one of a
nitrobenzyl-based PPG, a carbonyl-based PPG, or a benzyl-based PPG.
In some embodiments, the photocleavable protective group is removed
from one or more tagging probes by exposing the one or more tagging
probes to a light.
[1649] The present disclosure provides sample preparation
techniques that allow the enrichment of target analytes from a
library, based on a barcode corresponding to a set spatial region
of interest. Procedures for barcoding and isolating analytes from
single cells are herein discussed. Enrichment of select target
spatial barcodes corresponding to analytes of interest is more
efficient, as not all analytes from a library are subject to
downstream processes. Thus enrichment of analytes based on spatial
barcodes may allow downstream processes such as sequencing to be
more efficient or provide higher quality data from the desired
analytes.
[1650] In some embodiments provided is a method for enriching a
population of capture probes comprising: (a) providing a plurality
of capture probes wherein: (i) members of the plurality of capture
probes comprise a plurality of different spatial barcodes, and (ii)
each member of the population of capture probes to be enriched
comprises a target spatial barcode; and (b) enriching the
population of capture probes comprising the target spatial
barcode.
[1651] In some embodiments provided is a method for spatial
analysis of a tissue comprising: (a) providing a solid substrate
comprising a plurality of capture probes, wherein a subset of
capture probes is attached at a predetermined position on the solid
substrate, and wherein each capture probe of the subset comprises
an identical spatial barcode; (b) contacting a tissue sample with
the solid substrate such that the subset of capture probes
interacts with a plurality of analytes from the tissue sample to
generate a plurality of spatially barcoded analyte molecules,
wherein a member of the plurality of spatially barcoded analyte
molecules comprises the identical spatial barcode or a complement
thereof; (c) identifying a spatial region of interest within the
tissue sample, wherein the spatial region of interest comprises the
plurality of spatially barcoded analyte molecules; and (d)
enriching the plurality of spatially barcoded analyte molecules for
spatial analysis of the tissue sample.
[1652] In some embodiments, the solid substrate further comprises
an additional subset of capture probes attached at a predetermined
position on the solid substrate, wherein a member of the additional
subset of capture probes comprises a second identical spatial
barcode that is different from the spatial barcode.
[1653] In some embodiments, the solid substrate further comprises
an additional subset of capture probes attached at a predetermined
position on the solid substrate, wherein a member of the additional
subset of capture probes comprises a third identical spatial
barcode that is different from the previously used spatial
barcodes.
[1654] In some embodiments, the capture probe comprises an analyte
capture domain.
[1655] In some embodiments, the analyte comprises at least one of
RNA, DNA, and a cell labeling agent.
[1656] In some embodiments, the cell labelling agent comprises an
antigen binding molecule.
[1657] In some embodiments, the cell labelling agent comprises a
region configured to couple to the capture domain of the capture
probe.
[1658] In some embodiments, the spatial region of interest is
identified by performing shallow sequencing of at least a subset of
the plurality of spatially barcoded analyte molecules, and
identifying a target spatial barcode.
[1659] In some embodiments, the spatial region of interest is
identified by at least one of immunostaining, immunohistochemistry,
microscopy, visual inspection, and combinations thereof.
[1660] In some embodiments, the method further comprises performing
a nucleic acid extension reaction using (i) the plurality of
spatially barcoded analyte molecules and (ii) a primer comprising a
sequence specific for the identical spatial barcode or a complement
thereof to generate an enriched plurality of spatially barcoded
analyte molecules comprising the spatial barcode or a complement
thereof.
[1661] In some embodiments, the analyte is amplified by performing
a polymerase chain reaction (PCR).
[1662] In some embodiments, the method further comprises enriching
a population of capture probes present in a sub-spatial region of
interest: (a) wherein the sub-spatial region of interest is located
within the spatial region of interest, wherein the sub-spatial
region of interest comprises a sub-plurality of capture probes, and
wherein: (i) members of the sub-plurality of capture probes
comprise identical sub-spatial barcodes, (ii) members of the
sub-plurality of capture probes to be enriched comprise a second
target spatial barcode sequence; and (iii) the second target
spatial barcode sequence is associated with the sub-spatial region
of interest; and (b) enriching the population of capture probes
comprising the second target spatial barcode sequence.
[1663] In some embodiments, the method further comprises enriching
a population of capture probes present in a sub-sub-spatial region
of interest: (a) wherein the sub-sub-spatial region of interest is
located within the sub-spatial region of interest, wherein the
sub-sub-spatial region of interest comprises a third plurality of
capture probes, and wherein: (i) members of the third plurality of
capture probes comprise a third plurality of spatially barcoded
sequences, (ii) members of the third plurality of capture probes to
be enriched comprise a third target spatial barcode sequence; and
(iii) the third target spatial barcode sequence is associated with
the sub-sub-spatial region of interest; and (b) enriching the
population of capture probes comprising the third target spatial
barcode sequence.
[1664] In some embodiments, the subset of spatial barcodes comprise
one unique barcode.
[1665] In some embodiments, a spatial barcode sequence of the
subset of spatial barcodes identifies an analyte derived from a
single cell.
[1666] In some embodiments, the plurality of spatially barcoded
analyte molecules corresponds to one or more mRNAs in the tissue
sample.
[1667] In some embodiments, the plurality of spatially barcoded
analyte molecules corresponds to one or more proteins in the tissue
sample.
[1668] In some embodiments, the plurality of spatially barcoded
analyte molecules corresponds to one or more antibodies in the
tissue sample.
[1669] In some embodiments, the plurality of spatially barcoded
analyte molecules corresponds to one or more T-cell receptors in
the tissue sample.
[1670] In some embodiments, the plurality of spatially barcoded
analyte molecules corresponds to one or more regions of genomic DNA
in the tissue sample.
[1671] In some embodiments, the plurality of spatially barcoded
analyte molecules corresponds to one or more regions of chromatin
in the tissue sample.
[1672] In some embodiments, the plurality of spatially barcoded
analyte molecules corresponds to a clustered regularly interspaced
short palindromic repeat (CRISPR) ribonucleic acid (crRNA) or a
single guide ribonucleic acid (sgRNA) in the tissue sample. In some
embodiments, the plurality of spatially barcoded analyte molecules
corresponds to a microbiome or a portion thereof.
Shallow Sequencing
[1673] As used herein, the coverage or depth of sequencing is the
number of unique reads that include a given nucleotide in a
reconstructed sequence. As used herein, shallow sequencing is a
small or low number of unique reads of an identified analyte. In
some embodiments, shallow sequencing is performed to identify a
spatial region of interest. In some embodiments, shallow sequencing
is performed on an entire tissue sample. In some embodiments,
shallow sequencing is performed on an identified region of a tissue
sample. In some embodiments, shallow sequencing is performed after
identifying and/or selecting a region of a tissue sample by some
other method.
[1674] Spatial Analyte Capture
[1675] As used herein, spatial analyte capture may utilize nucleic
acid probes comprising spatial barcode sequences, which are
immobilized on a solid substrate to generate an array. The spatial
barcode sequences correspond to the location of the capture probes
on the array. In some embodiments, biological samples, such as
tissue sections, are placed onto the array and the DNA is
processed, e.g. fragmented and provided with a binding domain that
hybridizes to the probes on the array. In some embodiments, DNA
polymerase extension or ligation reaction is performed in the
tissue section on the array. In some embodiments, the probes to
which the processed DNA in the tissue section binds, are extended
using the bound DNA as a template to obtain complementary strands
of DNA, which is therefore immobilized on the surface of the array.
In some embodiments, probes to which the processed DNA in the
tissue section binds, template the ligation of the bound DNA to a
second strand of the probe or the strand of the DNA hybridized to
the probe may template the ligation of the other strand of DNA to
the probe, which is therefore immobilized on the surface of the
array. In some embodiments, the spatial barcode sequences in the
probes, the extended probes carry information about the position of
the "captured" DNA in the biological specimen, e.g. tissue section.
In some embodiments, the extended probes may be released from the
surface of the array and analyzed, e.g. sequenced, which results in
a genomic DNA "library" with exact positional information. In some
embodiments, the sequence data can then be matched to a position in
the biological specimen, e.g. tissue section. For instance, the
tissue section may be visualized or imaged, e.g. stained and
photographed, before or after the DNA synthesis or ligation step to
facilitate the correlation of the positional tag in the DNA
molecule with a position within the tissue section and the sequence
data may be overlaid on an image of the tissue section, e.g. using
a computer, to display the distribution pattern of any gene of
interest across the tissue section. The visualization step is not
essential as the tagged DNA molecule may be correlated with a
position in the tissue sample using other means, e.g. the unique
profile of the molecules captured with the same positional tag,
i.e. at the same on the array, may enable the molecule to be
correlated to a position within the tissue sample based on the
characteristics of cells or areas of cells within the tissue
sample. Other analytes may be captured by this method, including
but not limited to RNA, specific sub-types of RNA, proteins,
antibodies, genomic DNA, T-cell receptors, small molecules, or
other material.
Target Barcode Sequence
[1676] In some embodiments, a target barcode sequence is a barcode
sequence corresponding to a spatial region of interest. In some
embodiments, a target barcode sequence is or includes a spatial
barcode. In some embodiments, a target barcode sequence is
identified by identifying a spatial region of interest and
identifying the barcode associated with the spatial region of
interest on the array. In some embodiments, a target barcode
sequence is identified by performing shallow sequencing, and
selecting a barcode based on the captured analytes. In some
embodiments, a target barcode sequence is identified by a
co-localization of a target barcode sequence with one or more
additional barcode sequences. In some embodiments, a target barcode
sequence is identified manually. In some embodiments, a target
barcode sequence is identified using a computer. In some
embodiments, a target barcode sequence is identified
algorithmically. In some embodiments, the target barcode sequence
corresponds to a plurality of spatial barcode sequences that
identify a region, a sub-region, a sub-sub-region, or further
spatial demarcation. In some embodiments, a target barcode sequence
is a unique molecular identifier, which identifies one capture
probe. In some embodiments, a target barcode sequence is used to
identify and characterize captured analytes at single-cell
resolution. In some embodiments, the identifying can comprise
sequencing a plurality of capture probes to generate a plurality of
sequencing reads, and analyzing the plurality of sequencing reads
to identify the at least one target barcode sequence. In some
embodiments, each of the plurality of capture probes can further
comprise one or more functional sequences selected from the group
consisting of a sequencing primer sequence and a sequence
configured to attach to a flow cell of a sequencer. In some
embodiments, the plurality of capture probes can comprise, from 5'
to 3', a first functional domain (e.g., adapter sequence), a
sequence derived from a template nucleic acid, and a second adapter
sequence. The first adapter sequence may comprise a first sequence
configured to attach to a flow cell of a sequencer, a first
sequencing primer sequence, and a spatial barcode sequence. The
second adapter sequence may comprise a second sequencing primer
sequence and a second sequence configured to attach to a flow cell
of a sequencer. The second adapter sequence can further comprise an
index sequence.
Enriching Barcoded Nucleic Acids
[1677] In an aspect, the present disclosure provides methods of
enriching a sample for target barcoded nucleic acids. The methods
may comprise providing a plurality of capture probes comprising a
plurality of different spatial barcode sequences, identifying at
least one target barcode sequence from the plurality of different
barcode sequences, and enriching the analyte detected (e.g.,
specifically bound to the capture probe) by the capture probes
comprising the at least one target barcode sequence. Enriching may
be performed by purifying capture probes comprising the at least
one target barcode from a plurality of capture probes. Enriching
may be performed by amplifying (i.e. increasing the numbers of)
capture probes comprising the at least one target barcode from a
plurality of capture probes. The amplification may include
isothermal amplification. Enriching may be performed by both
purifying and amplifying the capture probe comprising the at least
one target barcode from a plurality of capture probes.
[1678] Spatial barcodes can be included in capture probes. For
example, the capture probes can be co-partitioned into a partition
with spatial barcodes to facilitate the barcoding reaction for
spatial analysis. The spatial barcodes may be attached to a bead
(e.g., in the context of a capture probe). The bead may be a gel
bead. In some embodiments, the spatial barcodes are releasably
attached to the bead or gel bead as described elsewhere herein
(e.g. via a cleavage domain). The plurality of capture probes may
be barcoded as described elsewhere herein.
[1679] In some embodiments, methods provided herein may comprise
enriching nucleic acids comprising the at least one target barcode
sequence. The enriching may comprise performing a nucleic acid
extension reaction using a capture probe comprising the at least
one target barcode sequence and a primer comprising a sequence
specific for the at least one target barcode sequence to generate
an enriched plurality of capture probes comprising the at least one
target barcode sequence. The nucleic acid extension reaction may be
a polymerase chain reaction (PCR). The nucleic acid extension
reaction may comprise a polymerase. The nucleic acid extension
reaction may result in amplification of the nucleic acid.
[1680] In some embodiments, methods provided herein may further
comprise performing an additional PCR on the enriched plurality of
capture probes comprising the at least one target barcode sequence.
The additional PCR may comprise use of an additional primer
comprising a sequence specific for the enriched plurality of
capture probes comprising the at least one target barcode sequence.
In some embodiments, the additional primer may comprise a sequence
specific for the spatial barcode sequence of the enriched plurality
of capture probes comprising the at least one target barcode
sequence. In some embodiments, the additional primer may comprise a
sequence substantially specific for the spatial barcode sequence of
the enriched plurality of capture probes comprising the at least
one target barcode sequence. For example, the additional primer may
comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,
99.9% or more sequence complementarity or sequence identity to the
spatial barcode sequence of the enriched plurality of capture
probes comprising the at least one target barcode sequence. In some
embodiments, the additional primer may comprise a sequence specific
for a 5' region of a spatial barcode sequence of the enriched
plurality of capture probes comprising the at least one target
barcode sequence. In some cases, the additional primer can comprise
a sequence that has no (or low) sequence complementarity or
identity to the spatial barcode sequence of the enriched plurality
of capture probes comprising the at least one target barcode
sequence.
[1681] In some embodiments, the additional primer may comprise one
or more functional sequences that facilitate sequencing of the
enriched plurality of capture probes. The one or more functional
sequences may comprise a sequencing primer sequence. The one or
more functional sequences may comprise a sequence configured to
attach to a flow cell of a sequencer.
[1682] In some embodiments, methods provided herein may further
comprise, subsequent to the nucleic acid extension reaction,
performing a size selection process to remove unincorporated
primers. The size selection process may comprise using a gel or
gel-like polymer. The size selection process may use gel
electrophoresis. In some cases, the size selection process may
comprise using a solid support. The solid support may be an
array.
[1683] In some embodiments, the enriched plurality of nucleic acids
may comprise an affinity group. The affinity group may be
incorporated to the enriched plurality of the nucleic acid via a
polymerase extension reaction with a primer comprising the affinity
group. The affinity group may be incorporated via enzymatic
reaction with a nucleotide and the affinity group. The affinity
group may be attached covalently or non-covalently. The method may
further comprise coupling the enriched plurality of capture probes
comprising the affinity group to a solid support specific for the
affinity group. The solid support may be an array. The solid
support may be a polymer matrix. The solid support may be a glass
slide. The solid support may be a resin. In some embodiments, the
solid support may comprise an antibody or antibody fragment. In
some embodiments, the solid support comprises avidin or
streptavidin and the affinity group comprises (or is) biotin. In
some embodiments, the solid support comprises maltose and the
affinity group comprises (or is) maltose binding protein. In some
embodiments, the solid support comprises maltose-binding protein
and the affinity group comprises (or is) maltose.
[1684] In some embodiments, prior to the nucleic acid extension
reaction, a first capture probe complementary to a first portion of
the at least one target barcode sequence and a second capture probe
complementary to a second portion of the at least one target
barcode sequence may be hybridized. The first capture probe can be
ligated to the second capture probe to generate the primer
comprising the sequence specific for the at least one target
barcode sequence. The ligating may comprise using a DNA ligase.
[1685] In some embodiments, the enriching can result in at least
10-fold enrichment of capture probes comprising the at least one
target barcode sequence. In some embodiments, the enriching can
result in at least 20-fold enrichment of capture probes comprising
the at least one target barcode sequence. The enrichment of nucleic
acids may be observed by an increase in the percent of nucleic
acids comprising the at least one target barcode sequence in the
enriched plurality of capture probes compared to the percent amount
of nucleic acids comprising the at least one target barcode
sequence in the plurality of capture probes. The enriching may
result in at least 50-fold enrichment of capture probes comprising
the at least one target barcode sequence. The enriching may result
in at least 100-fold enrichment of capture probes comprising the at
least one target barcode sequence. The enriching may result in at
least 200-fold enrichment of capture probes comprising the at least
one target barcode sequence. The enriching may results in at least
500-fold enrichment of capture probes comprising the at least one
target barcode sequence. The enriching may result in at least
1000-fold enrichment of capture probes comprising the at least one
target barcode sequence.
[1686] Primers are used for multiple reactions described elsewhere
herein. The primers may comprise a specific length of nucleotides.
For example, the primers may be at least about 5, 6, 7, 8, 9, 10,
12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 40, 50 or more
nucleotides long. Alternatively or in addition, the primers may be
no more than 50, 40, 30, 28, 26, 25, 24, 22, 20, 18, 16, 14, 12,
10, 9, 8, 7, 6, 5, or fewer nucleotides long.
[1687] The primers may have a specific melting temperature. The
melting temperature of the primer may be at least about 35 degrees
Celsius (.degree. C.), 40.degree. C., 41.degree. C., 42.degree. C.,
43.degree. C., 44.degree. C., 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C., 50.degree. C.,
51.degree. C., 52.degree. C., 53.degree. C., 54.degree. C.,
55.degree. C., 56.degree. C., 57.degree. C., 58.degree. C.,
59.degree. C. 60.degree. C., 61.degree. C., 62.degree. C.,
63.degree. C., 64.degree. C., 65.degree. C., or higher. The melting
temperature of the primer may be no more than about 35.degree. C.,
40.degree. C., 41.degree. C., 42.degree. C., 43.degree. C.,
44.degree. C., 45.degree. C., 46.degree. C., 47.degree. C.,
48.degree. C., 49.degree. C., 50.degree. C., 51.degree. C.,
52.degree. C., 53.degree. C., 54.degree. C., 55.degree. C.,
56.degree. C., 57.degree. C., 58.degree. C., 59.degree. C.
60.degree. C., 61.degree. C., 62.degree. C., 63.degree. C.,
64.degree. C., 65.degree. C., or lower.
[1688] In some cases, primers are ligated together to form a longer
primer. The longer primer may be subsequently used for any PCR
reactions, as described elsewhere herein. Ligating together shorter
primers into a longer primer may increase the stringency of binding
such that the targeted barcode is enriched. For example, the
shorter primers may be able to distinguish mismatches better and
thus bind only to sequences with perfect or near perfect
complementarity. The longer primer may be more able to accommodate
mismatches in sequences and bind to sequences that are not the
targeted barcode. In some cases, more than 2 primers are ligated
together to form a longer primer. For example, at least about 3, 4,
5, 6, 7, 8, 9, 10, or more primers may be ligated together to form
a longer primer.
[1689] In some embodiments, the primer comprises an affinity group
and the enriched plurality of capture probes comprises the affinity
group. The affinity group may comprise biotin. The affinity group
may comprise streptavidin or avidin. The affinity group may
comprise dioxigenin. The affinity group may comprise a protein or
polypeptide. The affinity group may comprise an antibody or
antibody fragment. The affinity group may comprise a receptor or
receptor ligand. The affinity group may comprise a sugar, a
monosaccharide, a disaccharide, or a polysaccharide. The affinity
group may comprise maltose.
[1690] Methods of the present disclosure may be used for various
applications, such as, processing a single analyte (e.g., RNA, DNA,
or protein) or multiple analytes (e.g., DNA and RNA, DNA and
protein, RNA and protein, or RNA, DNA, and protein) from a single
cell. For example, multiple analytes from the biological particle
can be processed. The multiple analytes may be from the single
cell. This may enable, for example, simultaneous proteomic,
transcriptomic and genomic analysis of the cell.
Hierarchal Barcode Enrichment
[1691] In an aspect, the present disclosure provides methods of
spatially designing or designating barcodes, such that the position
of the barcodes are specifically identified. In some embodiments, a
particular individual spot or bead may contain more than one
spatial barcode, wherein each spatial barcode corresponds to a
particular defined region on the spotted array. For example,
multiple spatial barcode sequences associated with one particular
spot on an array can provide a stronger address or attribution to a
given location by providing duplicate or independent confirmation
of the location. In some cases, the multiple spatial barcodes
represent increasing specificity of the location of the particular
array point. In a non-limiting example, a particular array point
may be coded with two different spatial barcodes, wherein each
spatial barcode identifies a particular defined region within the
array, and an array point possessing both spatial barcodes
identifies the sub-region where two defined regions overlap, e.g.,
such as the overlapping portion of a Venn diagram. In another
non-limiting example, a particular array point may be coded with
three or more different spatial barcodes, wherein the first spatial
barcode identifies a first region within the array, the second
spatial barcode identifies a second region, wherein the second
region is a subregion entirely within the first region (a
sub-spatial region), and the third spatial barcode identifies a
third region, wherein the third region is a subregion entirely
within the first and second subregions (a sub-spatial region), and
so on. In some embodiments, each capture probe has a unique spatial
barcode, or unique molecular identifier (UMI). In some embodiments,
each capture probe has a UMI, as well as one or more target
barcodes corresponding to one or more regions or sub-spatial
regions.
[1692] In some embodiments, an array has multiple regions (also
sometimes referred to as "spots" or "locations") and subregions. In
some embodiments, an array has 10 regions or more. In some
embodiments, an array has 20, 30, 40, 50, 60, 70, 80, 90, 100
regions, or more. In some embodiments, an array has 100, 200, 300,
400, 500, 600, 700, 800, 900, 1000 regions or more. In some
embodiments, an array has 1000, 2000, 3000, 4000, 5000, 6000, 7000,
8000, 9000, 1000 regions or more. In some embodiments, the regions
are identical in size. In some embodiments, the regions are not
identical in size. The regions may take any shape, including but
not limited to square, rectangle, circle, oval, pentagon, hexagon,
octagon arc, arches, lens, crescent, and any combinations thereof.
In some embodiments, a region of an array has multiple subregions.
In some embodiments, a region of an array has 10, 20, 30, 40, 50,
60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000
subregions or more. In some embodiments, the subregions are
identical in size to other subregions. In some embodiments, the
subregions are not identical in size to other subregions. In some
embodiments, the subregions are completely surrounded by a larger
region. In some embodiments, subregions are partially surrounded by
a larger region. In some embodiments, an array has multiple
sub-subregions. In some embodiments, an array has 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000 sub-subregions or more. In some embodiments, the
sub-subregions are identical in size to other sub-subregions. In
some embodiments, the sub-subregions are not identical in size to
other sub-subregions. In some embodiments, sub-subregions are
completely surrounded by a larger region. In some embodiments, the
larger region is a subregion. In some embodiments, the larger
region is a region. In some embodiments, sub-subregions are
partially surrounded by a larger subregion. In some embodiments,
sub-subregions are partially surrounded by a larger region. In some
embodiments, individual spots or beads comprise a region. In some
embodiments, individual spots or beads comprise a subregion. In
some embodiments, individual spots or beads comprise a
sub-subregion. In some embodiments, individual spots or beads
comprise a region corresponding to single cell resolution of the
sample.
[1693] In some embodiments, the present disclosure provides methods
of enriching a sample comprising more than one target barcoded
nucleic acid. The methods may comprise providing a plurality of
capture probes comprising a plurality of different spatial barcode
sequences, identifying more than one target barcode sequence from
the plurality of different spatial barcode sequences, and enriching
capture probes comprising the more than one target barcode
sequence. Enriching may be performed by purifying capture probes
comprising the more than one target barcode from a plurality of
capture probes. Enriching may be performed by amplifying (i.e.
increasing the numbers of) capture probes comprising the more than
one target barcode from a plurality of capture probes. Enriching
may be performed by both purifying and amplifying the capture probe
comprising the more than one target barcode from a plurality of
capture probes. Enriching may be performed using target barcodes
associated with smaller regions. Enriching may be performed using
target barcodes of increasing specificity to a particular spot or
location. For example, enriching may be performed by amplifying
and/or purifying a first plurality of capture probes comprising a
target barcode that is associated with a first region. The
enriching may further comprise amplifying and/or purifying a second
plurality of capture probes comprising a target barcode that is
associated with a second region that is partially or completely
within the first region. The enriching may further comprise
amplifying and/or purifying a second plurality of capture probes
comprising a target barcode that is associated with a second region
that is partially within the first region. The enriching may
further comprise amplifying and/or purifying a second plurality of
capture probes comprising a target barcode that is associated with
a second region that is completely within the first region. The
enriching may further comprise amplifying and/or purifying a third
plurality of capture probes comprising a target barcode that is
associated with a third region that is partially or completely
within the first and second regions. The enriching may further
comprise amplifying and/or purifying a third plurality of capture
probes comprising a target barcode that is associated with a third
region that is partially within the first and second regions. The
enriching may further comprise amplifying and/or purifying a third
plurality of capture probes comprising a target barcode that is
associated with a third region that is completely within the first
and second regions. The enriching may further comprise amplifying
and/or purifying a fourth plurality of capture probes comprising
additional target barcodes that are associated with four or more
regions that are partially or completely within the first, second,
and third regions. The enriching may further comprise amplifying
and/or purifying a fifth plurality of capture probes comprising
additional target barcodes that are associated with five or more
regions that are partially or completely within the first, second,
third, and fourth regions. The enriching may further comprise
amplifying and/or purifying additional pluralities of capture
probes comprising additional target barcodes that are associated
with 6, 7, 8, 9, 10, or more regions that are partially or
completely within the previously identified regions and/or
sub-spatial regions.
[1694] In some embodiments, the method may comprise enriching
nucleic acids comprising more than one target barcode sequence. The
enriching may comprise performing a nucleic acid extension reaction
using a capture probe comprising more than one target barcode
sequence and a primer comprising a sequence specific for more than
one target barcode sequence to generate an enriched plurality of
capture probes comprising more than one target barcode sequence.
The nucleic acid extension reaction may be a polymerase chain
reaction (PCR). The nucleic acid extension reaction may comprise a
polymerase. The nucleic acid extension reaction may result in
amplification of the nucleic acid.
[1695] In some embodiments, the method may further comprise
performing additional rounds of PCR on the enriched plurality of
capture probes corresponding to the number of spatial barcode
sequences of a given location. The additional rounds of PCR may
comprise use of additional primers comprising a sequence specific
for the additional target barcode sequences (e.g., target barcode
sequences associated with a subregion or sub-subregion of
interest). In some embodiments, the additional primers may comprise
a sequence specific for the spatial barcode sequence of the
enriched plurality of capture probes comprising more than one
target barcode sequence. In some embodiments, the additional
primers may comprise a sequence substantially specific for an
additional target barcode sequence of the enriched plurality of
capture probes comprising more than one target barcode sequence.
For example, the additional primer may comprises a sequence having
at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more sequence
complementarity or sequence identity to a target barcode sequence
of the enriched plurality of capture probes comprising the more
than one target barcode sequence. In some embodiments, the
additional primers may comprise a sequence specific for a 5' region
of a spatial barcode sequence of the enriched plurality of capture
probes comprising more than one target barcode sequence. In some
cases, the additional primer can comprise a sequence that has no
(or low) sequence complementarity or identity to one or more
spatial barcode sequences of the enriched plurality of capture
probes comprising more than one target barcode sequence.
[1696] In some embodiments, each spatial barcode sequence of the
plurality of different spatial barcode sequences can identify a
capture probe as derived from a single cell. Alternatively or in
addition, each spatial barcode sequence of the plurality of
different spatial barcode sequences may identify a capture probe as
derived from a single partition.
Identifying a Spatial Region of Interest
[1697] In some embodiments, identifying a spatial region of
interest includes any technique for observing and/or locating an
area within a tissue. In some embodiments, identifying a spatial
region of interest includes immunostaining techniques, including
but not limited to, immunohistochemistry techniques. In some
embodiments, identifying a spatial region of interest includes
microscopy techniques, including but not limited to, optical
microscopy, fluorescence microscopy, and confocal microscopy. In
some embodiments, identifying a spatial region of interest includes
visual inspection. In some embodiments, identifying a spatial
region of interest includes shallow sequencing a spatial region. In
some embodiments, identifying a spatial region of interest includes
shallow sequencing the entire tissue sample.
[1698] In at least one embodiment, the present disclosure provides
a method for preparing a composition comprising: providing a
substrate comprising a first oligonucleotide attached to the
substrate through its 3'-end or its 5'-end; and preparing by
spatially-directed in situ synthesis on the unattached end of the
first segment a plurality of second oligonucleotide segments of
8-mer to 30-mer each having a different sequence and located in a
different region of the substrate.
[1699] In at least one embodiment, the method further comprises:
attaching a plurality of third oligonucleotide segments to the
unattached ends of the plurality of second oligonucleotide
segments.
[1700] In at least one embodiment of the method of the present
disclosure the spatially-directed in situ synthesis is
light-directed spatial synthesis.
[1701] In at least one embodiment of the method of the present
disclosure the spatially-directed in situ synthesis is
mechanically-directed spatial synthesis.
[1702] In at least one embodiment of the method of the present
disclosure, the first oligonucleotide segment is attached to the
substrate through its 5'-end and the plurality of second
oligonucleotide segments are prepared (e.g., generated) by
spatially-directed in situ synthesis on the 3'-end of the first
segment.
[1703] In at least one embodiment of the method of the present
disclosure, the first oligonucleotide segment is attached to the
substrate through its 3'-end and the plurality of second
oligonucleotide segments are prepared (e.g., generated) by
spatially-directed in situ synthesis on the 5'-end of the first
segment.
[1704] In at least one embodiment, the present disclosure provides
a method for preparing a composition comprising: providing a
substrate comprising a first oligonucleotide segment attached
through its 5'-end to the substrate, wherein the first
oligonucleotide segment phosphodiester and exocyclic amine moieties
are protected; preparing by in situ synthesis a plurality of second
oligonucleotide segments of 8-mer to 20-mer attached through their
5'-ends to a 3'-end of a first oligonucleotide segment, wherein
each of the plurality of second segments comprises a different
sequence that identifies the region of the substrate where it is
located; and ligating a plurality of third oligonucleotide segments
through their 5'-ends to the 3'-ends of the plurality of second
segments.
[1705] In at least one embodiment of the method of the present
disclosure the regions of the substrate have an area of less than
about 2500 .mu.m.sup.2, less than about 1600 .mu.m.sup.2, less than
about 400 .mu.m.sup.2, less than about 100 .mu.m.sup.2, less than
about 50 .mu.m.sup.2, less than about 25 .mu.m.sup.2, less than
about 15 .mu.m.sup.2, less than about 10 .mu.m.sup.2, less than
about 5 .mu.m.sup.2, less than about 2 .mu.m.sup.2, less than about
1 .mu.m.sup.2, or smaller.
[1706] In at least one embodiment of the method of the present
disclosure the number of second oligonucleotide segment comprising
different sequences is at least about 100,000, at least about
250,000, at least about 500,000, at least about 750,000, at least
about 1,000,000, at least about 1,500,000, at least about
3,000,000, at least about 5,000,000, at least about 10,000,000, at
least about 100,000,000, at least about 500,000,000, or at least
about 1,000,000,000. In some embodiments, the number of second
oligonucleotide segment comprising different sequences is about
100,000 to about 1,000,000,000, about 750,000 to about 1,500,000,
about 1,000,000 to about 1,000,000,000, about 1,000,000 to about
10,000,000, or about 1,500,000 to about 5,000,000.
[1707] In at least one embodiment of the method of the present
disclosure the second oligonucleotide segments have a length
selected from 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer,
15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer,
23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, or
30-mer.
[1708] In at least one embodiment of the method of the present
disclosure the second oligonucleotide segment in each of the
different regions of the substrate has a purity of at least 75%, at
least 80%, at least 85%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or more.
[1709] In at least one embodiment of the method of the present
disclosure the first oligonucleotide segment further comprises
protected phosphodiester and exocyclic amine moieties.
[1710] In at least one embodiment of the method of the present
disclosure the substrate further comprises a complementary
oligonucleotide hybridized to the first oligonucleotide
segment.
[1711] In at least one embodiment of the method of the present
disclosure the first oligonucleotide segment has a length of at
least about 2-mer, at least about 5-mer, at least about 8-mer, at
least about 10-mer, or at least about 15-mer at least about 18-mer,
at least about 20-mer, at least about 22-mer, or at least about
25-mer, at least about 27-mer, at least about 30-mer, at least
about 33-mer, or at least about 35-mer. In some embodiments the
first oligonucleotide segment has a length of about 2-mer to about
45-mer, about 5-mer to about 40-mer, about 15-mer to about 40-mer
about 25-mer to about 40-mer, about 25-mer to about 35-mer, or
about 27-mer to about 33-mer.
[1712] In at least one embodiment of the method of the present
disclosure the method further comprises preparing the first
oligonucleotide segment off the substrate and subsequently
attaching it to the substrate. In some embodiments the
phosphodiester and exocyclic amine moieties of the first
oligonucleotide segment are protected prior to attaching it to the
substrate. In some embodiments the phosphodiester and exocyclic
amine moieties of the first oligonucleotide segment are protected
after attaching it to the substrate.
[1713] In at least one embodiment of the method of the present
disclosure the method further comprises preparing the first
oligonucleotide segment by synthesis on the substrate. In some
embodiments the synthesis of the first oligonucleotide segment on
the substrate is by light-directed spatial synthesis. In some
embodiments the synthesis of the first oligonucleotide segment on
the substrate is not spatially-directed.
[1714] In at least one embodiment of the method of the present
disclosure the first oligonucleotide segment sequence comprises a
cleavage site. In some embodiments, the cleavage site comprises a
poly-U sequence.
[1715] In at least one embodiment of the method of the present
disclosure the first oligonucleotide segment is attached to the
substrate through a linker. In some embodiments, the linker
comprises a chemical group selected from: silane, amine, peptide,
polyethylene glycol, biotin, desthiobiotin, streptavidin, thiol,
acrydite, azide, digoxigenin, hexynyl, or a combination thereof. In
some embodiments, the linker is a branched linker or a dendrimeric
linker.
[1716] In at least one embodiment of the method of the present
disclosure that further comprises attaching a plurality of third
oligonucleotide segments, the first oligonucleotide segment is
attached to the substrate through its 5'-end, the plurality of
second oligonucleotide segments are prepared by spatially-directed
in situ synthesis on the 3'-end of the first segment, and the
plurality of third oligonucleotide segments are attached through
their 5'-end to the 3'-end of the plurality of second segments.
[1717] In at least one embodiment of the method of the present
disclosure that further comprises attaching a plurality of third
oligonucleotide segments, the plurality of third oligonucleotide
segments are attached by ligation. In some embodiments, protected
moieties of the first and second oligonucleotide segments are
deprotected prior to ligating the plurality of third
oligonucleotide segments.
[1718] In at least one embodiment of the method of the present
disclosure that further comprises attaching a plurality of third
oligonucleotide segments, the plurality of third oligonucleotide
segments have a length of at least about 5-mer, at least about
7-mer, at least about 9-mer, at least about 13-mer, at least about
20-mer, at least about 25-mer, at least about 27-mer, at least
about 30-mer, at least about 33-mer, or at least about 35-mer. In
some embodiments, the plurality of third oligonucleotide segments
have a length of about 5-mer to about 40-mer, 5-mer to about
20-mer, 5-mer to about 10-mer, about 10-mer to about 35-mer, about
15-mer to about 30-mer, about 25-mer to about 35-mer, or about
27-mer to about 33-mer.
[1719] In at least one embodiment of the method of the present
disclosure that further comprises attaching a plurality of third
oligonucleotide segments, each of the plurality of third
oligonucleotide segments comprise a different 5-mer to 20-mer
random sequence at the 5'-end.
[1720] In at least one embodiment of the method of the present
disclosure that further comprises attaching a plurality of third
oligonucleotide segments, each of the plurality of third
oligonucleotide segments comprises an identical capture sequence of
at least 6-mer at the 3'-end. In some embodiments, the capture
sequence comprises a poly-T or a poly-U sequence.
[1721] In at least one embodiment the present disclosure also
provides a composition comprising a plurality of oligonucleotides
attached to a plurality of regions on a substrate, wherein each
oligonucleotide comprises: a first oligonucleotide segment of 2-mer
to 50-mer attached to the substrate through its 5'-end; and a
second oligonucleotide segment of 8-mer to 30-mer that is attached
to the 3'-end of the first segment through its 5'-end, wherein the
second segment comprises a sequence that identifies the region
where it is located.
[1722] In at least one embodiment of the composition each
oligonucleotide further comprises: a third oligonucleotide segment
of 11-mer to 50-mer that is attached to the 3'-end of the second
segment through its 5'-end, wherein the third segment comprises a
5-mer to 20-mer random sequence at its 5'-end and a 6-mer to 38-mer
capture sequence of at its 3'-end.
[1723] In at least one embodiment of the composition of the present
disclosure the regions of the substrate have an area of less than
about 2500 .mu.m2, less than about 1600 .mu.m2, less than about 400
.mu.m2, less than about 100 .mu.m2, less than about 50 .mu.m2, less
than about 25 .mu.m2, less than about 15 .mu.m2, less than about 10
.mu.m2, less than about 5 .mu.m2, less than about 2 .mu.m2, less
than about 1 .mu.m2, or smaller.
[1724] In at least one embodiment of the composition of the present
disclosure the number of second oligonucleotide segment comprising
different sequences is at least about 100,000, at least about
250,000, at least about 500,000, at least about 750,000, at least
about 1,000,000, at least about 1,500,000, at least about
3,000,000, at least about 5,000,000, at least about 10,000,000, at
least about 100,000,000, at least about 500,000,000, or at least
about 1,000,000,000. In some embodiments, the number of second
oligonucleotide segment comprising different sequences is about
100,000 to about 1,000,000,000, about 750,000 to about 1,500,000,
about 1,000,000 to about 1,000,000,000, about 1,000,000 to about
10,000,000, or about 1,500,000 to about 5,000,000.
[1725] In at least one embodiment of the composition of the present
disclosure the second oligonucleotide segments have a length
selected from 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer,
15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer,
23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, or
30-mer.
[1726] In at least one embodiment of the composition of the present
disclosure the second oligonucleotide segment in each of the
different regions of the substrate has a purity of at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or more.
[1727] In at least one embodiment of the composition of the present
disclosure the first and second oligonucleotide segments further
comprise protected phosphodiester and exocyclic amine moieties.
[1728] In at least one embodiment of the composition of the present
disclosure the length of the oligonucleotide in each region is at
least 10-mer, at least 20-mer, at least 30-mer, at least 40-mer, at
least 50-mer, or at least 60-mer. In some embodiments the length of
the oligonucleotide in each region is from 40-mer to 100-mer, from
50-mer to 90-mer, from 60-mer to 80-mer or 10-mer to 40-mer.
Generating Spatial Arrays
[1729] In at least one embodiment of the compositions and methods
of the present disclosure, the plurality of oligonucleotides is
attached to a substrate by any suitable means that immobilizes the
oligonucleotide by either its 3' or 5' end in a region of the
substrate and allows the unattached end to be available for further
synthesis. Further synthesis can include in situ base-by-base
step-wise synthesis (e.g., phosphoramidite chemistry) or
biosynthesis via enzyme reactions (e.g., DNA polymerization or
reverse transcription). Additionally, the means of attachment
should also be suitable to allow at least a portion of the
oligonucleotide to be capable of hybridization.
[1730] In at least one embodiment of the compositions and methods
of the present disclosure, the plurality of oligonucleotides is
attached to a substrate by a covalent linkage. Accordingly, in at
least one embodiment, the plurality of oligonucleotides are
attached to the substrate by a chemical reaction that forms one or
more covalent bonds between a functional group on the substrate (or
a linker group attached to the substrate) and a functional group on
the 3'- or 5'-end of the oligonucleotide (or a linker group
attached to the 3' or 5'-end of the oligonucleotide). Such
functional groups can include a naturally occurring nucleotide
functional group, e.g. a terminal hydroxyl group, or a non-natural
functional group introduced during oligonucleotide synthesis, such
as a 5'-amine group. Reagents and techniques for chemically
modifying substrate materials and/or nucleotides with functional
groups that are useful in the methods of the present disclosure are
well known in the art. For example, functional groups that may be
introduced on a substrate material that allow for attachment of an
oligonucleotide (e.g., 5'-amine modified oligonucleotide) to a
substrate include linkers with available carboxy, aldehyde or epoxy
groups. Other functionalized linker compounds are known to the
person skilled in the art.
[1731] The oligonucleotides of the plurality of oligonucleotides to
be attached to a substrate in preparing a spatial array of the
present disclosure can be understood as having at least three
segments: a first oligonucleotide segment of 2-mer to 40-mer that
is attached to the substrate through either its 3'-end or its
5'-end; a second oligonucleotide segment that is attached to the
unattached end of the first segment, and comprises a barcode
sequence of about 8-mer to 30-mer in length that indicates the
region on the substrate where the second segment is located; and a
plurality of third oligonucleotide segments of 5-mer to 50-mer
attached to the second segment, wherein the third segment comprises
a capture sequence at its 3'-end.
[1732] FIG. 31 depicts at least one embodiment of a method for
preparing an exemplary spatial array composition of the present
disclosure. First, as shown in FIG. 31A, a substrate is provided
that comprises a first oligonucleotide segment attached through its
5'-end to the substrate. In at least one embodiment, the first
oligonucleotide segment comprises a sequence that is identical
across all of the plurality of the different oligonucleotides
attached to the substrate. In some embodiments, the first
oligonucleotide segment can be understood as a common
oligonucleotide "lawn" across the surface of the substrate that
does not differ in sequence in the various regions of the
substrate. The free, unattached end of the first oligonucleotide
segment, thus, effectively acts as the starting point on the
spatial array for the spatially-directed in situ synthesis of the
second oligonucleotide segment that comprises the barcode
sequence.
[1733] In at least one embodiment, the length of the first
oligonucleotide segment is the same across the surface of the
substrate. While typically not critical to the function of the
spatial array, in at least one embodiment, the length of the first
oligonucleotide sequence should be at least about 2-mer.
Furthermore, in some embodiments the length of the first
oligonucleotide segment is at least 5-mer, at least about 10-mer,
at least about 15-mer, at least about 20-mer, at least about
25-mer, at least about 27-mer, at least about 30-mer, at least
about 33-mer, or at least about 35-mer. Similarly, in some
embodiments the first oligonucleotide segment has a length of about
2-mer to about 40-mer, about 5-mer to about 40-mer, about 10-mer to
about 30-mer, about 25-mer to about 35-mer, about 20-mer to about
40-mer, about 25-mer to about 40-mer, about 25-mer to about 35-mer,
or about 27-mer to about 33-mer.
[1734] As further depicted in FIG. 31A, in at least one embodiment,
the first oligonucleotide segment comprises phosphodiester and
exocyclic amine moieties that are protected. Protection of the
phosphodiester and exocyclic amine moieties of the first
oligonucleotide segment allows for optimal in situ synthesis of the
second oligonucleotide segments that comprise the barcode sequence.
Suitable protecting groups for phosphodiester and exocyclic amine
moieties are well-known in the art and include the protecting
groups employed in standard oligonucleotide synthesis using
phosphoramidite chemistry.
[1735] The present disclosure describes a variety of embodiments
for preparing a substrate having attached thereto a first
oligonucleotide segment, wherein the first oligonucleotide segment
comprises protected phosphodiester and exocyclic amine moieties
(see e.g., embodiments of FIGS. 32-36).
[1736] In at least one exemplary embodiment, depicted in FIG. 32,
the method further comprises preparing the first oligonucleotide
segment ex situ (e.g., on a separate solid support using solid
phase automated synthesis), cleaving it from the separate solid
support with the protecting groups intact (see FIG. 32A), and
subsequently attaching the ex situ synthesize oligonucleotide with
protecting groups it to the substrate (see FIG. 32B). A free
hydroxyl at the unattached end is available for subsequent
spatially-directed in situ synthesis of a second oligonucleotide
segment comprising a barcode sequence (see FIG. 32C).
[1737] In at least another exemplary embodiment, depicted in FIG.
33, the method further comprises preparing the first
oligonucleotide segment in situ directly the solid support using
base-by-base phosphoramidite synthesis chemistry with
phosphodiester and exocyclic amine protecting groups. Such a method
can be use standard solid-phase oligonucleotide synthesis chemistry
to provide a substrate with first oligonucleotide segments attached
thereto by the 3'-end and having protecting groups. In at least one
embodiment, as depicted in FIG. 33A, oligonucleotide synthesis
chemistry in a 5'-to-3' direction can be carried out to provide the
protected first oligonucleotide segments attached to the substrate
through the 5'-end. In both cases, the free hydroxyl at the
unattached end is available for subsequent spatially-directed in
situ synthesis of a second oligonucleotide segment comprising a
barcode sequence (see FIG. 33C).
[1738] Second, as depicted in FIG. 31B, a plurality of second
oligonucleotide segments of 8-mer to 30-mer are prepared by
spatially-directed in situ synthesis extending from the free end of
the first oligonucleotide segment. As shown in FIG. 31B, the
spatially-directed in situ synthesis is a light-directed spatial
synthesis which is carried out in the 5'-to-3' direction. This in
situ synthesis method results in a plurality of second
oligonucleotide segments each attached through its 5'-end to a
3'-end of a first oligonucleotide segment. FIG. 31B is a schematic
representation of only two distinct regions each having four
individual oligonucleotide molecules. Each region has a different
second oligonucleotide segment sequence, and all of the second
oligonucleotide segment molecules in a region ideally should have
the same sequence, although as discussed further below, less than
100% yield in the in situ synthesis process results in less than
perfect purity (or homogeneity) of oligonucleotide sequence in a
region.
[1739] In at least one embodiment of the present disclosure,
spatially-directed synthesis of a plurality of oligonucleotides on
a substrate in spatially distinct regions is carried out using
light-directed synthesis or by mechanically-directed synthesis
methods. A spatially-directed in situ synthesis method inherently
correlates each sequence synthesized with the location of
synthesis. In the schematic illustration of FIG. 31B, a
light-directed spatial synthesis is used to synthesize a second
oligonucleotide segment in a base-by-base step-wise fashion in each
region. The region where each nucleotide base is added is
controlled by the precise location (and duration) of
spatially-directed illumination of the substrate. The direction and
duration of illumination can be precisely controlled, for example
with maskless array synthesis (MAS) technology using
computer-controlled digital micromirror devices (DMD), to provide
distinct regions of 1 .mu.m.sup.2, or even smaller. It is only at
these precisely illuminated regions on the substrate where
deprotection of a photo-labile protecting group occurs, thereby
allowing coupling of a nucleoside phosphoramidite that provides the
next base in the oligonucleotide sequence.
[1740] Light-directed spatial in situ synthesis provides optimal
control in the synthesis of high-fidelity second oligonucleotide
segment barcode sequences at precise region locations on a
substrate. Light-directed spatial synthesis of oligonucleotides
refers to synthetic methods using photolabile nucleoside protecting
groups and spatially-directed illumination to effect selective
deprotection and base-by-base synthesis at specific locations on a
substrate. Light-directed spatial synthesis of oligonucleotides
uses an iterative cycle of phosphoramidite-based coupling chemistry
with photo-labile protecting groups rather than dimethoxytrityl
(DMT) protecting groups. The synthesis cycle, thus, is very similar
to that used in standard solid-phase phosphoramidite synthesis of
oligonucleotides with the following general steps: (1)
photo-deprotection; (2) activation and coupling; (3) capping; and
(4) drying. The drying step (4) can involve blowing helium to dry
the substrate surface after coupling and acetonitrile wash but
before the next photodeprotection step. Techniques, protocols,
reagents, materials, and other instrumentation useful for
light-directed spatial synthesis of high-density oligonucleotide
microarrays are well-known in the art and commercially available.
For example, U.S. Pat. Nos. 7,844,940, 8,030,477, PCT Publ. No. WO
99/63385A1, US Pat. Publ. No. 20150246336A1, Agbavwe et al.
"Efficiency, error and yield in light-directed maskless synthesis
of DNA microarrays." Journal of nanobiotechnology 9.1 (2011): 57;
and Sack et al. "Express photolithographic DNA microarray synthesis
with optimized chemistry and high-efficiency photolabile groups."
Journal of nanobiotechnology 14.1 (2016): 14, describe materials
and methods that can be used in the light-directed spatial
synthesis methods of the present disclosure.
[1741] The photo-deprotection step involves spatially-directed
illumination of the substrate and is the step that ultimately
defines the size and location of a region. Efficiency and accuracy
of the photo-deprotection step in defining the regions on the
substrate where specific oligonucleotides are synthesized depends
largely on the illumination techniques and the photo-labile groups
used. Photo-labile groups and illumination techniques useful for
light-directed spatial synthesis methods are well-known and
commercially available (e.g., from Sigma-Aldrich, St. Louis, Mo.,
USA). For example, photo-labile protecting groups that can be used
to prepare spatial array compositions of the present disclosure are
described in US Pat. Publ. No. 20150246336A1; Agbavwe et al.
"Efficiency, error and yield in light-directed maskless synthesis
of DNA microarrays." Journal of nanobiotechnology 9.1 (2011): 57;
Kretschy et al. "Next-Generation o-Nitrobenzyl Photolabile Groups
for Light-Directed Chemistry and Microarray Synthesis." Angewandte
Chemie International Edition 54.29 (2015): 8555-8559; Sack et al.
"Express photolithographic DNA microarray synthesis with optimized
chemistry and high-efficiency photolabile groups." Journal of
nanobiotechnology 14.1 (2016): 14; Lietard et al., "High-Density
RNA Microarrays Synthesized In Situ by Photolithography." Angew.
Chem. Int. Ed. 57 (2016): 15257-15261; and Holz et al.,
"High-Efficiency Reverse (5'.fwdarw.3') Synthesis of Complex DNA
Microarrays." Scientific reports 8.1 (2018): 15099.
[1742] In at least one embodiment, the photo-labile protecting
groups useful in the light-direct spatial synthesis methods of the
present disclosure include NPPOC, Bz-NPPOC, and SPh-NPPOC which are
shown in Table 1.
TABLE-US-00003 TABLE 1 Exemplary Photo-labile Protecting Groups
Structure Name (e.g., protecting 3'-OH of nucleoside) NPPOC
##STR00045## Bz--NPPOC ##STR00046## SPh--NPPOC ##STR00047##
[1743] The schematic illustration of FIG. 31B depicts
light-directed spatial synthesis of second oligonucleotide segments
in the 5'-to-3' direction. The resulting plurality of the second
oligonucleotide segments are thus attached through their 5'-ends to
the free 3'-ends of the first oligonucleotide segment on the
substrate. Accordingly, in at least one embodiment the
light-directed spatial synthesis of the second oligonucleotides is
carried out in the 5'-to-3' direction. Techniques, reagents, and
photo-labile protecting groups useful for synthesis in the 5'-to-3'
direction include those described in e.g., Holz et al.,
"High-Efficiency Reverse (5'.fwdarw.3') Synthesis of Complex DNA
Microarrays." Scientific reports 8.1 (2018): 15099. In at least one
embodiment, light-directed spatial synthesis of a plurality of
second oligonucleotide segments can be carried out using one or
more of the photo-labile protecting groups of Table 1 to protect
the 3'-OH. In at least one embodiment, the photo-labile protecting
group used in light-directed spatial synthesis of the second
oligonucleotide segment is Bz-NPPOC.
[1744] In at least one embodiment, the spatially-directed in situ
synthesis can be carried out using mechanically-directed spatial
synthesis. Mechanically-directed spatial synthesis of
oligonucleotides refers to methods that use computer-controlled
liquid dispensing and/or placement technology to mechanically
direct nucleoside phosphoramidite reagents at distinct locations on
a substrate, and thereby synthesizing an oligonucleotide using
standard solid-phase phosphoramidite chemistry at a distinct region
on a substrate. A range of technologies for mechanical
spatially-directed in situ synthesis of oligonucleotides are
well-known in the art.
[1745] The schematic illustration of FIG. 31B depicts only two
regions, however it is contemplated that a spatial array
composition made by the methods of the present disclosure can
include millions of different regions on a substrate, each
comprising a different second oligonucleotide segment barcode
sequence. In at least one embodiment, the number of second
oligonucleotide segments comprising different sequences is at least
about 100,000, at least about 250,000, at least about 500,000, at
least about 750,000, at least about 1,000,000, at least about
1,500,000, at least about 3,000,000, at least about 5,000,000, at
least about 10,000,000, at least about 100,000,000, at least about
500,000,000, or at least about 1,000,000,000. In some embodiments,
the number of second oligonucleotide segment comprising different
sequences is about 100,000 to about 1,000,000,000, about 750,000 to
about 1,500,000, about 1,000,000 to about 1,000,000,000, about
1,000,000 to about 10,000,000, or about 1,500,000 to about
5,000,000.
[1746] In order for a spatial array to accommodate these large
numbers of different oligonucleotides on a substrate, it also is
contemplated that the areas of the regions need to be small.
Accordingly, in at least one embodiment of the method of the
present disclosure the regions of the substrate have an area of
less than about 2500 .mu.m.sup.2, less than about 1600 .mu.m.sup.2,
less than about 400 .mu.m.sup.2, less than about 100 .mu.m.sup.2,
less than about 50 .mu.m.sup.2, less than about 25 .mu.m.sup.2,
less than about 15 .mu.m.sup.2, less than about 10 .mu.m.sup.2,
less than about 5 .mu.m.sup.2, less than about 2 .mu.m.sup.2, less
than about 1 .mu.m.sup.2, or smaller.
[1747] In at least one embodiment, the methods of the present
disclosure contemplate the spatially-directed in situ synthesis of
second oligonucleotide segments comprising millions of different
barcode sequences each in a different region. In order to
accommodate millions of distinct regions each with a distinct
barcode sequence, the present disclosure contemplates that the
second oligonucleotide segments have a length range of 8-mer to
30-mer. A length range of 8-mer to 30-mer allows for the plurality
of oligonucleotides to have extremely large number of distinct
oligonucleotides that are indexed for their location on the
substrate. For example, in the case of an 8-mer sequence, there are
48 or 65,536 different possible barcode sequences that can be used
to identify distinct locations on a substrate. In the case of a
12-mer sequence, there are 412 or 16,777,216 different possible
barcode sequences that can be used to identify distinct locations
on a substrate. Accordingly, in at least one embodiment of the
method of the present disclosure the second oligonucleotide
segments have a length selected from 8-mer, 9-mer, 10-mer, 11-mer,
12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer,
20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer,
28-mer, 29-mer, or 30-mer.
[1748] In order to provide for optimal functionality of a spatial
array, the correlation of the barcode sequences with the locations
should be high. Accordingly, the spatially-directed in situ
synthesis of the barcode sequence should result in high fidelity or
purity of barcode sequences in each of the regions. It is an
advantage of the spatially-directed synthesis methods of the
present disclosure that the second oligonucleotide segments are
prepared on a substrate with high fidelity. Generally, the fidelity
(or sequence purity) of oligonucleotide sequences prepared by
step-wise solid-phase synthesis decreases as the length of the
oligonucleotide being synthesized increases. For example, assuming
a spatially-directed in situ synthesis having a coupling efficiency
of 99.5%, the fidelity of 12-mer barcode sequences in a region is
.about.95%. In some embodiments (e.g., as in FIG. 32), the use of
spatially-directed in situ synthesis to prepare only the 8-mer to
30-mer second oligonucleotide segments results in barcode sequences
of high-fidelity. Accordingly, in at least one embodiment of the
methods of the present disclosure the resulting second
oligonucleotide segment synthesized in each of the different
regions of the substrate has a purity of at least 75%, at least
80%, at least 85%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or more.
[1749] In at least one embodiment, the second oligonucleotide
segment is unique for each region of the substrate. Additionally,
in some embodiments, the second oligonucleotide segment barcode
sequence comprises a sequence that uniquely identifies it from the
plurality of other barcode sequences on the spatial array but does
not interfere with the capture sequences of the oligonucleotides
that target binding to nucleic acids in a sample. Unique barcode
sequences may be generated using random sequence generation, along
with stringent filtering of sequences by mapping to the genomes of
all common reference species using desired Tm intervals, and GC
content, to minimize interference with capture sequence binding to
their targets, and to optimize the difference from the other
barcode sequences. Thus, in at least one embodiment, the barcode
sequences of the spatial array are selected such that desired
target nucleic acids of the sample applied to the array do not
hybridize specifically to the barcode sequence of the second
oligonucleotide segment. For example, in some embodiments, the
barcode sequences of the spatial array have less than 80% sequence
identity to the targeted nucleic acid sequences in the sample
applied to the spatial array. In some embodiments, barcode sequence
has less than 70% sequence identity, less than 60% sequence
identity, less than 50% sequence identity, or less than 40%
sequence identity to the nucleic acids in the sample targeted for
capture by the plurality oligonucleotide sequences.
[1750] Third, as depicted in FIG. 31C, a plurality of third
oligonucleotide segments are attached to the unattached end of the
second segment. In at least one embodiment, the third
oligonucleotide segment is in the same orientation as the second
segment. Thus, if the second segment is attached to the first
segment through its 5'-end (in a 5'-to-3' orientation relative to
the substrate) then the third oligonucleotide segment is attached
through its 5'-end to the 3'-end of the second segment. As
exemplified in FIG. 31C, in at least one embodiment, the third
oligonucleotide segments are attached through their 5'-ends to the
3'-ends of the plurality of second segments. In some embodiments,
the third oligonucleotide segment has a length of about 5-mer to
about 40-mer and comprises a capture sequence capable of
hybridizing to a nucleic acid in a sample. As described further
herein, in some embodiments, the third oligonucleotide segment
comprises a free 3'-OH and is capable of hybridizing to a
complementary nucleic acid in a sample and then undergoing
biosynthetic (e.g., polymerase catalyzed) extension from it
3'-OH.
[1751] In at least one embodiment, the plurality of third
oligonucleotide segments have a length of at least about 5-mer, at
least about 10-mer, at least about 15-mer, at least about 20-mer,
at least about 27-mer, at least about 30-mer, at least about
33-mer, or at least about 35-mer. In some embodiments, the
plurality of third oligonucleotide segments have a length of about
5-mer to about 40-mer, about 10-mer to about 40-mer, about 20-mer
to about 40-mer, about 25-mer to about 35-mer, or about 27-mer to
about 33-mer.
[1752] The 5-mer to 40-mer length of a third oligonucleotide
segment makes its attachment by in situ synthesis on the ends of
the second oligonucleotide segments problematic. The accumulating
errors during in situ synthesis would compromise the ability of any
capture sequences to function effectively on the spatial array.
Accordingly, in at least one embodiment of the methods of the
present disclosure, the third oligonucleotide segments are prepared
off the substrate and attached as a whole segment by chemical
coupling or ligation to the plurality of second oligonucleotide
segments. In at least one embodiment, the plurality of third
oligonucleotide segments are attached by ligation. In some
embodiments, protected moieties of the first and second
oligonucleotide segments are deprotected prior to ligating the
plurality of third oligonucleotide segments.
[1753] In at least one embodiment where the method of the present
disclosure further comprises attaching a plurality of third
oligonucleotide segments, the first oligonucleotide segment is
attached to the substrate through its 5'-end, the plurality of
second oligonucleotide segments are prepared by spatially-directed
in situ synthesis on the 3'-end of the first segment, and the
plurality of third oligonucleotide segments are attached through
their 5'-end to the 3'-end of the plurality of second segments.
[1754] Alternatively, in at least one embodiment where the method
of the present disclosure further comprises attaching a plurality
of third oligonucleotide segments, the first oligonucleotide
segment is attached to the substrate through its 3'-end, the
plurality of second oligonucleotide segments are prepared by
spatially-directed in situ synthesis on the 5'-end of the first
segment, and the plurality of third oligonucleotide segments are
attached through their 3'-end to the 5'-end of the plurality of
second segments.
[1755] In at least one embodiment, the third oligonucleotide
segment comprises a capture sequence capable of hybridizing to a
nucleic acid sequence in a sample. In at least one embodiment, the
capture sequence is located at the free 3'-end of the third
oligonucleotide segment which is oriented with its 5'-end attached
to the second oligonucleotide segment. In this orientation the free
3'-end can be extended (e.g. by template dependent polymerization).
It is contemplated that in at least one embodiment, the capture
sequence of the third segment comprises a sequence capable of
hybridizing to a specific type of target nucleic acid that is
present in a sample. For example, the capture sequence of the third
segment can be designed to selectively capture mRNA by
hybridization to the poly-A tail. Accordingly, in at least one
embodiment, the capture sequence of the third oligonucleotide
segment comprises a poly-T oligonucleotide. Alternatively, in some
embodiments, the capture sequence may comprise nucleotides which
are functionally analogous to poly-T and capable of binding
selectively to poly-A, such as a poly-U oligonucleotide. In at
least one embodiment, the capture sequence of the third
oligonucleotide comprises is a poly-T or poly-U sequence of at
least 5-mer to 40-mer in length. In some embodiments, the capture
sequence comprises a poly-T or poly-U sequence of at least 5-mer,
10-mer, at least 15-mer, at least 20-mer, at least 25-mer, at least
30-mer, at least 35-mer, or at least 40-mer in length.
[1756] In some embodiments, it is contemplated that the capture
sequence can be designed to selectively bind to a desired sub-type
or subset of nucleic acid targets suspected of being present in a
sample. For example, a particular type of RNA, such as mRNA,
microRNA or rRNA, or a particular mRNA species that corresponds to
a particular group of genes. Thus, capture sequence of the third
oligonucleotide segment may include a sequence that specifically
binds to a particular target gene or set of genes, and can be based
on a particular sequence motif sequence or conserved sequence
according to methods well known in the art.
[1757] In some embodiments, the third segments of the plurality of
the oligonucleotide can also comprise a random sequence. As is
well-known in the art, such random sequences (e.g. random hexamers
or nonamers) are effective for the capture nucleic acid targets by
selective binding. Accordingly, it is contemplated that the capture
sequence of the third segments of the plurality of oligonucleotides
can comprise a random sequence, where the random sequences are at
least 6-mers, at least 8-mers, at least 10-mers, at least 12-mers,
at least 14-mers, at least 16-mers, or longer. In some embodiments,
the plurality of third oligonucleotide segments can include random
sequences together with a poly-T or poly-U capture sequence.
Accordingly, where a third oligonucleotide comprises a poly-T or
poly-U capture sequence, it may also comprise a random sequence. In
some embodiments, where the third segment is in a 5'-to-3'
orientation, the random sequences are located at the 5'-end of the
third segment adjacent to the 3-end of the second segment
comprising the barcode sequence.
[1758] In at least one embodiment, the first, second, and third
oligonucleotide segments are linked via phosphodiester bonds.
Additionally, in some embodiments, it is contemplated that the
oligonucleotide segments can be attached via non-natural
oligonucleotide linkages, such as methylphosphonate or
phosphorothioate bonds, or via other non-natural biocompatible
linkages such as click-chemistry linkages. The bonds linking the
segments can be formed by synthetic phosphoramidite coupling
chemistry, by a synthetic click-chemistry, by a ligation reaction,
by enzymatic biosynthesis of nucleic acid polymers (e.g., using a
polymerase or transcriptase), or a combination of bond forming
reactions. For example, in at least one embodiment, the second
oligonucleotide segment is attached to the first oligonucleotide
segment by standard phosphoramidite chemistry forming a
phosphodiester linkage, and the third oligonucleotide segment is
attached to the unattached end of the second segment via a ligation
reaction.
[1759] The plurality of oligonucleotides on a spatial array can be
attached (e.g., covalently bound or immobilized) to the substrate
via either the 3'-end or 5'-end of the oligonucleotide. The
attachment of an oligonucleotide to a substrate of a spatial array
through a covalent bound can be either direct (e.g., substrate to
3'-end of oligonucleotide) or indirect (e.g., substrate to linker
molecule to 3'-end of oligonucleotide). Typically, for commercially
available DNA microarrays, the oligonucleotides are attached via a
3' linkage, thereby leaving a free 5'-end. The free 5'-end can be
used for further synthesis on the oligonucleotide strand. However,
oligonucleotides attached to the substrate via the 5'-end, provide
an advantage in having their 3'-ends available for further
biosynthetic extension (e.g., using polymerase or transcriptase) as
described elsewhere herein.
[1760] In at least one embodiment, the first oligonucleotide
segment is attached to the substrate through its 5'-end, the second
oligonucleotide segment is attached to the 3'-end of the first
segment through its 5'-end, and the third segment is attached to
the 3'-end of the second segment through its 5'-end.
[1761] In at least one embodiment, the first oligonucleotide
segment is attached to the substrate through its 3'-end; the second
oligonucleotide segment that is attached to the 5'-end of the first
segment through its 3'-end, and the third segment is attached to
the 5'-end of the second segment through its 3'-end.
[1762] In at least one embodiment of the composition of the present
disclosure the length of the oligonucleotide in each region is at
least 10-mer, at least 20-mer, at least 30-mer, at least 40-mer, at
least 50-mer, or at least 60-mer. In some embodiments the length of
the oligonucleotide in each region is from 5-mer to 100-mer, from
10-mer to 100-mer, from 20-mer to 100-mer, from 40-mer to 100-mer,
from 50-mer to 90-mer, or from 60-mer to 80-mer.
[1763] As described elsewhere herein, some embodiments of the
spatial arrays of the present disclosure, the first oligonucleotide
segment of the spatial array comprises a 2-mer to 40-mer having the
common sequence attached across all, or a substantial contiguous
portion, of the surface of the substrate. Thus, the step of
attaching the common first oligonucleotide segment to the substrate
does not need to be spatially directed. Accordingly, a variety of
different embodiments can be used to attach prepare a substrate
having a common first oligonucleotide segment of 2-mer to 40-mer
attached thereto.
[1764] As illustrated by FIG. 32, in at least one embodiment, a
first oligonucleotide segment is prepared ex situ by standard
automated solid-phase oligonucleotide synthesis in a 3'-to-5'
direction using phosphoramidite chemistry. However, as described
elsewhere herein, protection of the phosphodiester and exocyclic
amine moieties of the first oligonucleotide segment facilitates the
spatially-directed in situ synthesis of the second oligonucleotide
segment on the unattached end of the first oligonucleotide segment.
Thus, as shown in FIG. 32A, in at least one embodiment, the first
oligonucleotide segment is synthesized ex situ using standard
techniques, however, the synthesized oligonucleotide is cleaved
from the solid-support without deprotecting the phosphodiester and
exocyclic amine moieties that are protected during synthesis. The
protected common oligonucleotide used for the first segment is
attached to the substrate with the protecting groups intact as show
in FIG. 32B.
[1765] The standard oligonucleotide synthesis method using
phosphoramidite chemistry was developed in the 1980s and later
enhanced with solid-phase supports and automation. It the method
used by commercial synthesis of DNA and RNA oligonucleotides.
Generally, solid-phase oligonucleotide synthesis proceeds in the
3'.fwdarw.5' direction rather than 5'.fwdarw.3' direction of
natural biosynthesis. The solid-phase phosphoramidite synthesis
cycle generally comprises four steps: (1) detritylation; (2)
coupling; (3) oxidation; and (4) capping. Briefly, the four steps
in the synthesis cycle are as follows. In step (1),
"detritylation," a 5'-DMT protecting group is removed from the
first, solid-support-linked nucleoside. In step (2), "coupling,"
the de-protected 5'-OH of the first, solid-support-linked
nucleoside attacks the phosphorus of the incoming second nucleoside
to form a phosphite triester. In step (3), "oxidation," the
relatively unstable phosphite triester is converted to a stable
phosphate triester. In step (4), prior to moving to step (1) of the
next cycle, "capping," is carried out wherein unreacted 5'-OH
groups are acetylated, thereby preventing elongation of sequences
with deletion mutations.
[1766] In at least one embodiment of the ex situ synthesis method,
the final monomer unit added to the 5'-end during ex situ synthesis
of the first oligonucleotide segment can comprise a non-natural
nucleoside that facilitates attachment or immobilization of the
first oligonucleotide segment to the substrate or linkers attached
to the substrate via its 5'-end. Alternatively, in some embodiments
of the ex situ synthesis method, the first monomer unit added to
the 3'-end during ex situ synthesis of the first oligonucleotide
segment can comprise a non-natural nucleoside that upon cleavage
from the synthesis solid support can facilitate attachment or
immobilization of the first oligonucleotide segment to the
substrate or linkers attached to the substrate via the 3'-end.
[1767] In at least one embodiment, the first oligonucleotide
segment attached to the substrate is prepared by in situ synthesis
on the substrate. Generally, because the sequence of the first
oligonucleotide segment is the same across all regions of the
substrate, it is not necessary to use spatially-directed in situ
synthesis in such an embodiment. For example, in situ synthesis of
the first oligonucleotide can be carried out on the substrate using
standard solid-phase phosphoramidite chemistry and reagents. As
noted above, maintaining protection of the phosphodiester and
exocyclic amine moieties of the first oligonucleotide segment
facilitates the spatially-directed in situ synthesis of the second
oligonucleotide segment on the unattached end of the first
oligonucleotide segment. Accordingly, in at least one embodiment,
it is contemplated that the in situ synthesis first oligonucleotide
segment on the substrate is carried out without the final step of
deprotecting the phosphodiester and exocyclic amine moieties before
the second segment is synthesized.
[1768] FIG. 33 illustrates at least one embodiment of a method for
in situ synthesis of a common first oligonucleotide segment
directly on the substrate. The synthesis is carried out using
step-wise solid-phase phosphoramidite chemistry but without final
cleavage or deprotection steps. As shown in FIG. 33A, in this
embodiment, the synthesis would result in a first oligonucleotide
segment attached across the surface of the substrate with protected
phosphodiester and exocyclic amine moieties. Following this in situ
synthesis of the first oligonucleotide segment with protective
groups intact, light-directed spatial synthesis of the second
oligonucleotide segment to the 3'-ends of the first segments can be
carried out as shown in FIG. 33B.
[1769] In at least one embodiment, the first oligonucleotide
segment attached to the substrate is prepared by spatially-directed
in situ synthesis on the substrate. The spatially-directed
oligonucleotide synthesis method used can include light-directed
spatial synthesis and mechanically-directed spatial synthesis
described elsewhere herein. In this embodiment, the
spatially-directed synthesis can be used in essentially the same
way as the in situ standard oligonucleotide synthesis for the
preparation of the first oligonucleotide segment having a single
sequence across all regions of the substrate. It is contemplated,
however, that in some embodiments spatially-directed synthesis can
be used to prepare a substrate having the first oligonucleotide
attached in selected regions of the substrate. For example, in some
embodiments, a spatial array could be prepared having more than one
first oligonucleotide segment sequence in different locations of
the substrate.
[1770] Accordingly, as shown in FIG. 34 that in at least one
embodiment the common first oligonucleotide segment can be in situ
synthesized directly on the substrate however using light-directed
synthesis with photolabile groups, rather than standard protective
groups, such as dimethoxytrityl groups. The synthesized first
oligonucleotide segments would be prepared using light-directed
deprotection but would not be cleaved from the substrate or
deprotected as shown in FIG. 34A. Following this light-directed in
situ synthesis of the first oligonucleotide segment with protective
groups intact, light-directed spatial synthesis of the second
oligonucleotide segment to the 3'-ends of the first segments can be
carried out as shown in FIG. 34B.
[1771] Generally, during standard automated oligonucleotide
synthesis, following cleavage of the oligonucleotide from the
synthesis solid support, the protecting groups on the phosphates
and bases are removed (or deprotected) by treatment in concentrated
aqueous ammonia with heat. Accordingly, it is also contemplated in
at least one embodiment that a first oligonucleotide is synthesized
ex situ using standard automated synthesis, including cleavage and
deprotection of the phosphodiesters and/or exocyclic amine
moieties. As illustrated by the schematic of FIG. 35A, the cleaved
and deprotected first oligonucleotide segments synthesized ex situ
can be attached to the substrate surface through a linkage to the
5'-end as described elsewhere herein. Then, as depicted by FIG.
35B, the phosphodiester and/or exocyclic amine moieties can be
protected in situ on the substrate. Following this in situ
protection step, light-directed spatial synthesis of the second
oligonucleotide segment to the 3'-ends of the first segments can be
carried out as shown in FIG. 35C.
[1772] In another embodiment illustrated by the schematic of FIG.
36A, ex situ synthesized first oligonucleotide segments can be
cleaved from their synthesis support, deprotected, and then
attached to a substrate surface through a linkage to the 5'-end as
described elsewhere herein. Then, as depicted by FIG. 36B, a
complementary oligonucleotide can be hybridized to the common first
oligonucleotide segments located across the substrate. The
formation of a double-stranded structure by hybridization can
effectively protect the phosphodiester and/or exocyclic amine
moieties of the first oligonucleotide segment on the substrate.
Following this in situ protection-by-hybridization step,
light-directed spatial synthesis of the second oligonucleotide
segment to the 3'-ends of the first segments can be carried out as
shown in FIG. 36C.
[1773] In at least one embodiment, the spatial array compositions
prepared using the methods of the present disclosure can be useful
for spatial transcriptomics applications as described in e.g.,
US2014/0066318A1. Briefly, in spatial transcriptomics a complex
multi-cellular sample, such as a tissue sample, is immobilized on a
spatial array of oligonucleotides. Typically, the oligonucleotide
sequences are attached to the array substrate through their 5'-end
and include an mRNA capture sequence at their 3'-end. Each of the
oligonucleotides also includes 5'-adjacent to the capture sequence,
a random primer sequence of at least 16-mer that allows for
amplification of any captured sequences. The detection process
results in amplification of the captured mRNA sequence as well as
the barcode sequence. Sequencing of the amplicons thereby indicates
not only the identity of the transcribed gene but also the location
in the multi-cellular sample where the gene was transcribed.
[1774] Furthermore, in order to survey the genes undergoing
transcription in a complex multi-cellular sample, the spatial array
should include an extremely high density of different
oligonucleotides specifically located on the substrate. For
example, it is contemplated that spatial arrays useful in various
applications, such as spatial transcriptomics, can include 1.5
million, 10 million, 100 million, and even up to 1.0 billion
different oligonucleotides, each of which has a different barcode
sequence and is located in a different region on a substrate,
wherein the regions are less than 5 .mu.m.sup.2, even as small as
1.0 .mu.m.sup.2.
[1775] As noted elsewhere herein, each oligonucleotide of a spatial
array should comprise a different barcode sequence that correlates
with its location. In some embodiments, it is contemplated that
additional oligonucleotides may be present on the same substrate as
the spatial array, that comprise a sequence identical to a barcode
sequence of the spatial array. Such additional oligonucleotides may
arise due to errors in synthesis or can be prepared intentionally
on the substrate (e.g., for calibration or QC purposes). Generally,
these sequences located in a different location on the same
substrate are not considered part of the spatial array and are kept
to a level and/or location that avoids interference with the
intended function of the spatial array.
[1776] As illustrated by the particular exemplary application of
spatial transcriptomics, the ability to accurately correlate the
barcode sequence of each oligonucleotide on a spatial array with a
location (or region) on the substrate is critical to the utility of
the spatial array. Accordingly, the preparation of spatial arrays
requires methods for immobilizing up to a billion different
oligonucleotides, each with a specific barcode sequence, in a
specific identifiable region on a substrate.
[1777] Disclosed herein is a method of preparing a spatial array
comprising (a) providing a solid support; (b) immobilizing a
plurality of sets of primers on the solid support, wherein a first
set of the plurality of sets of primers comprises a first primer
and a second primer, wherein the first primer and the second primer
are at a 1:1 ratio; and wherein the first primer and the second
primer are less than 10 .mu.m apart; (c) providing a template
oligonucleotide comprising a reverse complement of a spatial
barcode domain, a reverse complement of a capture domain, a first
hybridization domain capable of hybridizing to the first primer,
and a second hybridization domain, wherein a reverse complement of
the second hybridization domain is capable of hybridizing to the
second primer; (d) hybridizing the template oligonucleotide to the
first primer; (e) extending the first primer using the template
oligonucleotide as a template to generate a capture probe
comprising the first primer, the spatial barcode, and the capture
domain, which capture probe is immobilized on the solid support;
and (f) releasing the template oligonucleotide from the spatial
array. In some aspects, the method further comprises amplifying the
capture probe to create a cluster of one or more capture probes. In
some aspects, amplifying comprises: (a) hybridizing the capture
probe to the second primer; (b) extending the second primer using
the capture probe as a template to generate a copy of the template
oligonucleotide that is immobilized on the solid support; (c)
hybridizing the copy of the template oligonucleotide to a first
primer of a second set of the plurality of primers; and (d)
extending the first primer of the second set of the plurality of
primers using the copy of template oligonucleotide as a template to
generate a copy of the capture probe that is immobilized on the
solid support, wherein the cluster of capture probes comprises the
capture probe and the copy of the capture probe.
[1778] In some aspects, the template oligonucleotide comprises a
reverse complement of a unique molecular identifier (UMI), and
wherein the generated capture probe comprises the UMI.
[1779] In some aspects, the first primer is affixed to the solid
support at a 5' end of the first primer.
[1780] In some aspects, the first primer comprises a sequence that
is complementary to the template oligonucleotide.
[1781] In some aspects, the first primer comprises poly (A)
domain.
[1782] In some aspects, the first primer comprises cleavage domain.
In some aspects, the second primer is affixed to the solid support
at a 5' end of the second primer. In some aspects, the second
primer comprises a cleavage domain. In some aspects, the copy of
the template oligonucleotide is released by cleaving the cleavage
domain after the copy of the capture probe is generated.
[1783] In some aspects, the template oligonucleotide is attached to
a bead. In some aspects, the bead is a gel bead. In some aspects,
the bead is 2 microns or less in diameter. In some aspects, the
bead is 1 micron or less in diameter. In some aspects, the bead is
removed prior to the step of extending the first primer. In some
aspects, the bead is removed after the step of extending the first
primer.
[1784] In some aspects, the identity of the generated capture probe
is determined. In some aspects, the identity of the generated
capture probe is determined after the template oligonucleotide is
released. In some aspects, the identity of the generated capture
probe is determined in situ. In some aspects, the identity of the
generated capture probe is determined by in situ sequencing. In
some aspects, in situ sequencing is performed via
sequencing-by-synthesis (SBS), sequential fluorescence
hybridization, sequencing by ligation, nucleic acid hybridization,
or high-throughput digital sequencing techniques.
[1785] In some aspects, the capture domain comprises a sequence
complementary to an analyte of interest. In some aspects, the
capture domain comprises a poly(T) domain.
[1786] In some aspects, the solid support comprises a glass slide.
In some aspects, the glass slide is a treated glass slide.
[1787] Also disclosed herein is a method for spatial analysis of a
biological analyte in a biological sample comprising: (a) preparing
a spatial array by a method as provided herein; (b) decoding the
one or more capture probes by determining the identity of the
spatial barcode of the one or more capture probes, thereby
associating the spatial barcode of the one or more capture probe
with a location on the spatial array; (c) contacting the biological
sample to the spatial array under conditions wherein the biological
analyte binds the cluster; (d) extending the one or more capture
probes via a polymerization reaction using the biological analyte
as a template to generate an extended one or more capture probes
comprising the one or more capture probes and the reverse
complement of the biological analyte; (e) determining the sequence
of the extended one or more capture probes, and associating the
spatial barcode of the one or more capture probes with the captured
biological analyte; thereby determining spatial location of the
biological analyte.
[1788] Also disclosed herein is a spatial array produced by any one
of the methods as provided herein.
[1789] Further, disclosed herein is a spatial array comprising (a)
a solid support; and (b) a plurality of sets of primers immobilized
on the solid support, wherein a first set of the plurality of sets
of primers comprises a first primer and a second primer, wherein
the first primer and the second primer are at a 1:1 ratio; wherein
the first primer and the second primer in each set are less than 10
.mu.m apart; and wherein the first primer comprises a sequence that
is complementary to a capture probe. In some aspects, the spatial
array further comprises a template oligonucleotide comprising a
reverse complement of a unique molecular identifier (UMI), and
wherein the capture probe comprises the UMI.
[1790] In some aspects, the first primer is affixed to the solid
support at a 5' end of the first primer. In some aspects, the first
primer comprises a sequence that is complementary to the template
oligonucleotide. In some aspects, the first primer comprises poly
(A) domain.
[1791] In some aspects, the first primer comprises cleavage domain.
In some aspects, the second primer is affixed to the solid support
at a 5' end of the second primer. In some aspects, the second
primer comprises a cleavage domain.
[1792] In some aspects, the template oligonucleotide is attached to
a bead. In some aspects, the bead is a gel bead. In some aspects,
the bead is 2 microns or less in diameter. In some aspects, the
bead is 1 micron or less in diameter.
[1793] In some aspects, the solid support comprises a glass slide.
In some aspects, the glass slide is a treated glass slide.
[1794] Provided herein are methods of preparing a spatial array. A
"spatial array" as disclosed herein is an array that allows
resolution equal to or less than 10 .mu.m, allowing single-cell
resolution. A spatial array comprises plurality of sets of primers
on the solid support. The sets of primers comprises a sets of a
first primer and a second primer at a 1:1 ratio. The second primer
has a sequence that is complementary to the first primer or to a
template oligonucleotide.
[1795] The methods of preparing a spatial array include providing a
solid support, immobilizing a plurality of sets of primers on the
solid support, providing a template oligonucleotide having a
reverse complement of a spatial barcode domain, a reverse
complement of a capture domain, a first hybridization domain
capable of hybridizing to the first primer, and a second
hybridization domain, wherein a reverse complement of the second
hybridization domain is capable of hybridizing to the second
primer; hybridizing the template oligonucleotide to the first
primer; extending the first primer using the template
oligonucleotide as a template to generate a capture probe
comprising the first primer, the spatial barcode, and the capture
domain, which capture probe is immobilized on the solid support;
and releasing the template oligonucleotide from the spatial
array.
[1796] The spatial array comprises a plurality of sets of primers
affixed to the solid support. The plurality of sets of primers
includes a first primer and a second primer that are at a 1:1 ratio
and that are less than 10 .mu.m apart. The first primer is affixed
to the solid support at the 5' end of the first primer. The second
primer is affixed to the solid support at the 5' end of the second
primer.
[1797] The first primer and/or the second primer can be a
nucleotide. In some aspects, the first primer is single stranded
DNA. In some aspects, the first primer is RNA. In some aspects, the
second primer is single stranded DNA. In some aspects, the second
primer is RNA.
[1798] The first primer on the solid support comprises a sequence
that is complementary to the template oligonucleotide and allows
the template oligonucleotide to bind to the first primer. The first
primer can also comprise a poly (A) or poly (T) domain that is
complementary to a poly(T) or poly(A) domain of the second primer.
The first primer can also comprise a cleavage domain. The second
primer can also comprise a cleavage domain.
[1799] The first primer and the second primer are used to amplify a
template oligonucleotide to create a cluster of one or more capture
probes. In some aspects, the template oligonucleotide includes a
reverse complement of a unique molecular identifier (UMI). Further,
the template oligonucleotide is attached to a bead. In some
aspects, the bead is a gel bead. In some aspects, the bead is less
than 10 .mu.m in diameter. In some aspects, the bead is less than 5
.mu.m in diameter. In some aspects, the bead is 2 .mu.m in
diameter. In some aspects, the bead is less than 2 .mu.m in
diameter. In some aspects, the bead is 1 .mu.m in diameter. In some
aspects, the bead is less than 1 .mu.m in diameter.
[1800] In some aspects, after binding of the template
oligonucleotide to the first primer, the sequence of the primer is
extended. In some aspects, the layer of beads that is attached to
the oligos is removed after extension of the primer. In some
aspects, the layer of beads that is attached to the oligos is
removed before extension of the primer.
[1801] After initial binding of the template oligonucleotide and
the primers, a capture probe is generated. In some aspects, the
capture probe is generated using cluster expansion.
[1802] In some aspects, the capture probe comprises a UMI. In some
aspects, the capture probe includes sequences for first primer, the
spatial barcode, and the capture domain. In some aspects, the
capture probe is immobilized on the solid support. In some aspects,
the capture domain comprises a sequence complementary to an analyte
of interest. In some aspects, the capture domain comprises a
poly(T) domain.
[1803] After a copy of the capture probe is generated, the copy of
the template oligonucleotide is released by cleaving the cleavage
domain after the copy of the capture probe is generated.
[1804] After cluster amplification, the identity of the generated
capture probe is determined. In some aspects, the identity of the
generated capture probe is determined after the template
oligonucleotide is released. In some aspects, the identity of the
generated capture probe is determined in situ. In some aspects, the
identity of the generated capture probe is determined by in situ
sequencing. In situ sequencing is performed via
sequencing-by-synthesis (SBS), sequential fluorescence
hybridization, sequencing by ligation, nucleic acid hybridization,
or high-throughput digital sequencing techniques.
[1805] After the probe is clonally expanded, spatial analysis of a
biological analyte in a biological sample can be performed. In some
aspects, method of performing spatial analysis of a biological
analyte in a biological sample include preparing the spatial array
as discussed above. In some aspects, the methods further include
decoding the one or more capture probes by determining the identity
of the spatial barcode of the one or more capture probes, thereby
associating the spatial barcode of the one or more capture probe
with a location on the spatial array; contacting the biological
sample to the spatial array under conditions wherein the biological
analyte binds the cluster; extending the one or more capture probes
via a polymerization reaction using the biological analyte as a
template to generate an extended one or more capture probes
comprising the one or more capture probes and the reverse
complement of the biological analyte; and determining the sequence
of the extended one or more capture probes, and associating the
spatial barcode of the one or more capture probes with the captured
biological analyte thereby determining spatial location of the
biological analyte.
Example 1--Method for Building an Array for Spatial Synthesis
[1806] In a non-limiting example, capture probes can be generated
using a plurality of oligonucleotides for spatially barcoding
analytes on an array (FIG. 24). The oligonucleotides "A" include a
constant sequence (A-i), and a spatial barcode sequence (A-ii), and
optionally a second constant sequence (A-iii). A primer
complementary to a portion of the oligonucleotide is hybridized
with and extended using the oligonucleotide as a template to
generate a first oligonucleotide. The first oligonucleotide is
further extended to create a 3' end overhang, where an enzyme
(e.g., template independent polymerase) is used to introduce
additional polynucleotides at the end of the first oligonucleotide.
A splint oligonucleotide "C" can be provided that hybridizes the 3'
end (e.g., overhang) of the first oligonucleotide. A second
oligonucleotide is ligated to the 3' end of the first
oligonucleotide to generate a capture probe.
[1807] As shown in FIG. 24 the second oligonucleotide "D" contains
several domains. D-i contains a sequence complementary to a portion
of the splint oligonucleotide, D-ii is a degenerate sequence that
can serve as a unique molecular identifier, and D-iii is a capture
domain that can be used to capture an analyte in a biological
sample. D-iii optionally contains a poly-T sequence. Furthermore,
D-iii can also be used to ligate to the analyte in a biological
sample. The capture probes can be used to spatially locate analytes
according to the methods described herein.
Example 2--Method for Spatial Analysis of a Biological Sample
[1808] In a non-limiting example, the array generated as described
in Example 1 is used for spatial analysis of a biological sample.
The capture domain (D-iii) is used to hybridize with the poly-A
tail of an mRNA molecule SEQ ID NO: 8 (FIG. 25). Reverse
transcription is carried out using a reverse transcriptase enzyme
to generate cDNA complementary to the captured mRNA. The sequence
and location of the captured mRNA are determined by sequencing the
capture probe that contains the spatial barcode as well as the
complementary cDNA.
Example 3--Methods for Preparing High-Density Spatially Barcoded
Arrays
[1809] An array including one or more pluralities of first
oligonucleotides is contacted with a hydrogel including one or more
pluralities of second oligonucleotides. A first oligonucleotide can
have a capture domain, a second oligonucleotide can have a capture
domain, or both first and second oligonucleotides can have a
capture domain. Members of the one or more pluralities of the first
oligonucleotides are attached to members of the one or more
pluralities of second oligonucleotides to form one or more
pluralities of coupled capture probes (e.g., a combinatorial oligo
array is built). The size of the hydrogel containing the one or
more pluralities of coupled capture probes is then reduced by
removing water, which forms a shrunken hydrogel array containing
the one or more pluralities of coupled capture probes (FIG. 26).
The shrunken hydrogel array can be contacted with a biological
sample under conditions sufficient to allow analytes present in the
biological sample to interact with the coupled capture probes of
the shrunken hydrogel array.
Example 4--Method for Building an Array Using a Two-Step Barcode
Construction Process and a Protective Group
[1810] In a non-limiting example, a two-step spatial barcode
construction process is used to build an array with high
complexity. An array that includes a plurality of features
immobilized on a substrate is provided, where each feature includes
a plurality of first oligonucleotides, and each first
oligonucleotide includes a first spatial barcode (FIG. 27). The
first spatial barcode is associated with a particular feature, such
that different features of the array are associated with different
spatial barcodes. The plurality of first oligonucleotides are
attached to a plurality of features or the substrate (e.g., direct
attachment) at one end, and include a photocleavable protective
group at the other end. A subset of the first oligonucleotides are
exposed to a light source and the protective group on the selected
first oligonucleotides are removed, leaving the first
oligonucleotides 3' end available for attaching a second
oligonucleotide. Next, a second oligonucleotide is ligated to the
first oligonucleotide that have had the protective groups removed,
where the second oligonucleotide includes a second spatial barcode,
a unique molecular identifier sequence, and a capture domain (e.g.,
a poly-T sequence). In some cases, the second oligonucleotide also
contains a protective group at the 3' end. The first
oligonucleotide and the second oligonucleotide can be ligated using
a ligase (e.g., App ligase). Alternatively, the first
oligonucleotide and the second oligonucleotide are ligated through
a chemical step (e.g., similar to oligonucleotide synthesis).
Capture probes are thereby generated that contain the first spatial
barcode and the second spatial barcode. Optionally, the protective
group at the 3' end of the capture probes may be removed to
generate a free 3'-OH end.
Example 5--Method for Building an Array by Building Separate
Sub-Arrays
[1811] In a non-limiting example, a two-step spatial barcode
construction process is used to build an array with high
complexity. An array that includes a plurality of sub-arrays is
provided, with each sub-array comprising a plurality of features
immobilized on a substrate, where each feature includes a plurality
of first oligonucleotides, and each first oligonucleotide includes
a first spatial barcode (BCA). The sub-array can include features
wherein each feature is associated with a first spatial barcode
unique to the feature of origin on the sub-array. Therefore, any
one particular feature of the sub-array associated with a first
spatial barcode, can be represented multiple times if the sub-array
or subtemplate is printed at multiple locations (see FIGS. 29 and
30). A plurality of second oligonucleotides are delivered to a
first sub-array, where each second oligonucleotide includes a
second spatial barcode (BCB), a unique molecular identifier
sequence, and a capture domain (e.g. a poly-T sequence). In some
cases, the second oligonucleotides also contain a protective group
at the 3' end. The second oligonucleotides are ligated to the first
oligonucleotides on the first subarray using a ligase, thereby
generating capture probes that include the first spatial barcode
and the second spatial barcode. Alternatively, the first
oligonucleotides and the second oligonucleotides are ligated
through a chemical step (e.g. similar to oligonucleotide
synthesis). Optionally, the protective group at the 3' end of the
capture probes may be removed to generate a 3'-OH end.
[1812] Next, a second plurality of third oligonucleotides may be
delivered to a second subarray, where each third oligonucleotide
includes a third spatial barcode, a unique molecular identifier
sequence, and a second capture domain (e.g., a poly-T sequence). In
some cases, the third oligonucleotides also contain a protective
group at the 3' end. The third oligonucleotides are ligated to a
fourth oligonucleotide containing the first spatial barcode on the
first sub-array using a ligase, thereby generating capture probes
that include the first spatial barcode, the third spatial barcode,
and the second capture domain. Optionally, the protective group at
the 3' end of the capture probes can be removed to generate a 3'-OH
end.
Example 6--Method for Preparing a Slide that Contains a High
Density Array
[1813] In a non-limiting example, the method of building a high
density array as described in Examples 4 and 5 involves building
the array on a substrate (e.g., a slide). A slide is provided that
includes a plurality of identical sub-arrays, where each sub-array
contains a plurality of first oligonucleotides immobilized on the
slide, and each first oligonucleotide includes a first spatial
barcode (BCA). In some cases, there are no gaps between the
sub-arrays.
[1814] In one example, the plurality of first oligonucleotides are
attached to the plurality of features or the substrate (e.g.,
direct attachment) at one end, and include a photocleavable
protective group at the other end. A first sub-array is exposed to
light while the remaining sub-arrays are masked from the light. The
protective group on the first oligonucleotides on the first
sub-array are thereby removed, leaving the first oligonucleotides
available for attaching additional polynucleotides. Second
oligonucleotides that contain a second spatial barcode (BCB) are
attached to first oligonucleotides on the first sub-array that have
had the protective group removed to generate capture probes that
include both the first spatial barcode and second spatial barcode.
The second oligonucleotides further include a unique molecular
identifier and a capture domain (e.g. a poly-T sequence), and
optionally a protective group attached to the 3' end. Next, a
second sub-array is exposed to light while the remaining sub-arrays
are masked from the light, and the above steps are repeated with
third oligonucleotides that contain a third barcode (FIG. 29). Upon
iteratively generating capture probes in all sub-arrays on the
slide, the 3' protective group on the capture probes are
removed.
[1815] In one example, one or more physical barriers are used to
isolate a first subarray and second oligonucleotides that contain a
second spatial barcode are delivered to the isolated first
sub-array using a microfluidic device to generate capture probes
that include both the first spatial barcode and the second spatial
barcode. A second sub-array is then isolated and third
oligonucleotides that contain a third spatial barcode are delivered
to the second sub-array to generate capture probes that include
both the first spatial barcode and the third spatial barcode. Upon
iteratively generating capture probes in all sub-arrays, the
protective group at the 3' end of the capture probes may be removed
to generate a 3'-OH end.
[1816] Fluidic isolation chambers can be used to isolate sub-arrays
on a slide, such that oligonucleotides containing different
barcodes can be applied to different sub-arrays, as shown in FIG.
30. The fluidic isolation chambers contain chamber walls that may
further include a tapered end that is highly hydrophobic and is
designed to minimize footprint and allow for array continuity.
Example 7--Enrichment of a Spatial Barcode
[1817] A sequencing library structure is shown in FIG. 38. A
capture probe including an analyte, at least one spatial barcode, a
region upstream of the at least one spatial barcode, and a region
downstream of the analyte is enriched by the following method. A
PCR reaction is initialized using a primer with complementarity to
the spatial barcode and another primer with complementarity to a
region (e.g., constant region 2) downstream of the analyte. The
region may be a sequencing index or handle which has a known and/or
ubiquitous sequence such that the primer does not enrich certain
sequences over other sequences. A second round of PCR is then run
by introducing another primer with complementarity to the region
upstream of the spatial barcode (e.g., constant region 1). This
region may be a sequencing index or handle which has a known and/or
ubiquitous sequence such that this primer does not enrich certain
sequences over other sequences (FIG. 38). This exemplary method
allows for the enrichment of the desired spatial barcode and
analyte sequence while retaining DNA regions upstream and
downstream of the spatial barcode that may be useful for any
downstream DNA processing.
Example 8--Hierarchical Enrichment
[1818] Spatial enrichment can be performed on different pluralities
of spatial barcodes, wherein the different spatial barcodes
correspond to regions, sub regions, and sub-sub regions. FIG. 37
shows an example wherein one region includes nine sub-regions, in
which each sub-region further includes fifteen sub-sub regions. In
this example, each of the 135 sub-sub regions possesses a spatial
barcode (e.g., sub-sub spatial barcode) unique to that sub-sub
region, specifically including the spatial barcode of one of the
nine subregions (sub spatial barcode), and the spatial barcode of
the region (first common sub spatial barcode).
[1819] An enrichment protocol can be performed using primers
directed to at least one, or more than one, spatial barcode (e.g.,
region spatial barcode, sub spatial barcode, sub-sub spatial
barcode). For example, a library can be created from the analytes
captured by the array. The library can contain constant regions and
spatial barcode regions. Primers can be employed to hybridize to
the regional spatial barcode and amplify members of the library
that contain the region specific spatial barcode. Primers can be
employed to hybridize to regional and sub-regional spatial barcodes
and amplify members of the library that contain region specific and
subregion specific spatial barcodes. Enriching multiple levels
(e.g., region, subregion, sub-sub region) of spatial barcodes can
provide sequencing regions of interest at a lower cost.
Example 9--Spatial Enrichment
[1820] Capture probes with individual spatial barcodes will be
linked to beads and the beads are attached to create a high density
bead array. The capture probes containing the spatial barcodes will
be printed in 16 identical arrays on a slide, wherein each array
contains a plurality of target barcode sequences in a pre-defined
pattern. Fresh, non-fixed mouse brain tissue will be trimmed and
frozen in -40.degree. C. cold isopentane and subsequently will be
mounted for sectioning with a cryostat at 10 .mu.m. A slice of
tissue will be applied onto each capture probe array.
[1821] The SuperScript.TM. III One-step RT-PCR System with
Platinum.RTM.Taq DNA Polymerase from Invitrogen can be used when
performing the cDNA synthesis. For each reaction 25 .mu.l 2.times.
reaction mix (SuperScript.TM. III One-step RT-PCR System with
Platinum.RTM.Taq DNA Polymerase, Invitrogen), 22.5 .mu.l H.sub.2O
and 0.5 .mu.l 100.times.BSA can be mixed and heated to 50.degree.
C. SuperScript III/Platinum Taq enzyme mix can be added to the
reaction mix, 2 .mu.l per reaction, and 50 .mu.l of the 10 reaction
mix can be added to each well on the chip. The chip can be
incubated at 50.degree. C. for 30 min.
[1822] Prior to tissue application, fluorescent marker probes will
be hybridized to spot or beads comprising marker oligonucleotides
printed on the capture probe array. The fluorescent marker probes
can aid in the orientation of the resulting image after tissue
visualization, making it possible to combine the image with the
resulting expression profiles for individual capture probe spatial
barcode sequences obtained after sequencing. To hybridize
fluorescent probes, a hybridization solution containing 4.times.SSC
and 0.1% SDS, 2 .mu.M detection probe (P) will be incubated for 4
min at 50.degree. C. Meanwhile, the high density bead array can be
attached to a ChipClip. The array can be subsequently incubated at
50.degree. C. for 30 min at 300 rpm shake with 50 .mu.L of
hybridization solution per well.
[1823] Capture probes can be released using an enzyme mixture in
TdT buffer or with heated TdT buffer. For example, 38 .mu.l of
cleavage mixture can be placed in a clean 0.2 ml PCR tube. The
mixture can contain: 1.times.TdT buffer (20 mM Tris-acetate (pH
7.9), 50 mM Potassium Acetate and 10 mM Magnesium Acetate), 0.1
.mu.g/.mu.l BSA; 0.1 U/.mu.l USER Enzyme; released cDNA; and
released surface probes. To the PCR tube, 0.5 .mu.l RNase H (5
U/.mu.l, final concentration of 0.06 U/.mu.l), 1 .mu.l TdT (20
U/.mu.l, final concentration of 0.5 U/.mu.l), and 0.5 .mu.l dATPs
(100 mM, final concentration of 1.25 mM), can be added. For dA
tailing, the tube can be incubated in a thermocycler at 37.degree.
C. for 15 min followed by an inactivation of TdT at 70.degree. C.
for 10 min. After dA tailing, a PCR master mix can be prepared. The
mix can contain: 1.times. Faststart HiFi PCR Buffer (pH 8.3) with
1.8 mM MgCl2; 0.2 mM of each dNTP; 0.2 .mu.M of each primer, A
(complementary to the amplification domain of the capture probe)
and B_(dT)24 (complementary to the poly-A tail to be added to the
3' end of the first cDNA strand); and 0.1 U/.mu.l Faststart HiFi
DNA polymerase. 23 .mu.l of PCR Master mix can be placed into nine
clean 0.2 ml PCR tubes. 2 .mu.l of dA tailing mixture can be added
to eight of the tubes, while 2 .mu.l water (RNase/DNase free) can
be added to the last tube (negative control). PCR amplification can
be carried out with the following program: Hot start at 95.degree.
C. for 2 minutes, second strand synthesis at 50.degree. C. for 2
minutes and 72.degree. C. for 3 5 minutes, amplification with 30
PCR cycles at 95.degree. C. for 30 seconds, 65.degree. C. for 1
minutes, 72.degree. C. for 3 minutes, and a final extension at
72.degree. C. for 10 minutes.
[1824] A dsDNA library for Illumina.RTM. sequencing using sample
indexing can be carried out according to manufacturer's
instructions. Sequencing can be carried out on a HiSeq2000
platform. The sequencing data can be converted from FASTQ format to
FASTA format using the FastX toolkit FASTQ-to-FASTA converter. The
sequencing reads can be aligned to the capture probe positional
domain (tag) sequences using Blastn and the reads with hits better
than 1e-6 to one of tag sequences were sorted out to individual
files for each tag sequence respectively. The file of tag sequence
reads can then be aligned using Blastn to the mouse transcriptome,
and hits collected.
[1825] The expression profiles for individual capture probe spatial
barcode sequences can be combined with the spatial information
obtained from the tissue sections through staining. The
transcriptomic data from the cellular compartments of the tissue
section can be analyzed in a directly comparative fashion, with the
availability to distinguish distinct expression features for
different cellular subtypes in a given structural context.
Example 10--Generating Gene Specific Capture Probes
[1826] Capture probes containing various functional sequences and
gene specific or general (e.g., poly(T)) capture domains were
generated with a ligation strategy. FIG. 41 shows an immobilized
oligonucleotide, acceptor oligonucleotide, on a substrate, from 3'
to 5' containing an amino modifier C6 at its 3' end, followed by 12
uracils that can function as a cleavage domain, a sequencing handle
(e.g., R1), a 16 base pair spatial barcode, a 12 base pair UMI, and
a ligation handle (SEQ ID NO: 9). A splint oligonucleotide (SEQ ID
NO: 10) with a sequence complementary to the ligation handle and an
additional sequence (e.g., poly(A) or gene specific sequence)
facilitates the ligation of oligonucleotide and the capture domain
(SEQ ID NO: 11). Ligation of both poly(T) (e.g., mRNA) and a
specific mRNA (e.g., Penk) capture domains to the immobilized
oligonucleotide was completed using a mixture containing 30 .mu.M
Poly dT or capture sequence (e.g., Penk), 30 .mu.M splint
oligonucleotide, and 100 units of ligase. The mixture was placed on
top of the slide for 60 minutes then removed before washing and
adding tissue. All tissues were permeabilized without
pre-permeabilization for a duration of 3, 6, or 9 minutes (FIG.
43). The data show Poly dT was successfully ligated to the
immobilized oligonucleotide and capable of capturing mRNA in a
biased manner. Additionally, a capture domain for the Penk mRNA was
successfully ligated and capable of capturing Penk mRNA. As shown
in the table below, ligating a Poly dT capture domain did not
affect sequencing efficiency. The reads mapped to the transcriptome
for gene specific data are relatively high for a single gene
capture (e.g., Penk), but the ligation strategy and subsequent gene
specific mRNA capture is possible.
TABLE-US-00004 Reads Mapped Reads Mapped Fraction read with
Fraction reads Fraction Fraction Confidently to Confidently to
primer or homopolymer with any polyA Name Transcriptome Genome
sequence sequence UMI counts counts Ligated PolyT(N = .delta.)
83.42% 90.94% 13.58% 4.30% 9.84% 25.80% No Ligate Average (N =
.delta.) 83.32% 92.67% 33.37% 2.52% 10.18% 26.15% Penk Average (N =
.delta.) 23.33% 53.07% 10.78% 0.28% 1.18% 2.78% indicates data
missing or illegible when filed
Example 11--Ligation of Capture Domains to Immobilized
Oligonucleotides
[1827] The following ligation protocol and reagents provide for the
ligation of capture domains to existing arrays. A portion of a
splint oligonucleotide is complementary to the ligation handle of
an acceptor oligonucleotide, acceptor oligonucleotide 2, on the
surface of the array. Another portion of the splint oligonucleotide
is complementary to a second ligation handle sequence present in an
oligonucleotide that also includes the capture domain. The splint
oligonucleotide facilitates the ligation of the capture domain
(e.g., via hybridization to the second ligation handle) to the
immobilized oligonucleotide (e.g., ligation handle of the
immobilized oligonucleotide).
Ligation Protocol:
[1828] Equal amounts of a splint oligonucleotide and a capture
domain are incubated at room temperature overnight. Alternatively,
equal amounts of a splint oligonucleotide and a ligation probe are
mixed and warmed up to 98.degree. C. for 2 minutes, and then cooled
down to 4.degree. C. at a rate of 0.1.degree. C./second.
[1829] Next, 100 .mu.l probe and ligase mix are added to each well,
and incubated at 22.degree. C. for 60 minutes. After 60 minutes,
100 .mu.l of volume is removed and discard. 100 .mu.l of
2.times.SSC buffer that has been heated to 50.degree. C. is added
to each well and incubated for 2 minutes. After 2 minutes, 100
.mu.l of the 2.times.SSC buffer is removed from the well and
discard. This process is repeated 2 times for a total of 3 washes
in 2.times.SSC buffer at 50.degree. C.
[1830] After the 3 washes in 2.times.SSC buffer, 100 .mu.l of room
temperature 0.2.times.SSC buffer is added to each well and
incubated for 1 minute. 100 .mu.l of 0.2.times.SSC buffer is then
removed, followed by addition of 100 .mu.l of 0.1.times.SSC
buffer.
[1831] The slide can be stored at 4.degree. C. until ready for
fluorescent probe hybridization and imaging.
[1832] Ligation Reaction Reagent Mix:
TABLE-US-00005 Stock Desired Volume Concentration Concentration
(.mu.l) Desired final 100 volume H.sub.2O 55 ATP 100 2.5 2.5 5X
Ligation 5X 1X 20 Buffer Ligase 1000 100 10 PolyT + splint 250 30
12 oligonucleotide
[1833] Reagents in the Ligation Reaction Reagent Mix Table:
TABLE-US-00006 10x Part Item Number Origin Composition 5X Ligation
210007 10x Manufactured 35% Glycerol, Buffer (bulk formulation) 250
mM Tris-HCl pH 7.5, 50 mM MgCl.sub.2 Bulk, ATP 210010 10x
Manufactured 100 mM (powder 100 mM (bulk formulation) disodium salt
dissolved in Nuclease-Free Water). pH adjusted to 6.8-7.2 using 10M
NaOH DTT-Free 310280 Enzymatics Buffer: 10 mM Tris- T4 Ligase
(Qiagen) HCl, 50 mM KCl, 0.1 mM EDTA, 50% glycerol, pH 7.5
Sequence Listing
TABLE-US-00007 [1834] Synthetic PURAMATRIX polypeptide sequence SEQ
ID NO: 1 RADARADARADARADA Synthetic EAK16 polypeptide sequence SEQ
ID NO: 2 AEAEAKAKAEAEAKAK Synthetic KLD12 polypeptide sequence SEQ
ID NO: 3 KLDLKLDLKLDL 18s cDNA Probe 1 (P1) SEQ ID NO: 4
GAGGAATTCCCAGTAAGT 18s cDNA Probe 2 (P2) SEQ ID NO: 5
GAGATTGAGCAATAACAG 18s cDNA Probe 3 (P3) SEQ ID NO: 6
GTAGTTCCGACCATAAAC 18s cDNA Probe 4 (P4) SEQ ID NO: 7
GGTGACTCTAGATAACCT Analyte poly(A) tail from a biological sample
SEQ ID NO: 8 AAAAAAAAAAANNNNNNNNNNNNNNNNNNNNNNNN Acceptor
Oligonucleotide SEQ ID NO: 9
UUUUUUUUUUUUCTACACGACTCTTCCGATCTACGTACGTACGTACGTN
NNNNNNNNNNNGCGGCCCG Splint oligonucleotide SEQ ID NO: 10
AAAAAAAAAACGGGCCGC Capture domain poly(T) sequence SEQ ID NO: 11
TTTTTTTTTTTTTTTTTTTTTTTTTTT Acceptor Oligonucleotide 2 SEQ ID NO:
12 TTTTTCTACACGACGCTCTTCCGATCTACGTACGTACGTACGTNNNNNN NNNNNNCGCTACG
Sequence CWU 1
1
12116PRTArtificialSynthetic Trademark of PURAMATRIX polypeptide
sequence 1Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala
Asp Ala1 5 10 15216PRTArtificialSynthetic EAK16 polypeptide
sequence 2Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys
Ala Lys1 5 10 15312PRTArtificialSynthetic KLD12 polypeptide
sequence 3Lys Leu Asp Leu Lys Leu Asp Leu Lys Leu Asp Leu1 5
10418DNAArtificial18s cDNA Probe 1 4gaggaattcc cagtaagt
18518DNAArtificial18S cDNA Probe 2 5gagattgagc aataacag
18618DNAArtificial18s cDNA Probe 3 6gtagttccga ccataaac
18718DNAArtificial18s cDNA Probe 4 7ggtgactcta gataacct
18835RNAUnknownAnalyte poly(A) tail from biological
samplemisc_feature(12)..(35)n is a, c, g, or u 8aaaaaaaaaa
annnnnnnnn nnnnnnnnnn nnnnn 35968DNAArtificialCapture
Probemisc_feature(49)..(60)n is a, c, g, t or u 9uuuuuuuuuu
uuctacacga ctcttccgat ctacgtacgt acgtacgtnn nnnnnnnnnn 60gcggcccg
681018DNAArtificialSplint Oligonucleotide 10aaaaaaaaaa cgggccgc
181127DNAArtificialCapture domain poly(T) sequence 11tttttttttt
tttttttttt ttttttt 271262DNAArtificialCapture probe
2misc_feature(44)..(55)n is a, c, g, or t 12tttttctaca cgacgctctt
ccgatctacg tacgtacgta cgtnnnnnnn nnnnncgcta 60cg 62
* * * * *