U.S. patent application number 16/435393 was filed with the patent office on 2020-01-30 for systems and methods for metabolome analysis.
The applicant listed for this patent is 10X GENOMICS, INC.. Invention is credited to Luigi Jhon Alvarado Martinez.
Application Number | 20200032335 16/435393 |
Document ID | / |
Family ID | 69179086 |
Filed Date | 2020-01-30 |
![](/patent/app/20200032335/US20200032335A1-20200130-D00000.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00001.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00002.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00003.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00004.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00005.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00006.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00007.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00008.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00009.png)
![](/patent/app/20200032335/US20200032335A1-20200130-D00010.png)
View All Diagrams
United States Patent
Application |
20200032335 |
Kind Code |
A1 |
Alvarado Martinez; Luigi
Jhon |
January 30, 2020 |
SYSTEMS AND METHODS FOR METABOLOME ANALYSIS
Abstract
The disclosure provides systems and methods for detecting and
analyzing the metabolome of a cell. Such methods and systems can
include or make use of barcoding of nucleic acid molecules and
their sequencing.
Inventors: |
Alvarado Martinez; Luigi Jhon;
(Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
10X GENOMICS, INC. |
Pleasanton |
CA |
US |
|
|
Family ID: |
69179086 |
Appl. No.: |
16/435393 |
Filed: |
June 7, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16434099 |
Jun 6, 2019 |
|
|
|
16435393 |
|
|
|
|
62756495 |
Nov 6, 2018 |
|
|
|
62711351 |
Jul 27, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G16B 25/10 20190201;
C12Q 1/6806 20130101; C12Q 1/6876 20130101; C12Q 1/6806 20130101;
C12Q 2531/113 20130101; C12Q 2535/122 20130101; C12Q 2563/149
20130101; C12Q 2563/159 20130101; C12Q 2563/179 20130101; C12Q
2565/629 20130101 |
International
Class: |
C12Q 1/6876 20060101
C12Q001/6876 |
Claims
1. A method for processing or analyzing a metabolite from a cell,
comprising: (a) generating a partition comprising (i) said
metabolite, (ii) a bead comprising a plurality of nucleic acid cell
barcode molecules each comprising a cell barcode sequence and a
capture sequence, which cell barcode sequence uniquely corresponds
to said cell, and (iii) a molecular complex that is capable of
coupling to said metabolite, wherein said molecular complex
comprises a molecular complex capture sequence and a molecular
complex identifier sequence, wherein said molecular complex capture
sequence is inaccessible to said capture sequence in the absence of
said metabolite coupled to said molecular complex; (b) in said
partition, providing conditions sufficient for said metabolite to
couple to said molecular complex to (i) render said molecular
complex capture sequence accessible to said capture sequence of a
given nucleic acid cell barcode molecule of said plurality of
nucleic acid cell barcode molecules and (ii) permit said capture
sequence to couple to said molecular complex capture sequence; and
(c) with said capture sequence coupled to said molecular complex
capture sequence, using said given nucleic acid cell barcode
molecule and said molecular complex to synthesize a nucleic acid
molecule comprising (1) a first nucleic acid sequence corresponding
to said molecular complex identifier sequence, and (2) a second
nucleic acid sequence corresponding to said cell barcode sequence,
wherein said first nucleic acid sequence and said second nucleic
acid sequence permit said metabolite to be identified as
corresponding to said cell.
2. The method of claim 1, further comprising sequencing at least a
portion of said nucleic acid molecule or a derivative thereof, to
identify said molecular complex identifier sequence and said cell
barcode sequence, and using said molecular complex identifier
sequence and said cell barcode sequence to identify said metabolite
as originating from said cell.
3. (canceled)
4. The method of claim 1, wherein said molecular complex is a
riboswitch.
5. The method of claim 1, wherein said partition comprises said
cell.
6. The method of claim 5, further comprising, after (a), releasing
said metabolite from said cell.
7. The method of claim 1, further comprising, prior to (c),
releasing said nucleic acid cell barcode molecule from said
bead.
8. The method of claim 7, wherein said nucleic acid cell barcode
molecule is released from said bead upon exposure to a chemical
stimulus in said partition.
9. The method of claim 1, wherein (c) is performed in said
partition.
10. (canceled)
11. The method of claim 1, wherein in (c), said nucleic acid
molecule is synthesized using one or more of a nucleic acid
amplification reaction, a reverse transcription reaction and a
template switching reaction.
12. The method of claim 1, further comprising subjecting said
nucleic acid molecule to one or more additional reactions, wherein
said additional reactions comprise a primer extension reaction and
an addition of one or more functional sequences to said nucleic
acid molecule, wherein said one or more functional sequences are
configured to permit attachment of said nucleic acid molecule or a
derivative thereof to a flow cell of a sequencer.
13. (canceled)
14. (canceled)
15. The method of claim 1, wherein said bead is a gel bead.
16. (canceled)
17. The method of claim 1, wherein said partition comprises an
additional molecular complex that is capable of coupling to an
additional metabolite that is different than said metabolite.
18. The method of claim 17, wherein said bead comprises an
additional plurality of nucleic acid cell barcode molecules each
comprising said cell barcode sequence and an additional capture
sequence capable of coupling to an additional molecular complex
capture sequence of said additional molecular complex when said
additional metabolite is coupled to said additional molecular
complex.
19. The method of claim 18, further comprising: in said partition,
providing conditions sufficient for said additional metabolite to
couple to said additional molecular complex to (iii) render said
additional molecular complex capture sequence accessible to said
additional capture sequence of a given additional nucleic acid cell
barcode molecule of said additional plurality of nucleic acid cell
barcode molecules and (iv) permit said additional capture sequence
to couple to said additional molecular complex capture sequence;
and with said additional capture sequence coupled to said
additional molecular complex capture sequence, using said given
additional nucleic acid cell barcode molecule and said additional
molecular complex to synthesize an additional nucleic acid molecule
comprising (3) a third nucleic acid sequence corresponding to said
additional molecular complex identifier sequence, and (4) a fourth
additional nucleic acid sequence corresponding to said cell barcode
sequence, wherein said third nucleic acid sequence and said fourth
nucleic acid sequence permit said additional metabolite to be
identified as corresponding to the cell.
20. (canceled)
21. The method of claim 1, wherein said partition is a droplet
among a plurality of droplets or a well among a plurality of
wells.
22. (canceled)
23. The method of claim 1, wherein said metabolite is selected from
the group consisting of adenosine triphosphate (ATP), adenosine
diphosphate (ADP), adenosine monophosphate (AMP), guanosine
triphosphate (GTP), guanosine monophosphate (GMP), ribose, glucose,
mannose, glycerol, phosphatidyl choline, phosphoryl choline,
glyceryl phosphoryl choline, phosphatidyl serine, phosphatidyl
ethanolamine, diglyceride, nicotinamide adenine dinucleotide
phosphate, nicotinamide adenine dinucleotide, glycine, glutamine,
aspartic acid, citrate, glycerine, acetone, acetoacetic acid and
lysine.
24. The method of claim 1, wherein each of said plurality of
nucleic acid cell barcode molecules comprises an identifier
sequence separate from said cell barcode sequence, and wherein said
identifier sequence is different for each nucleic acid cell barcode
molecule of said plurality of nucleic acid cell barcode
molecules.
25. The method of claim 1, wherein, in (a), each of said plurality
of nucleic acid cell barcode molecules comprises said cell barcode
sequence, wherein said bead is from a plurality of beads, and
wherein said cell barcode sequence is different from cell barcode
sequences of nucleic acid cell barcode molecules of other beads of
said plurality of beads.
26. The method of claim 1, wherein said bead comprises a plurality
of additional nucleic acid cell barcode molecules each comprising
said cell barcode sequence and a binding sequence, which binding
sequence is capable of coupling to an additional nucleic acid
molecule different from said molecular complex.
27. The method of claim 26, wherein said additional nucleic acid
molecule is selected from the group consisting of a ribonucleic
acid molecule of said cell, a deoxyribonucleic acid molecule of
said cell, and an editing nucleic acid molecule capable of
participating in a gene editing reaction.
28. The method of claim 26, further comprising, prior to (a),
exposing a protein of said cell to an antibody coupled to said
additional nucleic acid molecule, wherein said antibody binds to
said protein.
29. The method of claim 26, wherein said partition comprises said
additional nucleic acid molecule, and said method further
comprises, permitting a given additional nucleic acid cell barcode
molecule of said additional plurality of nucleic acid cell barcode
molecules to bind to said additional nucleic acid molecule via said
binding sequence.
30. The method of claim 29, further comprising, using said
additional nucleic acid molecule and said given additional nucleic
acid cell barcode molecule of said additional plurality of nucleic
acid cell barcode molecules to synthesize a reporter nucleic acid
molecule comprising (3) a third nucleic acid sequence corresponding
to a sequence of said reporter nucleic acid molecule, and (4) a
fourth nucleic acid sequence corresponding to said cell barcode
sequence.
31. The method of claim 30, further comprising sequencing at least
a portion of said reporter nucleic acid molecule or a derivative
thereof, to identify said additional nucleic acid molecule and as
associated with said cell.
Description
CROSS-REFERENCE
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 16/434,099, filed Jun. 6, 2019, which claims
the benefit U.S. Provisional Patent Application No. 62/756,495,
filed Nov. 6, 2018, and U.S. Provisional Patent Application No.
62/711,351, filed Jul. 27, 2018, each of which is entirely
incorporated herein by reference.
BACKGROUND
[0002] A sample may be processed for various purposes, such as
identification of a type of moiety within the sample. The sample
may be a biological sample. Biological samples may be processed,
such as for detection of a disease (e.g., cancer) or identification
of a particular species. There are various approaches for
processing samples, such as polymerase chain reaction (PCR) and
sequencing.
[0003] Biological samples may be processed within various reaction
environments, such as partitions. Partitions may be wells or
droplets. Droplets or wells may be employed to process biological
samples in a manner that enables the biological samples to be
partitioned and processed separately. For example, such droplets
may be fluidically isolated from other droplets, enabling accurate
control of respective environments in the droplets.
[0004] Biological samples in partitions may be subjected to various
processes, such as chemical processes or physical processes.
Samples in partitions may be subjected to heating or cooling, or
chemical reactions, such as to yield species that may be
qualitatively or quantitatively processed.
[0005] Partitioning of cells and/or cellular materials can be
useful for analyzing nucleic acid molecules that are endogenous to
the cell.
SUMMARY
[0006] While partitioning of cells and/or cellular materials may be
useful in the analysis of endogenous nucleic acids, analysis of
other cellular components, such that analysis can be linked to a
particular cell, remains challenging. Such analyses include the
analysis of cellular metabolites. Profiling the array of
metabolites of a cell, the metabolome, can be useful in evaluating
a host of cellular characteristics, including cellular processes,
function and viability. Accordingly, recognized herein is a need
for improved systems and methods for detecting and analyzing
cellular compounds. In particular, systems and methods described
herein are useful in detecting and analyzing the metabolome of a
cell.
[0007] An aspect of the present disclosure provides a method for
processing or analyzing a metabolite from a cell. The method
comprises: (a) generating a partition comprising (i) the
metabolite, (ii) one or more beads (e.g., a single bead, a
plurality of beads) comprising a plurality of nucleic acid cell
barcode molecules each comprising a cell barcode sequence and a
capture sequence, which cell barcode sequence uniquely corresponds
to the cell, and (iii) a molecular complex that is capable of
coupling to the metabolite, where the molecular complex comprises a
molecular complex capture sequence and a molecular complex
identifier sequence, where the molecular complex capture sequence
is inaccessible to the capture sequence in the absence of the
metabolite coupled to the molecular complex; (b) in the partition,
providing conditions sufficient for the metabolite to couple to the
molecular complex to (i) render the molecular complex capture
sequence accessible to the capture sequence of a given nucleic acid
cell barcode molecule of the plurality of nucleic acid cell barcode
molecules and (ii) permit the capture sequence to couple to the
molecular complex capture sequence; and (c) with the capture
sequence coupled to the molecular complex capture sequence, using
the given nucleic acid cell barcode molecule and the molecular
complex to synthesize a nucleic acid molecule comprising (1) a
first nucleic acid sequence corresponding to the molecular complex
identifier sequence, and (2) a second nucleic acid sequence
corresponding to the cell barcode sequence, where the first nucleic
acid sequence and the second nucleic acid sequence permit the
metabolite to be identified as corresponding to the cell. In some
cases, a corresponding sequence may be a complementary
sequence.
[0008] In some embodiments, the method further comprises sequencing
at least a portion of the nucleic acid molecule or a derivative
thereof, to identify the molecular complex identifier sequence and
the cell barcode sequence. In some embodiments, the method further
comprises using the molecular complex identifier sequence and the
cell barcode sequence to identify the metabolite as originating
from the cell. In some embodiments, the molecular complex is a
riboswitch. In some embodiments, the partition comprises the cell.
In some embodiments, the method further comprises, after (a),
releasing the metabolite from the cell.
[0009] In some embodiments, the method further comprises, prior to
(c), releasing the nucleic acid cell barcode molecule from the
bead. In some embodiments, the nucleic acid cell barcode molecule
is released from the bead upon exposure to a chemical stimulus in
the partition. In some embodiments, (c) is performed in the
partition. In some embodiments, the method further comprises
releasing or recovering the nucleic acid molecule or a derivative
thereof from the partition. In some embodiments, in (c), the
nucleic acid molecule is synthesized using one or more of a nucleic
acid amplification reaction, a reverse transcription reaction and a
template switching reaction.
[0010] In some embodiments, the method further comprises subjecting
the nucleic acid molecule to one or more additional reactions. In
some embodiments, the one or more additional reactions comprise a
primer extension reaction. In some embodiments, the one or more
additional reactions comprise addition of one or more functional
sequences to the nucleic acid molecule, where the one or more
functional sequences are configured to permit attachment of the
nucleic acid molecule or a derivative thereof to a flow cell of a
sequencer.
[0011] In some embodiments, the bead is a gel bead. In some
embodiments, while the nucleic acid molecule is synthesized, the
nucleic acid cell barcode molecule is attached to the bead. In some
embodiments, the partition comprises an additional molecular
complex that is capable of coupling to an additional metabolite
that is different than the metabolite. In some embodiments, the
bead comprises an additional plurality of nucleic acid cell barcode
molecules each comprising the cell barcode sequence and an
additional capture sequence capable of coupling to an additional
molecular complex capture sequence of the additional molecular
complex when the additional metabolite is coupled to the additional
molecular complex. In some embodiments, the method further
comprises in the partition, permitting the additional metabolite to
couple to the additional molecular complex to (iii) render the
additional molecular complex capture sequence accessible to the
additional capture sequence of a given additional nucleic acid cell
barcode molecule of the additional plurality of nucleic acid cell
barcode molecules and (iv) permit the additional capture sequence
to couple to the additional molecular complex capture sequence; and
with the additional capture sequence coupled to the additional
molecular complex capture sequence, using the given additional
nucleic acid cell barcode molecule and the additional molecular
complex to synthesize an additional nucleic acid molecule
comprising (3) a third nucleic acid sequence corresponding to the
additional molecular complex identifier sequence, and (4) a fourth
additional nucleic acid sequence corresponding to the cell barcode
sequence, where the third nucleic acid sequence and the fourth
nucleic acid sequence permit the additional metabolite to be
identified with the cell.
[0012] In some embodiments, the plurality of nucleic acid cell
barcode molecules comprises at least 100,000 nucleic acid cell
barcode molecules. In some embodiments, the partition is a droplet
among a plurality of droplets. In some embodiments, the partition
is a well among a plurality of wells. In some embodiments, the
metabolite is selected from the group consisting of adenosine
triphosphate (ATP), adenosine diphosphate (ADP), adenosine
monophosphate (AMP), guanosine triphosphate (GTP), guanosine
monophosphate (GMP), ribose, glucose, mannose, glycerol,
phosphatidyl choline, phosphoryl choline, glyceryl phosphoryl
choline, phosphatidyl serine, phosphatidyl ethanolamine,
diglyceride, nicotinamide adenine dinucleotide phosphate,
nicotinamide adenine dinucleotide, glycine, glutamine, aspartic
acid, citrate, glycerine, acetone, acetoacetic acid and lysine.
[0013] In some embodiments, each of the plurality of nucleic acid
cell barcode molecules comprises an identifier sequence separate
from the cell barcode sequence, and where the identifier sequence
is different for each nucleic acid cell barcode molecule of the
plurality of nucleic acid cell barcode molecules. In some
embodiments, where, in (a), each of the plurality of nucleic acid
cell barcode molecules comprises the cell barcode sequence, where
the bead is from a plurality of beads, and where the cell barcode
sequence is different from cell barcode sequences of nucleic acid
cell barcode molecules of other beads of the plurality of
beads.
[0014] In some embodiments, where the bead comprises a plurality of
additional nucleic acid cell barcode molecules each comprising the
cell barcode sequence and a binding sequence, which binding
sequence is capable of coupling to an additional nucleic acid
molecule different from the molecular complex. In some embodiments,
the additional nucleic acid molecule is selected from the group
consisting of a ribonucleic acid molecule of the cell, a
deoxyribonucleic acid molecule of the cell, and an editing nucleic
acid molecule capable of participating in a gene editing reaction.
In some embodiments, the method further comprises, prior to (a),
exposing a protein of the cell to an antibody coupled to the
additional nucleic acid molecule, where the antibody binds to the
protein. In some embodiments, the partition comprises the
additional nucleic acid molecule, and the method further comprises,
permitting a given additional nucleic acid cell barcode molecule of
the additional plurality of nucleic acid cell barcode molecules to
bind to the additional nucleic acid molecule via the binding
sequence. In some embodiments, the method further comprises using
the additional nucleic acid molecule and the given additional
nucleic acid cell barcode molecule of the additional plurality of
nucleic acid cell barcode molecules to synthesize a reporter
nucleic acid molecule comprising (3) a third nucleic acid sequence
corresponding to a sequence of the additional nucleic acid
molecule, and (4) a fourth nucleic acid sequence corresponding to
the cell barcode sequence. In some embodiments, the method further
comprises sequencing at least a portion of the reporter nucleic
acid molecule or a derivative thereof, to identify the species and
as associated with the cell.
[0015] Another aspect of the present disclosure provides a
non-transitory computer readable medium comprising machine
executable code that, upon execution by one or more computer
processors, implements any of the methods above or elsewhere
herein.
[0016] Another aspect of the present disclosure provides a system
comprising one or more computer processors and computer memory
coupled thereto. The computer memory comprises machine executable
code that, upon execution by the one or more computer processors,
implements any of the methods above or elsewhere herein.
[0017] Additional aspects and advantages of the present disclosure
will become readily apparent to those skilled in this art from the
following detailed description, wherein only illustrative
embodiments of the present disclosure are shown and described. As
will be realized, the present disclosure is capable of other and
different embodiments, and its several details are capable of
modifications in various obvious respects, all without departing
from the disclosure. Accordingly, the drawings and description are
to be regarded as illustrative in nature, and not as
restrictive.
INCORPORATION BY REFERENCE
[0018] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference. To the extent publications and patents
or patent applications 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings (also "Figure" and
"FIG." herein), of which:
[0020] FIG. 1 shows an example of a microfluidic channel structure
for partitioning individual biological particles or analyte
carriers;
[0021] FIG. 2 shows an example of a microfluidic channel structure
for delivering barcode carrying beads to droplets;
[0022] FIG. 3 shows an example of a microfluidic channel structure
for co-partitioning biological particles or analyte carriers and
reagents;
[0023] FIG. 4 shows an example of a microfluidic channel structure
for the controlled partitioning of beads into discrete
droplets;
[0024] FIG. 5 shows an example of a microfluidic channel structure
for increased droplet generation throughput;
[0025] FIG. 6 shows another example of a microfluidic channel
structure for increased droplet generation throughput;
[0026] FIG. 7A shows a cross-section view of another example of a
microfluidic channel structure with a geometric feature for
controlled partitioning. FIG. 7B shows a perspective view of the
channel structure of FIG. 7A;
[0027] FIG. 8 illustrates an example of a barcode carrying
bead;
[0028] FIG. 9 shows a computer system that is programmed or
otherwise configured to implement methods provided herein;
[0029] FIG. 10 schematically illustrates an example molecular
complex, a riboswitch, and its function upon binding of a
metabolite;
[0030] FIG. 11A schematically illustrates an example method for
detecting and analyzing a metabolite of a cell;
[0031] FIG. 11B schematically illustrates an example scheme for
generating barcoded sequencing constructs derived from a molecular
complex;
[0032] FIG. 11C schematically illustrates an example scheme for
adding additional sequences to barcoded sequencing constructs;
and
[0033] FIG. 12 schematically illustrates an example bead comprising
nucleic acid cell barcode molecules that can be used to detect and
analyze a plurality of different types of species.
DETAILED DESCRIPTION
[0034] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
[0035] Where values are described as ranges, it will be understood
that such disclosure 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.
[0036] The terms "a," "an," and "the," as used herein, generally
refers to singular and plural references unless the context clearly
dictates otherwise.
[0037] The term "barcode," as used herein, generally refers to a
label, or identifier, that conveys or is capable of conveying
information about an analyte. A barcode can be part of an analyte.
A barcode can be independent of an analyte. A barcode can be a tag
attached to an analyte (e.g., nucleic acid molecule) or a
combination of the tag in addition to an endogenous characteristic
of the analyte (e.g., size of the analyte or end sequence(s)). A
barcode may be unique. 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 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, during, and/or after sequencing of the sample. Barcodes can
allow for identification and/or quantification of individual
sequencing-reads.
[0038] The term "real time," as used herein, can refer to a
response time of less than about 1 second, a tenth of a second, a
hundredth of a second, a millisecond, or less. The response time
may be greater than 1 second. In some instances, real time can
refer to simultaneous or substantially simultaneous processing,
detection or identification.
[0039] The term "subject," as used herein, generally refers to an
animal, such as a mammal (e.g., human) or avian (e.g., bird), or
other organism, such as a plant. For example, the subject can be a
vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian
or a human. Animals may include, but are not limited to, farm
animals, sport animals, and pets. A subject can be a healthy or
asymptomatic individual, an individual that has or is suspected of
having a disease (e.g., cancer) or a pre-disposition to the
disease, and/or an individual that is in need of therapy or
suspected of needing therapy. A subject can be a patient. A subject
can be a microorganism or microbe (e.g., bacteria, fungi, archaea,
viruses).
[0040] The term "genome," as used herein, generally refers to
genomic information from a subject, which may be, for example, at
least a portion or an entirety of a subject's hereditary
information. A genome can be encoded either in DNA or in RNA. A
genome can comprise coding regions (e.g., that code for proteins)
as well as non-coding regions. A genome can include the sequence of
all chromosomes together in an organism. For example, the human
genome ordinarily has a total of 46 chromosomes. The sequence of
all of these together may constitute a human genome.
[0041] The terms "adaptor(s)", "adapter(s)" and "tag(s)" may be
used synonymously. An adaptor or tag can be coupled to a
polynucleotide sequence to be "tagged" by any approach, including
ligation, hybridization, or other approaches.
[0042] The term "sequencing," as used herein, generally refers to
methods and technologies for determining the sequence of nucleotide
bases in one or more polynucleotides. The 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). Sequencing can be performed by
various systems currently available, such as, without limitation, a
sequencing system by Illumina.RTM., Pacific Biosciences
(PacBio.RTM.), Oxford Nanopore.RTM., or Life Technologies (Ion
Torrent.RTM.). Alternatively or in addition, sequencing may be
performed using nucleic acid amplification, polymerase chain
reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time
PCR), or isothermal amplification. Such systems may provide a
plurality of raw genetic data corresponding to the genetic
information of a subject (e.g., human), as generated by the systems
from a sample provided by the subject. In some examples, such
systems provide sequencing reads (also "reads" herein). A read may
include a string of nucleic acid bases corresponding to a sequence
of a nucleic acid molecule that has been sequenced. In some
situations, systems and methods provided herein may be used with
proteomic information.
[0043] The term "bead," as used herein, generally refers to a
particle. The bead may be a solid or semi-solid particle. The bead
may be a gel bead. The gel bead may include a polymer matrix (e.g.,
matrix formed by polymerization or cross-linking). The polymer
matrix may include one or more polymers (e.g., polymers having
different functional groups or repeat units). Polymers in the
polymer matrix may be randomly arranged, such as in random
copolymers, and/or have ordered structures, such as in block
copolymers. Cross-linking can be via covalent, ionic, or inductive,
interactions, or physical entanglement. The bead may be a
macromolecule. The bead may be formed of nucleic acid molecules
bound together. The bead may be formed via covalent or non-covalent
assembly of molecules (e.g., macromolecules), such as monomers or
polymers. Such polymers or monomers may be natural or synthetic.
Such polymers or monomers may be or include, for example, nucleic
acid molecules (e.g., DNA or RNA). The bead may be formed of a
polymeric material. The bead may be magnetic or non-magnetic. The
bead may be rigid. The bead may be flexible and/or compressible.
The bead may be disruptable or dissolvable. The bead may be a solid
particle (e.g., a metal-based particle including but not limited to
iron oxide, gold or silver) covered with a coating comprising one
or more polymers. Such coating may be disruptable or
dissolvable.
[0044] The term "sample," as used herein, generally refers to a
biological sample of a subject. The biological sample may comprise
any number of macromolecules, for example, cellular macromolecules.
The sample may be a cell sample. The sample may be a cell line or
cell culture sample. The sample can include one or more cells. The
sample can include one or more microbes. The biological sample may
be a nucleic acid sample or protein sample. The biological sample
may also be a carbohydrate sample or a lipid sample. The biological
sample may be derived from another sample. The sample may be a
tissue sample, such as a biopsy, core biopsy, needle aspirate, or
fine needle aspirate. The sample may be a fluid sample, such as a
blood sample, urine sample, or saliva sample. The sample may be a
skin sample. The sample may be a cheek swab. The sample may be a
plasma or serum sample. The sample may be a cell-free or cell free
sample. A cell-free sample may include extracellular
polynucleotides. Extracellular polynucleotides may be isolated from
a bodily sample that may be selected from the group consisting of
blood, plasma, serum, urine, saliva, mucosal excretions, sputum,
stool and tears.
[0045] The terms "biological particle" or "analyte carrier," as
used herein, generally refers to a discrete biological system
derived from a biological sample. The biological particle or
analyte carrier may comprise, or carry therein, an analyte (e.g.,
biological analyte) of interest. In some embodiments, the analyte
carrier is itself the analyte of interest. The biological particle
or analyte carrier may be a macromolecule. The biological particle
or analyte carrier may be a small molecule. The biological particle
or analyte carrier may be a virus. The biological particle or
analyte carrier may be a cell or derivative of a cell. The
biological particle or analyte carrier may be an organelle. The
biological particle or analyte carrier may be a rare cell from a
population of cells. The biological particle or analyte carrier may
be any type of cell, including without limitation prokaryotic
cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or
other animal cell type, mycoplasmas, normal tissue cells, tumor
cells, or any other cell type, whether derived from single cell or
multicellular organisms. The biological or analyte carrier particle
may be a constituent of a cell. The biological particle or analyte
carrier may be or may include DNA, RNA, organelles, proteins, or
any combination thereof. The biological particle or analyte carrier
may be or may include a matrix (e.g., a gel or polymer matrix)
comprising a cell or one or more constituents from a cell (e.g.,
cell bead), such as DNA, RNA, organelles, proteins, or any
combination thereof, from the cell. The biological particle or
analyte carrier may be obtained from a tissue of a subject. The
biological particle or analyte carrier may be a hardened cell. Such
hardened cell may or may not include a cell wall or cell membrane.
The biological particle or analyte carrier may include one or more
constituents of a cell, but may not include other constituents of
the cell. An example of such constituents is a nucleus or an
organelle. A cell may be a live cell. The live cell may be capable
of being cultured, for example, being cultured when enclosed in a
gel or polymer matrix, or cultured when comprising a gel or polymer
matrix. As used herein, the terms "biological particle" and
"analyte carrier" may be used interchangeably.
[0046] The term "macromolecular constituent," as used herein,
generally refers to a macromolecule contained within or from a
biological particle or analyte carrier. The macromolecular
constituent may comprise a nucleic acid. In some cases, the
biological particle or analyte carrier may be a macromolecule. The
macromolecular constituent may comprise DNA. The macromolecular
constituent may comprise RNA. The RNA may be coding or non-coding.
The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) or
transfer RNA (tRNA), for example. The RNA may be a transcript. The
RNA may be small RNA that are less than 200 nucleic acid bases in
length, or large RNA that are greater than 200 nucleic acid bases
in length. Small RNAs may 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 may be double-stranded RNA or single-stranded RNA.
The RNA may be circular RNA. The macromolecular constituent may
comprise a protein. The macromolecular constituent may comprise a
peptide. The macromolecular constituent may comprise a
polypeptide.
[0047] The term "molecular tag," as used herein, generally refers
to a molecule capable of binding to a macromolecular constituent.
The molecular tag may bind to the macromolecular constituent with
high affinity. The molecular tag may bind to the macromolecular
constituent with high specificity. The molecular tag may comprise a
nucleotide sequence. The molecular tag may comprise a nucleic acid
sequence. The nucleic acid sequence may be at least a portion or an
entirety of the molecular tag. The molecular tag may be a nucleic
acid molecule or may be part of a nucleic acid molecule. The
molecular tag may be an oligonucleotide or a polypeptide. The
molecular tag may comprise a DNA aptamer. The molecular tag may be
or comprise a primer. The molecular tag may be, or comprise, a
protein. The molecular tag may comprise a polypeptide. The
molecular tag may be a barcode.
[0048] The term "partition," as used herein, generally, refers to a
space or volume that may be suitable to contain one or more species
or conduct one or more reactions. A partition may be a physical
compartment, such as a droplet or well. The partition may isolate
space or volume from another space or volume. The droplet may be a
first phase (e.g., aqueous phase) in a second phase (e.g., oil)
immiscible with the first phase. The droplet may be a first phase
in a second phase that does not phase separate from the first
phase, such as, for example, a capsule or liposome in an aqueous
phase. A partition may comprise one or more other (inner)
partitions. In some cases, a partition may be a virtual compartment
that can be defined and identified by an index (e.g., indexed
libraries) across multiple and/or remote physical compartments. For
example, a physical compartment may comprise a plurality of virtual
compartments.
Analysis of Cellular Metabolomes
[0049] Provided are systems and methods for analyzing the
metabolome of a cell. A metabolite of a cell can be partitioned
into a partition such that its cellular origin can be identified.
The partition can also include molecular complexes that can
uniquely couple with the metabolite and have nucleic acid sequences
that identify the molecular complex and, thus, the metabolite. Such
sequences can be determined and used to identify the metabolite as
originating from the cell.
[0050] In an aspect, the disclosure provides a method for
processing or analyzing a metabolite from a cell. The method
comprises: (a) generating a partition comprising (i) the
metabolite, (ii) one or more beads (e.g., a single bead, a
plurality of beads) comprising a plurality of nucleic acid cell
barcode molecules each comprising a cell barcode sequence and a
capture sequence, which cell barcode sequence uniquely corresponds
to the cell, and (iii) a molecular complex that is capable of
coupling to the metabolite, where the molecular complex comprises a
molecular complex capture sequence and a molecular complex
identifier sequence, where the molecular complex capture sequence
is inaccessible to the capture sequence in the absence of the
metabolite coupled to the molecular complex; (b) in the partition,
providing conditions sufficient for the metabolite to couple to the
molecular complex to (i) render the molecular complex capture
sequence accessible to the capture sequence of a given nucleic acid
cell barcode molecule of the plurality of nucleic acid cell barcode
molecules and (ii) permit the capture sequence to couple to the
molecular complex capture sequence; and (c) with the capture
sequence coupled to the molecular complex capture sequence, using
the given nucleic acid cell barcode molecule and the molecular
complex to synthesize a nucleic acid molecule comprising (1) a
first nucleic acid sequence corresponding to the molecular complex
identifier sequence, and (2) a second nucleic acid sequence
corresponding to the cell barcode sequence, where the first nucleic
acid sequence and the second nucleic acid sequence permit the
metabolite to be identified with the cell.
[0051] In some cases, the plurality of nucleic acid cell barcode
molecules comprises at least about 1,000 nucleic acid cell barcode
molecules, at least about 5,000 nucleic acid cell barcode
molecules, at least about 10,000 nucleic acid cell barcode
molecules, at least about 50,000 nucleic acid cell barcode
molecules, at least about 100,000 nucleic acid cell barcode
molecules, at least about 500,000 nucleic acid cell barcode
molecules, at least about 1,000,000 nucleic acid cell barcode
molecules, at least about 5,000,000 nucleic acid cell barcode
molecules, at least about 10,000,000 nucleic acid cell barcode
molecules, at least about 100,000,000 nucleic acid cell barcode
molecules, at least about 1,000,000,000 nucleic acid cell barcode
molecules, or more. In some cases, the nucleic acid cell barcode
molecules comprise the same cell barcode sequence.
[0052] Each of the plurality of nucleic acid cell barcode molecules
can include an identifier sequence separate from the cell barcode
sequence, where the identifier sequence is different for each
nucleic acid cell barcode molecule of the plurality of nucleic acid
cell barcode molecules. In some cases, such an identifier sequence
is a unique molecular identification sequence (UMI) as described
elsewhere herein. As described elsewhere herein, UMI sequences can
uniquely identify a particular nucleic acid molecule that is
barcoded, which may be identifying particular nucleic acid
molecules that are analyzed, counting particular nucleic acid
molecules that are analyzed, etc. Furthermore, in some cases,
including (a), each of the plurality of nucleic acid cell barcode
molecules can comprise the cell barcode sequence and the bead can
be from a plurality of beads, such as a population of barcoded
beads as described elsewhere herein. Each of the cell barcode
sequences can be different from cell barcode sequences of nucleic
acid barcode molecules of other beads of the plurality of beads.
Where this is the case, a population of barcoded beads, with each
bead comprising a different cell barcode sequence can be
obtained.
[0053] The method can process or analyze any suitable metabolite
from the cell. Non-limiting examples of such metabolites include
adenosine triphosphate (ATP), adenosine diphosphate (ADP),
adenosine monophosphate (AMP), guanosine triphosphate (GTP),
guanosine monophosphate (GMP), ribose, glucose, mannose, glycerol,
phosphatidyl choline, phosphoryl choline, glyceryl phosphoryl
choline, phosphatidyl serine, phosphatidyl ethanolamine,
diglyceride, nicotinamide adenine dinucleotide phosphate,
nicotinamide adenine dinucleotide, glycine, glutamine, aspartic
acid, citrate, glycerine, acetone, acetoacetic acid and lysine.
[0054] In general, the molecular complex comprises, at least in
part, a nucleic acid component that provides the capture sequence
and molecular complex identifier sequence. In some cases, though,
the molecular complex comprises a plurality of nucleic acid
components, with each of the molecular complex capture sequence and
the molecular complex identifier sequence on different nucleic acid
components of the molecular complex. The molecular complex
identifier sequence can be determined and used to identify the
metabolite and the molecular complex capture sequence can be used
to aid in generating barcoded molecules corresponding to the
molecular complex (e.g., via its molecular complex identifier
sequence).
[0055] An example of a molecular complex is a riboswitch. A
riboswitch can refer to a regulatory segment of a nucleic acid
molecule (e.g., a ribonucleic acid molecule (RNA), a messenger RNA,
etc.) that can bind a species (including small molecules,
metabolites, etc.), resulting in a change in production of proteins
encoded by nucleic acid molecules in the cell. Additional details
regarding riboswitches are provided in Ruff & Strobel, RNA,
2014 November; 20(11): 1775-88 and Butler et al., Chem Biol. 2011
Mar. 25; 18(3): 293-298, which are both herein entirely
incorporated by reference in their entireties for all purposes. A
particular riboswitch may uniquely bind to a particular species
(such as a particular metabolite) and may also have a unique
sequence among other riboswitches. Such a riboswitch identifier
sequence can be determined and used to determine binding of the
particular species. Moreover, when the species binds to its
respective riboswitch, the riboswitch can change its secondary
structure and/or tertiary structure such that one or more sequences
of the riboswitch, inaccessible in a species-free state, become
accessible in a species-bound state. Such a sequence can be used as
a capture sequence that can bind a nucleic acid barcode molecule
and, via one or more reactions (e.g., one or more primer extension
reactions), add a complementary sequence corresponding to the
riboswitch (including the riboswitch identifier sequence) to the
nucleic acid barcode molecule.
[0056] An example riboswitch in species-free and species-bound
conformations is schematically depicted in FIG. 10. As shown,
riboswitch 1001 comprises an inaccessible capture sequence 1002
when it is not bound to its respective species 1003 (e.g.,
metabolite) that binds to the riboswitch. In its inaccessible
conformation, the capture sequence 1002 cannot bind with other
oligonucleotides. When the species 1003 binds to the riboswitch
1001, the capture sequence 1002 becomes accessible. As shown in the
example, the riboswitch also includes a riboswitch identifier
sequence 1004 that is 5' to the accessible capture sequence. A
barcode nucleic acid molecule can bind to the accessible capture
sequence and be extended to add a sequence complementary to the
riboswitch identifier sequence 1004 to the nucleic acid barcode
molecule.
[0057] In some cases, a riboswitch may comprise an aptamer sequence
in addition to a capture sequence. The aptamer sequence can engage
with the capture sequence when not bound to a ligand, and disengage
from the capture sequence when bound to the ligand. The aptamer
sequence can also provide one or more binding sites for a ligand
such as a metabolite. In some examples, the aptamer sequence and
capture sequence are configured in 5' to 3' order (e.g., 5'-aptamer
sequence-capture sequence-3'). In other examples, they are
configured in an opposite configuration (e.g., 5'-capture
sequence-aptamer sequence-3'). Moreover, the aptamer sequence may
comprise the riboswitch's identifier sequence.
[0058] A riboswitch may comprise any suitable number of
nucleotides. For example, a riboswitch may comprise at least about
2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,
375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950, 1000 or more nucleotides. In some examples, a riboswitch
may comprise at most about 1000, 950, 900, 850, 800, 750, 700, 650,
600, 550, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250,
225, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55,
50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less nucleotides.
Moreover, an aptamer sequence of a riboswitch may comprise at least
about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,
350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, 1000 or more nucleotides. In some examples, an
aptamer sequence of riboswitch may comprise at most about 1000,
950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 475, 450, 425,
400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15,
10, 5 or less nucleotides. Furthermore, a capture sequence of a
riboswitch may comprise at least about 2, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,
175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more
nucleotides. In some examples, a capture sequence of riboswitch may
comprise at most about 1000, 950, 900, 850, 800, 750, 700, 650,
600, 550, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250,
225, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55,
50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less nucleotides.
[0059] Additionally, in some cases, the partition comprises the
cell (which can be intact) and, after the partition is generated in
(a), the method can also include releasing the metabolite from the
cell. Release can be performed via any suitable strategy including
cell lysis. In such cases, the partition may comprise a cell lysis
reagent. In some cases, the partition may comprise a cell bead
comprising the cell or at least a portion of its cellular material
and including the metabolite. Cell beads and useful applications of
them are described in U.S. patent application Ser. No. 15/887,947,
which is entirely herein incorporated by reference in its entirety
for all purposes. Where cell beads are used, release of the
metabolite from the cell may be performed prior to the generation
of the partition in (a). In some cases, such as when wells are
implemented in completing the method, release of a metabolite from
a cell may occur prior to partitioning, such that the wells
implemented in partitions contain cells or cellular extracts
comprising the metabolite.
[0060] Prior to synthesizing the nucleic acid molecule, in (c), the
nucleic acid cell barcode molecule can be released from the bead,
such as upon exposure of the bead to a chemical stimulus in the
partition as described elsewhere herein. In some cases, though, the
nucleic acid cell barcode molecule is attached to the bead, such
that the synthesized nucleic acid molecule is coupled to the
bead.
[0061] Moreover, in (c), the nucleic acid molecule can be
synthesized using one or more of nucleic acid amplification
reaction, a reverse transcription and a template switching reaction
as described elsewhere herein. Any suitable type of nucleic acid
amplification can be used for synthesis, including
amplification-based barcoding methods including any described
elsewhere herein and those described in U.S. Patent Publication No.
2014/0378349, U.S. Patent Publication No. 2016/0257984, and U.S.
Patent Publication No. 2015/0376609, which are entirely herein
incorporated by reference for all purposes.
[0062] The method may also include subjecting the nucleic acid
molecule to one or more additional reactions, which may take place
in the partition and/or which may take place once the nucleic acid
molecule or a derivative of the nucleic acid molecule (e.g., a
subsequent nucleic acid molecule that includes at least a portion
of the nucleic acid molecule or its complement, etc.) is released
or recovered from the partition. Accordingly, the method may
further comprise releasing or recovering the nucleic acid molecule
from the partition, such as where synthesis of the nucleic acid
molecule is performed as in (c), in the partition. Where the
partition is a droplet among a plurality of droplets (e.g. droplets
of an emulsion as described elsewhere herein), removal or recovery
of the nucleic acid molecule or derivative thereof can be achieved
by droplet disruption, such as the breaking of an emulsion in which
the droplet is situated. Where the partition is a well amount a
plurality of wells, the nucleic acid molecule or derivative thereof
can be removed from the well, such as with standard fluid transport
techniques, like pipetting or other forced fluid flow.
[0063] In some cases, the one or more additional reactions may
comprise a primer extension reaction or a plurality of primer
extension reactions. Such reactions include nucleic acid
amplification reactions, like polymerase chain reaction (PCR).
Primer extension reactions can be used to add additional sequences
to the nucleic acid molecule or derivative thereof via priming of
primer binding sites on the nucleic acid molecule or derivative
thereof and further extension of the nucleic acid molecule, using
the primer and/or a sequence associated with the primer as a
template. Primer extension reactions can also be employed to
increase the number of copies of nucleic acid molecule or
derivative thereof. Any number of primer extension reactions can be
implemented depending on the particular construct to be used for
analysis.
[0064] In some cases, the one or more additional reactions may
comprise a ligation reaction (or a ligation reaction in combination
with one or more primer extension reactions). Via the action of a
ligase, additional sequences can be appended onto the nucleic
molecule or derivative thereof.
[0065] Furthermore, the one or more additional reactions can add
one or more functional sequences to the nucleic acid molecules,
which can render the nucleic acid molecule or derivative thereof
suitable for nucleic acid sequencer and sequencing analysis. For
example, the one or more functional sequences may be configured to
permit hybridization of a sequencing primer to nucleic acid
molecule or derivative thereof, may be configured as a sample
index, or may be configured to permit attachment of the nucleic
acid molecule or derivative thereof to a flow cell of sequencer
(e.g., an Illumina sequencer or other type of sequencer in which
oligonucleotides for sequencing are immobilized).
[0066] Moreover, the method can further comprise sequencing at
least a portion of the nucleic acid molecule or a derivative
thereof, to identify the molecular complex identifier sequence and
the cell barcode sequence. The molecular complex identifier
sequence can be determined and used to back-determine the
particular metabolite bound to the molecular complex. As bound
molecular complexes are those that can interact with nucleic acid
cell barcode molecules, the molecular complex identifier sequence
can uniquely identify particular metabolites present in the
partition (and, thus, the underlying cell). Additionally, the cell
barcode sequence can be used to identify a particular partition
(and thus its contents, which include cellular material from a
particular cell) and, thus, the underlying cell from which the
metabolite is obtained. Sequencing may be performed using any
suitable platform, including next-generation sequencing platforms,
such as Illumina.
[0067] An example method for processing or analyzing a metabolite
from a cell is schematically depicted in FIGS. 11A-C. With
reference to FIG. 11A, a barcoded bead 1101, among a plurality of
barcoded beads 1107, comprises a plurality of nucleic acid cell
barcode molecules 1102. Each of the nucleic acid cell barcode
molecules 1102 comprises a functional sequence 1103 that permits
attachment of the nucleic acid cell barcode molecules 1102 to the
flow cell (or other component) of a sequencer, a cell barcode
sequence 1104 and a functional sequence 1105 that permits binding
of a sequencing primer to the nucleic acid cell barcode molecules
1102. The nucleic acid cell barcode molecules also comprise a
capture sequence 1106 that hybridizes with a molecular complex
capture sequence. While not shown in FIG. 11, the nucleic acid cell
barcode molecules may also comprise an identifier sequence, such as
a UMI.
[0068] The barcoded bead 1101 is provided in an aqueous mixture to
a double-junction microfluidic device where the beads are mixed at
a first junction of the microfluidic device with an aqueous stream
1109 comprising molecular complex 1108 that can bind to a
particular metabolite, reagents necessary for barcoding, a cell
1111, a cell lysis reagent(s) and a reagent(s) that are capable of
releasing the nucleic acid cell barcode molecules 1102 from the
bead. The resulting mixture is then passed to a second junction of
the microfluidic device where it is contacted with an immiscible
stream (e.g., a stream comprising an oil, such as a fluorinated oil
and optionally a surfactant), such that a droplet is generated. The
droplet comprises the molecular complex 1108, the barcoded bead
1101, the cell 1110, the cell lysis reagents and the reagents
necessary for barcoding. The cell lysis reagents lyse 1112 the cell
1110 such that the contents, including a metabolite, of the cell
1110 are released to the droplet.
[0069] The metabolite 1113 released from the cell 1110 then binds
with the molecular complex 1108, which changes the conformation of
the molecular complex as in FIG. 10, and renders its molecular
complex capture sequence accessible to the capture sequence 1106 of
the nucleic acid cell barcode molecules 1102. The nucleic acid cell
barcode molecules 1102 are released from the barcoded bead with the
aid of respective release reagent(s) (e.g., such as a reducing
agent that degrades disulfide bonds of the bead, breaks disulfide
bonds between the bead and the nucleic acid cell barcode molecules
1102, etc.). A given nucleic acid cell barcode molecule of the
released nucleic acid cell barcode molecules 1102 bind with the
molecular complex 1108 for barcoding of the molecular complex
identifier sequence of the molecular complex 1108.
[0070] Barcoding of the molecular complex identifier sequence adds
a sequence complementary to the given nucleic acid cell barcode
molecule, as schematically depicted in FIG. 11B. With reference to
FIG. 11B, the given nucleic acid cell barcode molecule binds via
its capture sequence 1106 to the molecular complex capture sequence
1114. The given nucleic acid cell barcode molecule is extended via
the action of a polymerase to add a sequence complementary to a
sequence of the molecular complex identifier sequence 1115 of the
molecular complex 1108. After extension, a terminal transferase
adds three cytosine nucleotides to the extended nucleic acid
molecule, which allows the extended nucleic acid molecule to
participate in a template switch reaction. A template switch oligo
1116, comprising a functional sequence (e.g., a sequencing primer
binding site), then hybridizes with the terminal cytosine
nucleotides and the extended nucleic acid molecule is then further
extended. The resulting product can then be released from the
droplet (e.g., breaking of an emulsion comprising the droplet) and
further amplified to generate a double-stranded product 1117.
[0071] Additional sequences (e.g., additional functional sequences,
sample index sequences, etc.) can be added to the double-stranded
nucleic acid product 1117. In some cases, such sequences can be
added via additional rounds of primer extension reactions and/or
amplification, via binding of templates that comprise the
additional sequences. An example is shown schematically in FIG.
11C. With reference to FIG. 11C, nucleic acid molecule 1118 can add
a first sequence to a strand of double-stranded product 1117 via
hybridization and extension/amplification. This added first
sequence can then hybridize with another nucleic acid molecule 1119
to add a second sequence via hybridization and
extension/amplification. Any number of additional sequences can be
added. Likewise, nucleic acid molecule 1120 can be used to add
additional sequences to the other end of the product 1117 via
hybridization and extension. The completed product 1121 can then be
isolated and sequenced for analysis. Sequencing analysis can
determine the cell barcode sequence and molecular complex
identifier sequence and, thus, identify the metabolite and as
originating from the cell.
[0072] While not shown in FIG. 11C, other schemes exist for adding
additional sequences, including, for example, ligation. For
example, any of nucleic acid molecules 1118, 1119 and 1120 can be
added to a strand of product 1117 via ligation. In some cases,
shearing of product 1117 may be first completed, prior to
ligation.
[0073] Also, while the example shown in FIGS. 11A and 11B
implements a droplet as a partition, the method shown can be
analogously performed in another type of partition such as a well.
In such cases, the cell, barcoded bead and all other reagents can
be provided to the well for cell lysis and barcoding and the
contents removed from wells and processed in bulk after barcoding
to add further sequences to barcoded products and/or amplify the
products as shown in FIG. 11C.
[0074] The method can also be used to process or analyze a
plurality of different metabolites. Determination of different
metabolites can be used to elucidate the cell's metabolome.
Accordingly, the partition can include an additional molecular
complex that is capable of coupling to an additional metabolite
that is different than the metabolite. In some cases, the bead
comprises an additional plurality of nucleic acid cell barcode
molecules that each comprises the cell barcode sequence and an
additional capture sequence capable of coupling to an additional
molecular complex capture sequence of the additional molecular
complex when the additional metabolite is coupled to the additional
molecular complex. In some cases, the additional capture sequence
is the same sequence as the capture sequence. In other cases, the
additional capture is a different capture sequence.
[0075] Where an additional molecular complex that binds with the
additional metabolite is implemented, the method can further
comprise permitting the additional metabolite to couple to the
additional molecular complex to (iii) render the additional
molecular complex capture sequence accessible to the additional
capture sequence of a given additional nucleic acid cell barcode
molecule of the additional plurality of nucleic acid cell barcode
molecules and (iv) permit the additional capture sequence to couple
to the additional molecular complex capture sequence. With the
additional capture sequence coupled to the additional molecular
complex capture sequence, the given additional nucleic acid cell
barcode molecule and the additional molecular complex to can be
used to synthesize an additional nucleic acid molecule comprising
(3) a third nucleic acid sequence corresponding to the additional
molecular complex identifier sequence, and (4) a fourth additional
nucleic acid sequence corresponding to the cell barcode sequence.
The third nucleic acid sequence and the fourth nucleic acid
sequence can permit the additional metabolite to be identified with
the cell.
[0076] As an example, the example method shown in FIGS. 11A-C can
be modified such that a library of different molecular complexes is
provided to each droplet. Each synthesized nucleic acid molecule
would comprise the cell barcode sequence and a sequence
corresponding to a particular molecular complex where its
corresponding metabolite was present in the cell. Identification of
the molecular complex identifier sequences of the various molecular
complexes can then be used to identify an array of metabolites
present in the cell. The resulting analysis can then be used to
construct a metabolome of the cell.
[0077] Metabolite processing and analysis can be combined with
other analyses in the partition, with non-limiting examples that
include transcript analysis, cell surface feature analysis, cell
protein analysis and analysis of cellular gene-editing processes
and associated nucleic acid molecules. Suitable methods for
multi-analyte analysis in a partition are described in U.S. patent
application Ser. No. 15/720,085 and PCT International Application
No. PCT/US2017/068320, each of which is herein incorporated by
reference in its entirety for all purposes.
[0078] In multi-analyte analysis, a barcoded bead used for analysis
can comprise barcode molecules that can capture different analytes.
Accordingly, the bead may comprise a plurality of additional
nucleic acid cell barcode molecules each comprising the cell
barcode sequence and a binding sequence, which binding sequence is
capable of coupling to an additional nucleic acid molecule
different from the molecular complex (e.g., a ribonucleic acid
molecule of the cell (such as messenger ribonucleic acid (mRNA), a
deoxyribonucleic acid (such as genomic deoxyribonucleic acid
(gDNA)), and an editing nucleic acid molecule capable of
participating in a gene editing reaction). The partition can
comprise an additional nucleic acid molecule, and the method can
further comprising permitting a given additional nucleic acid cell
barcode molecule of the additional plurality of nucleic acid
barcode molecules to bind to the additional nucleic acid molecule
via the binding sequence. For example, the given additional nucleic
acid cell barcode molecule may bind to a nucleic acid molecule
coupled to an agent (e.g., an antibody) that binds a cellular
protein, whether on the cell surface or inside the cell. In such
cases, prior to partitioning, the protein can be exposed to an
antibody coupled to the additional nucleic acid molecule, where the
antibody binds to the protein.
[0079] Moreover, the additional nucleic acid molecule and the given
additional nucleic acid cell barcode molecule of the additional
plurality of nucleic acid cell barcode molecules can be used to
synthesize a reporter nucleic acid molecule comprising (3) a third
nucleic acid sequence corresponding to a sequence of the additional
nucleic acid molecule, and (4) a fourth nucleic acid sequence
corresponding to the cell barcode sequence. At least a portion of
the reporter nucleic acid molecule or a derivative thereof can be
sequenced to identify the additional nucleic acid molecule as
originating from the cell. Where the additional nucleic acid
molecule is coupled to an agent (e.g., antibody) that binds a
cellular protein, barcoded nucleic acid molecules can then be
generated from the additional nucleic acid molecule, sequenced and
the antibody and its target protein identified as originating from
the particular cell.
[0080] An example barcoded bead that can be provided to a partition
for multi-analyte analysis is schematically depicted in FIG. 12.
With reference to FIG. 12, the barcoded bead 1201 comprises two
nucleic acid cell barcode molecules 1202 and 1203. Nucleic acid
cell barcode molecule 1202 comprises a functional sequence 1204
that permits attachment of nucleic acid cell barcode molecule 1202
to a sequencer component; a cell barcode sequence 1205, a UMI
sequence 1206, a functional sequence that can bind a sequencing
primer 1207 and a capture sequence 1208. The capture sequence 1208
can bind a particular cellular analyte, such as mRNA, gDNA, or a
nucleic acid molecule coupled to an agent that binds a cellular
protein, etc. Barcoded molecules can be generated in a partition
and sequenced (or derivatives sequenced) to identify target
molecules as originating from the underlying cell from which
cellular material is provided with the barcoded bead 1201 in a
partition.
[0081] Additionally, nucleic acid cell barcode molecule 1203
comprises sequences 1204, 1205 and 1207 as in nucleic acid cell
barcode molecule and also comprises a capture sequence 1209. In
some cases, the capture sequence 1209 is the same sequence as
capture sequence 1208. In other cases, the capture sequence 1209 is
a different sequence as the capture sequence 1208. Capture sequence
1209 can couple with a molecular complex having an accessible
binding sequence (e.g., as a result of metabolite binding) and
processing/analysis of a metabolite performed as described
elsewhere herein.
Systems and Methods for Sample Compartmentalization
[0082] In an aspect, the systems and methods described herein
provide for the compartmentalization, depositing, or partitioning
of one or more particles (e.g., biological particles or analyte
carriers, macromolecular constituents of biological particles or
analyte carriers, beads, reagents, etc.) into discrete compartments
or partitions (referred to interchangeably herein as partitions),
where each partition maintains separation of its own contents from
the contents of other partitions. The partition can be a droplet in
an emulsion. A partition may comprise one or more other
partitions.
[0083] A partition may include one or more particles. A partition
may include one or more types of particles. For example, a
partition of the present disclosure may comprise one or more
biological particles or analyte carriers and/or macromolecular
constituents thereof. A partition may comprise one or more gel
beads. A partition may comprise one or more cell beads. A partition
may include a single gel bead, a cell bead, or both a cell bead and
single gel bead. A partition may include one or more reagents.
Alternatively, a partition may be unoccupied. For example, a
partition may not comprise a bead. A cell bead can be a biological
particle or analyte carrier and/or one or more of its
macromolecular constituents encased inside of a gel or polymer
matrix, such as via polymerization of a droplet containing the
biological particle or analyte carrier and precursors capable of
being polymerized or gelled. Unique identifiers, such as barcodes,
may be injected into the droplets previous to, subsequent to, or
concurrently with droplet generation, such as via a microcapsule
(e.g., bead), as described elsewhere herein. Microfluidic channel
networks (e.g., on a chip) can be utilized to generate partitions
as described herein. Alternative mechanisms may also be employed in
the partitioning of individual biological particles or analyte
carriers, including porous membranes through which aqueous mixtures
of cells are extruded into non-aqueous fluids.
[0084] The partitions can be flowable within fluid streams. The
partitions may comprise, for example, micro-vesicles that have an
outer barrier surrounding an inner fluid center or core. In some
cases, the partitions may comprise 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 may 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, which is entirely
incorporated herein by reference for all purposes. Emulsion systems
for creating stable droplets in non-aqueous or oil continuous
phases are described in, for example, U.S. Patent Application
Publication No. 2010/0105112, which is entirely incorporated herein
by reference for all purposes.
[0085] In the case of droplets in an emulsion, allocating
individual particles to discrete partitions may in one non-limiting
example be accomplished 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
may be adjusted to control the occupancy of the resulting
partitions (e.g., number of biological particles or analyte
carriers 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
particles. To generate single biological particle or analyte
carrier partitions, the relative flow rates of the immiscible
fluids can be selected such that, on average, the partitions may
contain less than one biological particle or analyte carrier per
partition in order to ensure that those partitions that are
occupied are primarily singly occupied. In some cases, partitions
among a plurality of partitions may contain at most one biological
particle or analyte carrier (e.g., bead, DNA, cell or cellular
material). In some embodiments, the various parameters (e.g., fluid
properties, particle properties, microfluidic architectures, etc.)
may 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.
[0086] FIG. 1 shows an example of a microfluidic channel structure
100 for partitioning individual biological particles or analyte
carriers. The channel structure 100 can include channel segments
102, 104, 106 and 108 communicating at a channel junction 110. In
operation, a first aqueous fluid 112 that includes suspended
biological particles or analyte carriers (or cells) 114 may be
transported along channel segment 102 into junction 110, while a
second fluid 116 that is immiscible with the aqueous fluid 112 is
delivered to the junction 110 from each of channel segments 104 and
106 to create discrete droplets 118, 120 of the first aqueous fluid
112 flowing into channel segment 108, and flowing away from
junction 110. The channel segment 108 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 or analyte carrier 114 (such as
droplets 118). A discrete droplet generated may include more than
one individual biological particle or analyte carrier 114 (not
shown in FIG. 1). A discrete droplet may contain no biological
particle or analyte carrier 114 (such as droplet 120). Each
discrete partition may maintain separation of its own contents
(e.g., individual biological particle or analyte carrier 114) from
the contents of other partitions.
[0087] The second fluid 116 can comprise 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 118, 120. Examples of
particularly useful partitioning fluids and fluorosurfactants are
described, for example, in U.S. Patent Application Publication No.
2010/0105112, which is entirely incorporated herein by reference
for all purposes.
[0088] As will be appreciated, the channel segments described
herein may 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 100 may have other
geometries. For example, a microfluidic channel structure can have
more than one channel junction. For example, a microfluidic channel
structure can have 2, 3, 4, or 5 channel segments each carrying
particles (e.g., biological particles or analyte carriers, cell
beads, and/or gel beads) that meet at a channel junction. Fluid may
be directed to flow along one or more channels or reservoirs via
one or more fluid flow units. A fluid flow unit can comprise
compressors (e.g., providing positive pressure), pumps (e.g.,
providing negative pressure), actuators, and the like to control
flow of the fluid. Fluid may also or otherwise be controlled via
applied pressure differentials, centrifugal force, electrokinetic
pumping, vacuum, capillary or gravity flow, or the like.
[0089] The generated droplets may comprise two subsets of droplets:
(1) occupied droplets 118, containing one or more biological
particles or analyte carriers 114, and (2) unoccupied droplets 120,
not containing any biological particles or analyte carriers 114.
Occupied droplets 118 may comprise singly occupied droplets (having
one biological particle or analyte carrier) and multiply occupied
droplets (having more than one biological particle or analyte
carrier). As described elsewhere herein, in some cases, the
majority of occupied partitions can include no more than one
biological particle or analyte carrier per occupied partition and
some of the generated partitions can be unoccupied (of any
biological particle or analyte carrier). In some cases, though,
some of the occupied partitions may include more than one
biological particle or analyte carrier. In some cases, the
partitioning process may be controlled such that fewer than about
25% of the occupied partitions contain more than one biological
particle or analyte carrier, and in many cases, fewer than about
20% of the occupied partitions have more than one biological
particle or analyte carrier, while in some cases, fewer than about
10% or even fewer than about 5% of the occupied partitions include
more than one biological particle or analyte carrier per
partition.
[0090] In some cases, it may be useful to minimize the creation of
excessive numbers of empty partitions, such as to reduce costs
and/or increase efficiency. While this minimization may be achieved
by providing a sufficient number of biological particles or analyte
carriers (e.g., biological particles or analyte carriers 114) at
the partitioning junction 110, such as to ensure that at least one
biological particle or analyte carrier is encapsulated in a
partition, the Poissonian distribution may expectedly increase the
number of partitions that include multiple biological particles or
analyte carriers. As such, where singly occupied partitions are to
be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the
generated partitions can be unoccupied.
[0091] In some cases, the flow of one or more of the biological
particles or analyte carriers (e.g., in channel segment 102), or
other fluids directed into the partitioning junction (e.g., in
channel segments 104, 106) can be controlled such that, in many
cases, no more than about 50% of the generated partitions, no more
than about 25% of the generated partitions, or no more than about
10% of the generated partitions are unoccupied. These flows can be
controlled so as to present a non-Poissonian distribution of
single-occupied partitions while providing lower levels of
unoccupied partitions. The above noted ranges of unoccupied
partitions can be achieved while still providing any of the single
occupancy rates described above. For example, in many cases, the
use of the systems and methods described herein can create
resulting partitions that have multiple occupancy rates of less
than about 25%, less than about 20%, less than about 15%, less than
about 10%, and in many cases, less than about 5%, while having
unoccupied partitions of less than about 50%, less than about 40%,
less than about 30%, less than about 20%, less than about 10%, less
than about 5%, or less.
[0092] As will be appreciated, the above-described occupancy rates
are also applicable to partitions that include both biological
particles or analyte carriers and additional reagents, including,
but not limited to, microcapsules or beads (e.g., gel beads)
carrying barcoded nucleic acid molecules (e.g., oligonucleotides)
(described in relation to FIG. 2). The occupied partitions (e.g.,
at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
99% of the occupied partitions) can include both a microcapsule
(e.g., bead) comprising barcoded nucleic acid molecules and a
biological particle or analyte carrier.
[0093] In another aspect, in addition to or as an alternative to
droplet based partitioning, biological particles or analyte
carriers may be encapsulated within a microcapsule that comprises
an outer shell, layer or porous matrix in which is entrained one or
more individual biological particles or analyte carriers or small
groups of biological particles or analyte carriers. The
microcapsule may include other reagents. Encapsulation of
biological particles or analyte carriers may be performed by a
variety of processes. Such processes may combine an aqueous fluid
containing the biological particles or analyte carriers with a
polymeric precursor material that may be capable of being formed
into a gel or other solid or semi-solid matrix upon application of
a particular stimulus to the polymer precursor. Such stimuli can
include, for example, thermal stimuli (e.g., either heating or
cooling), photo-stimuli (e.g., through photo-curing), chemical
stimuli (e.g., through crosslinking, polymerization initiation of
the precursor (e.g., through added initiators)), mechanical
stimuli, or a combination thereof.
[0094] Preparation of microcapsules comprising biological particles
or analyte carriers may be performed by a variety of methods. For
example, air knife droplet or aerosol generators may be used to
dispense droplets of precursor fluids into gelling solutions in
order to form microcapsules that include individual biological
particles or analyte carriers or small groups of biological
particles or analyte carriers. Likewise, membrane based
encapsulation systems may be used to generate microcapsules
comprising encapsulated biological particles or analyte carriers as
described herein. Microfluidic systems of the present disclosure,
such as that shown in FIG. 1, may be readily used in encapsulating
cells as described herein. In particular, and with reference to
FIG. 1, the aqueous fluid 112 comprising (i) the biological
particles or analyte carriers 114 and (ii) the polymer precursor
material (not shown) is flowed into channel junction 110, where it
is partitioned into droplets 118, 120 through the flow of
non-aqueous fluid 116. In the case of encapsulation methods,
non-aqueous fluid 116 may also include an initiator (not shown) to
cause polymerization and/or crosslinking of the polymer precursor
to form the microcapsule that includes the entrained biological
particles or analyte carriers. Examples of polymer
precursor/initiator pairs include those described in U.S. Patent
Application Publication No. 2014/0378345, which is entirely
incorporated herein by reference for all purposes.
[0095] For example, in the case where the polymer precursor
material comprises a linear polymer material, such as a linear
polyacrylamide, PEG, or other linear polymeric material, the
activation agent may comprise a cross-linking agent, or a chemical
that activates a cross-linking agent within the formed droplets.
Likewise, for polymer precursors that comprise polymerizable
monomers, the activation agent may comprise a polymerization
initiator. For example, in certain cases, where the polymer
precursor comprises a mixture of acrylamide monomer with a
N,N'-bis-(acryloyl)cystamine (BAC) comonomer, an agent such as
tetraethylmethylenediamine (TEMED) may be provided within the
second fluid streams 116 in channel segments 104 and 106, which can
initiate the copolymerization of the acrylamide and BAC into a
cross-linked polymer network, or hydrogel.
[0096] Upon contact of the second fluid stream 116 with the first
fluid stream 112 at junction 110, during formation of droplets, the
TEMED may diffuse from the second fluid 116 into the aqueous fluid
112 comprising the linear polyacrylamide, which will activate the
crosslinking of the polyacrylamide within the droplets 118, 120,
resulting in the formation of gel (e.g., hydrogel) microcapsules,
as solid or semi-solid beads or particles entraining the cells 114.
Although described in terms of polyacrylamide encapsulation, other
`activatable` encapsulation compositions may also be employed in
the context of the methods and compositions described herein. For
example, formation of alginate droplets followed by exposure to
divalent metal ions (e.g., Ca.sup.2+ ions), can be used as an
encapsulation process using the described processes. Likewise,
agarose droplets may also be transformed into capsules through
temperature based gelling (e.g., upon cooling, etc.).
[0097] In some cases, encapsulated biological particles or analyte
carriers can be selectively releasable from the microcapsule, such
as through passage of time or upon application of a particular
stimulus, that degrades the microcapsule sufficiently to allow the
biological particles or analyte carriers (e.g., cell), or its other
contents to be released from the microcapsule, such as into a
partition (e.g., droplet). For example, in the case of the
polyacrylamide polymer described above, degradation of the
microcapsule may be accomplished through the introduction of an
appropriate reducing agent, such as DTT or the like, to cleave
disulfide bonds that cross-link the polymer matrix. See, for
example, U.S. Patent Application Publication No. 2014/0378345,
which is entirely incorporated herein by reference for all
purposes.
[0098] The biological particle or analyte carrier can be subjected
to other conditions sufficient to polymerize or gel the precursors.
The conditions sufficient to polymerize or gel the precursors may
comprise exposure to heating, cooling, electromagnetic radiation,
and/or light. The conditions sufficient to polymerize or gel the
precursors may comprise any conditions sufficient to polymerize or
gel the precursors. Following polymerization or gelling, a polymer
or gel may be formed around the biological particle or analyte
carrier. The polymer or gel may be diffusively permeable to
chemical or biochemical reagents. The polymer or gel may be
diffusively impermeable to macromolecular constituents of the
biological particle or analyte carrier. In this manner, the polymer
or gel may act to allow the biological particle or analyte carrier
to be subjected to chemical or biochemical operations while
spatially confining the macromolecular constituents to a region of
the droplet defined by the polymer or gel. The polymer or gel may
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 may comprise any other
polymer or gel.
[0099] The polymer or gel may be functionalized to bind to targeted
analytes, such as nucleic acids, proteins, carbohydrates, lipids or
other analytes. The polymer or gel may be polymerized or gelled via
a passive mechanism. The polymer or gel may be stable in alkaline
conditions or at elevated temperature. The polymer or gel may have
mechanical properties similar to the mechanical properties of the
bead. For instance, the polymer or gel may be of a similar size to
the bead. The polymer or gel may have a mechanical strength (e.g.
tensile strength) similar to that of the bead. The polymer or gel
may be of a lower density than an oil. The polymer or gel may be of
a density that is roughly similar to that of a buffer. The polymer
or gel may have a tunable pore size. The pore size may be chosen
to, for instance, retain denatured nucleic acids. The pore size may
be chosen to maintain diffusive permeability to exogenous chemicals
such as sodium hydroxide (NaOH) and/or endogenous chemicals such as
inhibitors. The polymer or gel may be biocompatible. The polymer or
gel may maintain or enhance cell viability. The polymer or gel may
be biochemically compatible. The polymer or gel may be polymerized
and/or depolymerized thermally, chemically, enzymatically, and/or
optically.
[0100] The polymer may comprise poly(acrylamide-co-acrylic acid)
crosslinked with disulfide linkages. The preparation of the polymer
may comprise a two-step reaction. In the first activation step,
poly(acrylamide-co-acrylic acid) may be exposed to an acylating
agent to convert carboxylic acids to esters. For instance, the
poly(acrylamide-co-acrylic acid) may be exposed to
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to other
salts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium.
In the second cross-linking step, the ester formed in the first
step may be exposed to a disulfide crosslinking agent. For
instance, the ester may be exposed to cystamine
(2,2'-dithiobis(ethylamine)). Following the two steps, the
biological particle or analyte carrier may be surrounded by
polyacrylamide strands linked together by disulfide bridges. In
this manner, the biological particle or analyte carrier may be
encased inside of or comprise a gel or matrix (e.g., polymer
matrix) to form a "cell bead." A cell bead can contain biological
particles or analyte carriers (e.g., a cell) or macromolecular
constituents (e.g., RNA, DNA, proteins, etc.) of biological
particles or analyte carriers. A cell bead may include a cell or
multiple cells, or a derivative of the cell or multiple cells. For
example after lysing and washing the cells, inhibitory components
from cell lysates can be washed away and the macromolecular
constituents can be bound as cell beads. Systems and methods
disclosed herein can be applicable to both cell beads (and/or
droplets or other partitions) containing biological particles or
analyte carriers and cell beads (and/or droplets or other
partitions) containing macromolecular constituents of biological
particles or analyte carriers.
[0101] Encapsulated biological particles or analyte carriers can
provide certain potential advantages of being more storable and
more portable than droplet-based partitioned biological particles
or analyte carriers. Furthermore, in some cases, it may be useful
to allow biological particles or analyte carriers to incubate for a
select period of time before analysis, such as in order to
characterize changes in such biological particles or analyte
carriers over time, either in the presence or absence of different
stimuli. In such cases, encapsulation may allow for longer
incubation than partitioning in emulsion droplets, although in some
cases, droplet partitioned biological particles or analyte carriers
may also be incubated for different periods of time, e.g., at least
10 seconds, at least 30 seconds, at least 1 minute, at least 5
minutes, at least 10 minutes, at least 30 minutes, at least 1 hour,
at least 2 hours, at least 5 hours, or at least 10 hours or more.
The encapsulation of biological particles or analyte carriers may
constitute the partitioning of the biological particles or analyte
carriers into which other reagents are co-partitioned.
Alternatively or in addition, encapsulated biological particles or
analyte carriers may be readily deposited into other partitions
(e.g., droplets) as described above.
Beads
[0102] A partition may comprise one or more unique identifiers,
such as barcodes. Barcodes may be previously, subsequently or
concurrently delivered to the partitions that hold the
compartmentalized or partitioned biological particle or analyte
carrier. For example, barcodes may 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 or analyte carrier to the particular
partition. Barcodes may 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 comprise a bead. Beads are described in further
detail below.
[0103] In some cases, 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 may 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.
[0104] FIG. 2 shows an example of a microfluidic channel structure
200 for delivering barcode carrying beads to droplets. The channel
structure 200 can include channel segments 201, 202, 204, 206 and
208 communicating at a channel junction 210. In operation, the
channel segment 201 may transport an aqueous fluid 212 that
includes a plurality of beads 214 (e.g., with nucleic acid
molecules, oligonucleotides, molecular tags) along the channel
segment 201 into junction 210. The plurality of beads 214 may be
sourced from a suspension of beads. For example, the channel
segment 201 may be connected to a reservoir comprising an aqueous
suspension of beads 214. The channel segment 202 may transport the
aqueous fluid 212 that includes a plurality of biological particles
or analyte carriers 216 along the channel segment 202 into junction
210. The plurality of biological particles or analyte carriers 216
may be sourced from a suspension of biological particles or analyte
carriers. For example, the channel segment 202 may be connected to
a reservoir comprising an aqueous suspension of biological
particles or analyte carriers 216. In some instances, the aqueous
fluid 212 in either the first channel segment 201 or the second
channel segment 202, or in both segments, can include one or more
reagents, as further described below. A second fluid 218 that is
immiscible with the aqueous fluid 212 (e.g., oil) can be delivered
to the junction 210 from each of channel segments 204 and 206. Upon
meeting of the aqueous fluid 212 from each of channel segments 201
and 202 and the second fluid 218 from each of channel segments 204
and 206 at the channel junction 210, the aqueous fluid 212 can be
partitioned as discrete droplets 220 in the second fluid 218 and
flow away from the junction 210 along channel segment 208. The
channel segment 208 may deliver the discrete droplets to an outlet
reservoir fluidly coupled to the channel segment 208, where they
may be harvested.
[0105] As an alternative, the channel segments 201 and 202 may meet
at another junction upstream of the junction 210. At such junction,
beads and biological particles or analyte carriers may form a
mixture that is directed along another channel to the junction 210
to yield droplets 220. The mixture may provide the beads and
biological particles or analyte carriers in an alternating fashion,
such that, for example, a droplet comprises a single bead and a
single biological particle or analyte carrier.
[0106] Beads, biological particles or analyte carriers and droplets
may flow along channels at substantially regular flow profiles
(e.g., at regular flow rates). Such regular flow profiles may
permit a droplet to include one or more beads (e.g., a single bead,
a plurality of beads) and a single biological particle or analyte
carrier. Such regular flow profiles may permit the droplets to have
an occupancy (e.g., droplets having beads and biological particles
or analyte carriers) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, or 95%. Such regular flow profiles and devices that
may be used to provide such regular flow profiles are provided in,
for example, U.S. Patent Publication No. 2015/0292988, which is
entirely incorporated herein by reference.
[0107] The second fluid 218 can comprise 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 220.
[0108] A discrete droplet that is generated may include an
individual biological particle or analyte carrier 216. A discrete
droplet that is generated may include a barcode or other reagent
carrying bead 214. A discrete droplet generated may include both an
individual biological particle or analyte carrier and a barcode
carrying bead, such as droplets 220. In some instances, a discrete
droplet may include more than one individual biological particle or
analyte carrier or no biological particle or analyte carrier. In
some instances, a discrete droplet may include more than one bead
or no bead. A discrete droplet may be unoccupied (e.g., no beads,
no biological particles or analyte carriers).
[0109] Beneficially, a discrete droplet partitioning a biological
particle or analyte carrier and a barcode carrying bead may
effectively allow the attribution of the barcode to macromolecular
constituents of the biological particle or analyte carrier within
the partition. The contents of a partition may remain discrete from
the contents of other partitions.
[0110] As will be appreciated, the channel segments described
herein may 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 200 may have other
geometries. For example, a microfluidic channel structure can have
more than one channel junctions. For example, a microfluidic
channel structure can have 2, 3, 4, or 5 channel segments each
carrying beads that meet at a channel junction. Fluid may be
directed flow along one or more channels or reservoirs via one or
more fluid flow units. A fluid flow unit can comprise compressors
(e.g., providing positive pressure), pumps (e.g., providing
negative pressure), actuators, and the like to control flow of the
fluid. Fluid may also or otherwise be controlled via applied
pressure differentials, centrifugal force, electrokinetic pumping,
vacuum, capillary or gravity flow, or the like.
[0111] A bead may be porous, non-porous, solid, semi-solid,
semi-fluidic, fluidic, and/or a combination thereof. In some
instances, a bead may be dissolvable, disruptable, and/or
degradable. In some cases, a bead may not be degradable. In some
cases, the bead may be a gel bead. A gel bead may be a hydrogel
bead. A gel bead may be formed from molecular precursors, such as a
polymeric or monomeric species. A semi-solid bead may be a
liposomal bead. Solid beads may comprise metals including iron
oxide, gold, and silver. In some cases, the bead may be a silica
bead. In some cases, the bead can be rigid. In other cases, the
bead may be flexible and/or compressible.
[0112] A bead may be of any suitable shape. Examples of bead shapes
include, but are not limited to, spherical, non-spherical, oval,
oblong, amorphous, circular, cylindrical, and variations
thereof.
[0113] Beads may be of uniform size or heterogeneous size. In some
cases, the diameter of a bead may be at least about 10 nanometers
(nm), 100 nm, 500 nm, 1 micrometer (.mu.m), 5 .mu.m, 10 .mu.m, 20
.mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m, 60 .mu.m, 70 .mu.m, 80 .mu.m,
90 .mu.m, 100 .mu.m, 250 .mu.m, 500 .mu.m, 1 mm, or greater. In
some cases, a bead may have a diameter of less than about 10 nm,
100 nm, 500 nm, l.mu.m, 5 .mu.m, 10 .mu.m, 20 .mu.m, 30 .mu.m, 40
.mu.m, 50 .mu.m, 60 .mu.m, 70 .mu.m, 80 .mu.m, 90 .mu.m, 100 .mu.m,
250 .mu.m, 500 .mu.m, 1 mm, or less. In some cases, a bead may have
a diameter in the range of about 40-75 .mu.m, 30-75 .mu.m, 20-75
.mu.m, 40-85 .mu.m, 40-95 .mu.m, 20-100 .mu.m, 10-100 .mu.m, 1-100
.mu.m, 20-250 .mu.m, or 20-500 .mu.m.
[0114] In certain aspects, beads can be provided as a population or
plurality of beads having a relatively monodisperse size
distribution. Where relatively consistent amounts of reagents
within partitions are provided, maintaining relatively consistent
bead characteristics, such as size, can contribute to the overall
consistency. In particular, the beads described herein may 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%, and in some cases less than 15%, less than
10%, less than 5%, or less.
[0115] A bead may comprise natural and/or synthetic materials. For
example, a bead can comprise a natural polymer, a synthetic polymer
or both natural and synthetic polymers. Examples of natural
polymers include proteins and sugars such as deoxyribonucleic acid,
rubber, cellulose, starch (e.g., amylose, amylopectin), proteins,
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 acrylics, nylons,
silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl
acetate, polyacrylamide, polyacrylate, polyethylene glycol,
polyurethanes, polylactic acid, silica, polystyrene,
polyacrylonitrile, polybutadiene, polycarbonate, 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 may also be formed
from materials other than polymers, including lipids, micelles,
ceramics, glass-ceramics, material composites, metals, other
inorganic materials, and others.
[0116] In some instances, the bead may contain molecular precursors
(e.g., monomers or polymers), which may form a polymer network via
polymerization of the molecular precursors. In some cases, a
precursor may be an already polymerized species capable of
undergoing further polymerization via, for example, a chemical
cross-linkage. In some cases, a precursor can comprise one or more
of an acrylamide or a methacrylamide monomer, oligomer, or polymer.
In some cases, the bead may comprise prepolymers, which are
oligomers capable of further polymerization. For example,
polyurethane beads may be prepared using prepolymers. In some
cases, the bead may contain individual polymers that may be further
polymerized together. In some cases, beads may be generated via
polymerization of different precursors, such that they comprise
mixed polymers, co-polymers, and/or block co-polymers. In some
cases, the bead may comprise covalent or ionic bonds between
polymeric precursors (e.g., monomers, oligomers, linear polymers),
nucleic acid molecules (e.g., oligonucleotides), primers, and other
entities. In some cases, the covalent bonds can be carbon-carbon
bonds, thioether bonds, or carbon-heteroatom bonds.
[0117] Cross-linking may be permanent or reversible, depending upon
the particular cross-linker used. Reversible cross-linking may
allow for the polymer to linearize or dissociate under appropriate
conditions. In some cases, reversible cross-linking may also allow
for reversible attachment of a material bound to the surface of a
bead. In some cases, a cross-linker may form disulfide linkages. In
some cases, the chemical cross-linker forming disulfide linkages
may be cystamine or a modified cystamine.
[0118] In some cases, 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). Cystamine (including modified
cystamines), for example, is an organic agent comprising a
disulfide bond that may be used as a crosslinker agent between
individual monomeric or polymeric precursors of a bead.
Polyacrylamide may be polymerized in the presence of cystamine or a
species comprising cystamine (e.g., a modified cystamine) to
generate polyacrylamide gel beads comprising disulfide linkages
(e.g., chemically degradable beads comprising chemically-reducible
cross-linkers). The disulfide linkages may permit the bead to be
degraded (or dissolved) upon exposure of the bead to a reducing
agent.
[0119] In some cases, chitosan, a linear polysaccharide polymer,
may be crosslinked with glutaraldehyde via hydrophilic chains to
form a bead. Crosslinking of chitosan polymers may be achieved by
chemical reactions that are initiated by heat, pressure, change in
pH, and/or radiation.
[0120] In some cases, a bead may comprise an acrydite moiety, which
in certain aspects may be used to attach one or more nucleic acid
molecules (e.g., barcode sequence, barcoded nucleic acid molecule,
barcoded oligonucleotide, primer, or other oligonucleotide) to the
bead. In some cases, an acrydite moiety can refer to an acrydite
analogue generated from the reaction of acrydite with one or more
species, such as, the reaction of acrydite with other monomers and
cross-linkers during a polymerization reaction. Acrydite moieties
may be modified to form chemical bonds with a species to be
attached, such as a nucleic acid molecule (e.g., barcode sequence,
barcoded nucleic acid molecule, barcoded oligonucleotide, primer,
or other oligonucleotide). Acrydite moieties may be modified with
thiol groups capable of forming a disulfide bond or may be modified
with groups already comprising a disulfide bond. The thiol or
disulfide (via disulfide exchange) may be used as an anchor point
for a species to be attached or another part of the acrydite moiety
may be used for attachment. In some cases, 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 other cases, an acrydite moiety can comprise a
reactive hydroxyl group that may be used for attachment.
[0121] Functionalization of beads for attachment of nucleic acid
molecules (e.g., oligonucleotides) may be achieved through a wide
range of different approaches, including 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.
[0122] For example, precursors (e.g., monomers, cross-linkers) that
are polymerized to form a bead may comprise acrydite moieties, such
that when a bead is generated, the bead also comprises acrydite
moieties. The acrydite moieties can be attached to a nucleic acid
molecule (e.g., oligonucleotide), which may 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 barcode sequences. The one more barcode sequences may include
sequences that are the same for all nucleic acid molecules coupled
to a given bead and/or sequences that are different across all
nucleic acid molecules coupled to the given bead. The nucleic acid
molecule may be incorporated into the bead.
[0123] In some cases, the nucleic acid molecule can comprise a
functional sequence, for example, for attachment to a sequencing
flow cell, such as, for example, a P5 sequence for Illumina.RTM.
sequencing. In some cases, the nucleic acid molecule or derivative
thereof (e.g., oligonucleotide or polynucleotide generated from the
nucleic acid molecule) can comprise another functional sequence,
such as, for example, a P7 sequence for attachment to a sequencing
flow cell for Illumina sequencing. In some cases, the nucleic acid
molecule can comprise a barcode sequence. In some cases, the primer
can further comprise a unique molecular identifier (UMI). In some
cases, the primer can comprise an R1 primer sequence for Illumina
sequencing. In some cases, the primer can comprise an R2 primer
sequence for Illumina sequencing. Examples of such nucleic acid
molecules (e.g., oligonucleotides, polynucleotides, etc.) and uses
thereof, as may be used with compositions, devices, methods and
systems of the present disclosure, are provided in U.S. Patent Pub.
Nos. 2014/0378345 and 2015/0376609, each of which is entirely
incorporated herein by reference.
[0124] FIG. 8 illustrates an example of a barcode carrying bead. A
nucleic acid molecule 802, such as an oligonucleotide, can be
coupled to a bead 804 by a releasable linkage 806, such as, for
example, a disulfide linker. The same bead 804 may be coupled
(e.g., via releasable linkage) to one or more other nucleic acid
molecules 818, 820. The nucleic acid molecule 802 may be or
comprise a barcode. As noted elsewhere herein, the structure of the
barcode may comprise a number of sequence elements. The nucleic
acid molecule 802 may comprise a functional sequence 808 that may
be used in subsequent processing. For example, the functional
sequence 808 may 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 802 may comprise a barcode sequence 810 for use in
barcoding the sample (e.g., DNA, RNA, protein, etc.). In some
cases, the barcode sequence 810 can be bead-specific such that the
barcode sequence 810 is common to all nucleic acid molecules (e.g.,
including nucleic acid molecule 802) coupled to the same bead 804.
Alternatively or in addition, the barcode sequence 810 can be
partition-specific such that the barcode sequence 810 is common to
all nucleic acid molecules coupled to one or more beads that are
partitioned into the same partition. The nucleic acid molecule 802
may comprise a specific priming sequence 812, 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 802 may comprise an anchoring sequence 814 to ensure
that the specific priming sequence 812 hybridizes at the sequence
end (e.g., of the mRNA). For example, the anchoring sequence 814
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.
[0125] The nucleic acid molecule 802 may comprise a unique
molecular identifying sequence 816 (e.g., unique molecular
identifier (UMI)). In some cases, the unique molecular identifying
sequence 816 may comprise from about 5 to about 8 nucleotides.
Alternatively, the unique molecular identifying sequence 816 may
compress less than about 5 or more than about 8 nucleotides. The
unique molecular identifying sequence 816 may be a unique sequence
that varies across individual nucleic acid molecules (e.g., 802,
818, 820, etc.) coupled to a single bead (e.g., bead 804). In some
cases, the unique molecular identifying sequence 816 may be a
random sequence (e.g., such as a random N-mer sequence). For
example, the UMI may provide a unique identifier of the starting
mRNA molecule that was captured, in order to allow quantitation of
the number of original expressed RNA. As will be appreciated,
although FIG. 8 shows three nucleic acid molecules 802, 818, 820
coupled to the surface of the bead 804, an individual bead may 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 comprise both common
sequence segments or relatively common sequence segments (e.g.,
808, 810, 812, etc.) and variable or unique sequence segments
(e.g., 816) between different individual nucleic acid molecules
coupled to the same bead.
[0126] In operation, a biological particle or analyte carrier
(e.g., cell, DNA, RNA, etc.) can be co-partitioned along with a
barcode bearing bead 804. The barcoded nucleic acid molecules 802,
818, 820 can be released from the bead 804 in the partition. By way
of example, in the context of analyzing sample RNA, the poly-T
segment (e.g., 812) of one of the released nucleic acid molecules
(e.g., 802) can hybridize to the poly-A tail of a mRNA molecule.
Reverse transcription may result in a cDNA transcript of the mRNA,
but which transcript includes each of the sequence segments 808,
810, 816 of the nucleic acid molecule 802. Because the nucleic acid
molecule 802 comprises an anchoring sequence 814, it will more
likely hybridize to and prime reverse transcription at the sequence
end of the poly-A tail of the mRNA. Within any given partition, all
of the cDNA transcripts of the individual mRNA molecules may
include a common barcode sequence segment 810. However, the
transcripts made from the different mRNA molecules within a given
partition may vary at the unique molecular identifying sequence 812
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, and thus from the biological
particle or analyte carrier (e.g., cell). 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 may 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 (e.g., gel bead) may 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.
[0127] In some cases, precursors comprising 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 comprising the activated or activatable functional group. The
functional group may then be used to attach additional species
(e.g., disulfide linkers, primers, other oligonucleotides, etc.) to
the gel beads. For example, some precursors comprising a carboxylic
acid (COOH) group can co-polymerize with other precursors to form a
gel bead that also comprises 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 gel bead comprising free COOH groups. The
COOH groups of the gel 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.
[0128] Beads comprising disulfide linkages in their polymeric
network may be functionalized with additional species via reduction
of some of the disulfide linkages to free thiols. The disulfide
linkages may 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 comprising 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 cases, free thiols of the beads may react with any
other suitable group. For example, free thiols of the beads may
react with species comprising an acrydite moiety. The free thiol
groups of the beads can react with the acrydite via Michael
addition chemistry, such that the species comprising the acrydite
is linked to the bead. In some cases, uncontrolled reactions can be
prevented by inclusion of a thiol capping agent such as
N-ethylmalieamide or iodoacetate.
[0129] Activation of disulfide linkages within a bead can be
controlled such that only a small number of disulfide linkages are
activated. Control may 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 cases, a low concentration
(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, less than or equal to about 1:10,000) of
reducing agent may be used for reduction. Controlling the number of
disulfide linkages that are reduced to free thiols may be useful in
ensuring bead structural integrity during functionalization. In
some cases, optically-active agents, such as fluorescent dyes may
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.
[0130] In some cases, addition of moieties to a gel bead after gel
bead formation may be advantageous. For example, addition of an
oligonucleotide (e.g., barcoded oligonucleotide) after gel bead
formation may avoid loss of the species during chain transfer
termination that can occur during polymerization. Moreover, smaller
precursors (e.g., monomers or cross linkers that do not comprise
side chain groups and linked moieties) may be used for
polymerization and can be minimally hindered from growing chain
ends due to viscous effects. In some cases, functionalization after
gel 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 cases,
the generated gel may possess an upper critical solution
temperature (UCST) that can permit temperature driven swelling and
collapse of a bead. Such functionality may aid in oligonucleotide
(e.g., a primer) infiltration into the bead during subsequent
functionalization of the bead with the oligonucleotide.
Post-production functionalization may also be useful in controlling
loading ratios of species in beads, such that, for example, the
variability in loading ratio is minimized. Species loading may also
be performed in a batch process such that a plurality of beads can
be functionalized with the species in a single batch.
[0131] A bead injected or otherwise introduced into a partition may
comprise releasably, cleavably, or reversibly attached barcodes. A
bead injected or otherwise introduced into a partition may comprise
activatable barcodes. A bead injected or otherwise introduced into
a partition may be degradable, disruptable, or dissolvable
beads.
[0132] Barcodes can be releasably, cleavably or reversibly attached
to the beads such that barcodes can be released or be releasable
through cleavage of a linkage between the barcode molecule and the
bead, or released through degradation of the underlying bead
itself, allowing the barcodes to be accessed or be accessible by
other reagents, or both. In non-limiting examples, cleavage may be
achieved through reduction of di-sulfide bonds, use of restriction
enzymes, photo-activated cleavage, or cleavage via other types of
stimuli (e.g., chemical, thermal, pH, enzymatic, etc.) and/or
reactions, such as described elsewhere herein. Releasable barcodes
may sometimes be referred to as being activatable, in that they are
available for reaction once released. Thus, for example, an
activatable barcode may be activated by releasing the barcode from
a bead (or other suitable type of partition described herein).
Other activatable configurations are also envisioned in the context
of the described methods and systems.
[0133] In addition to, or as an alternative to the cleavable
linkages between the beads and the associated molecules, such as
barcode containing nucleic acid molecules (e.g., barcoded
oligonucleotides), the beads may be degradable, disruptable, or
dissolvable spontaneously or upon exposure to one or more stimuli
(e.g., temperature changes, pH changes, exposure to particular
chemical species or phase, exposure to light, reducing agent,
etc.). In some cases, a bead may be dissolvable, such that material
components of the beads are solubilized when exposed to a
particular chemical species or an environmental change, such as a
change temperature or a change in pH. In some cases, a gel bead can
be degraded or dissolved at elevated temperature and/or in basic
conditions. In some cases, a bead may be thermally degradable such
that when the bead is exposed to an appropriate change in
temperature (e.g., heat), the bead degrades. Degradation or
dissolution of a bead bound to a species (e.g., a nucleic acid
molecule, e.g., barcoded oligonucleotide) may result in release of
the species from the bead.
[0134] As will be appreciated from the above disclosure, the
degradation of a bead may 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, the degradation of
the bead may involve cleavage of a cleavable linkage via one or
more species and/or methods described elsewhere herein. In another
example, entrained species may be released from beads through
osmotic pressure differences due to, for example, changing chemical
environments. By way of example, alteration of bead pore sizes due
to osmotic pressure differences can generally occur without
structural degradation of the bead itself. In some cases, an
increase in pore size due to osmotic swelling of a bead can permit
the release of entrained species within the bead. In other cases,
osmotic shrinking of a bead may cause a bead to better retain an
entrained species due to pore size contraction.
[0135] A degradable bead may be introduced into a partition, such
as a droplet of an emulsion or a well, 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) may interact with other
reagents contained in the partition. For example, a polyacrylamide
bead comprising cystamine and linked, via a disulfide bond, to a
barcode sequence, may 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 comprising a bead-bound barcode sequence in
basic solution may also result in bead degradation and release of
the attached barcode sequence into the aqueous, inner environment
of the droplet.
[0136] Any suitable number of molecular tag molecules (e.g.,
primer, barcoded oligonucleotide) can be associated with a bead
such that, upon release from the bead, the molecular tag molecules
(e.g., primer, e.g., barcoded oligonucleotide) are present in the
partition at a pre-defined concentration. Such pre-defined
concentration may 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.
[0137] In some cases, 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. The swelling of the
beads may 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. The swelling of the beads may be accomplished by various
swelling methods. The de-swelling of the beads may be accomplished,
for instance, by transferring the beads in a thermodynamically
unfavorable solvent, subjecting the beads to lower or high
temperatures, subjecting the beads to a lower or higher ion
concentration, and/or removing an electric field. The de-swelling
of the beads may be accomplished by various de-swelling methods.
Transferring the beads may cause pores in the bead to shrink. The
shrinking may then hinder reagents within the beads from diffusing
out of the interiors of the beads. The hindrance may be due to
steric interactions between the reagents and the interiors of the
beads. The transfer may be accomplished microfluidically. For
instance, the transfer may be achieved by moving the beads from one
co-flowing solvent stream to a different co-flowing solvent stream.
The swellability and/or pore size of the beads may be adjusted by
changing the polymer composition of the bead.
[0138] In some cases, an acrydite moiety linked to a precursor,
another species linked to a precursor, or a precursor itself can
comprise a labile bond, such as chemically, thermally, or
photo-sensitive bond e.g., disulfide bond, UV sensitive bond, or
the like. Once acrydite moieties or other moieties comprising a
labile bond are incorporated into a bead, the bead may also
comprise the labile bond. The labile bond may be, for example,
useful in reversibly linking (e.g., covalently linking) species
(e.g., barcodes, primers, etc.) to a bead. In some cases, a
thermally labile bond may include a nucleic acid hybridization
based attachment, e.g., where an oligonucleotide is hybridized to a
complementary sequence that is attached to the bead, such that
thermal melting of the hybrid releases the oligonucleotide, e.g., a
barcode containing sequence, from the bead or microcapsule.
[0139] The addition of multiple types of labile bonds to a gel bead
may result in the generation of a bead capable of responding to
varied stimuli. Each type of labile bond may be sensitive to an
associated stimulus (e.g., chemical stimulus, light, temperature,
enzymatic, etc.) such that release of species attached to a bead
via each labile bond may be controlled by the application of the
appropriate stimulus. Such functionality may be useful in
controlled release of species from a gel bead. In some cases,
another species comprising a labile bond may be linked to a gel
bead after gel bead formation via, for example, an activated
functional group of the gel bead as described above. As will be
appreciated, barcodes that are releasably, cleavably or reversibly
attached to the beads described herein include barcodes that are
released or releasable through cleavage of a linkage between the
barcode molecule and the bead, or that are released through
degradation of the underlying bead itself, allowing the barcodes to
be accessed or accessible by other reagents, or both.
[0140] The barcodes that are releasable as described herein may
sometimes be referred to as being activatable, in that they are
available for reaction once released. Thus, for example, an
activatable barcode may be activated by releasing the barcode from
a bead (or other suitable type of partition described herein).
Other activatable configurations are also envisioned in the context
of the described methods and systems.
[0141] In addition to thermally cleavable bonds, disulfide bonds
and UV sensitive bonds, other non-limiting examples of labile bonds
that may 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 may be cleavable via other nucleic acid molecule
targeting enzymes, such as restriction enzymes (e.g., restriction
endonucleases), as described further below.
[0142] Species may be encapsulated in beads during bead generation
(e.g., during polymerization of precursors). Such species may or
may not participate in polymerization. Such species may be entered
into polymerization reaction mixtures such that generated beads
comprise the species upon bead formation. In some cases, such
species may be added to the gel beads after formation. Such species
may include, for example, nucleic acid molecules (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,
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 species may 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 species may include one or more
reagents described elsewhere herein (e.g., lysis agents,
inhibitors, inactivating agents, chelating agents, stimulus).
Trapping of such species may be controlled by the polymer network
density generated during polymerization of precursors, control of
ionic charge within the gel bead (e.g., via ionic species linked to
polymerized species), or by the release of other species.
Encapsulated species may be released from a bead upon bead
degradation and/or by application of a stimulus capable of
releasing the species from the bead. Alternatively or in addition,
species may be partitioned in a partition (e.g., droplet) during or
subsequent to partition formation. Such species may include,
without limitation, the abovementioned species that may also be
encapsulated in a bead.
[0143] A degradable bead may comprise one or more species with a
labile bond such that, when the bead/species is exposed to the
appropriate stimuli, the bond is broken and the bead degrades. The
labile bond may be a chemical bond (e.g., covalent bond, ionic
bond) or may be another type of physical interaction (e.g., van der
Waals interactions, dipole-dipole interactions, etc.). In some
cases, a crosslinker used to generate a bead may comprise 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 comprising cystamine crosslinkers to a
reducing agent, the disulfide bonds of the cystamine can be broken
and the bead degraded.
[0144] A degradable bead may 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
may have greater mobility and accessibility to other species in
solution upon degradation of the bead. In some cases, a species may
also be attached to a degradable bead via a degradable linker
(e.g., disulfide linker). The degradable linker may respond to the
same stimuli as the degradable bead or the two degradable species
may respond to different stimuli. For example, a barcode sequence
may 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.
[0145] As will be appreciated from the above disclosure, while
referred to as degradation of a bead, in many instances as noted
above, that degradation may 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 may be released from beads through osmotic
pressure differences due to, for example, changing chemical
environments. By way of example, alteration of bead pore sizes due
to osmotic pressure differences can generally occur without
structural degradation of the bead itself. In some cases, an
increase in pore size due to osmotic swelling of a bead can permit
the release of entrained species within the bead. In other cases,
osmotic shrinking of a bead may cause a bead to better retain an
entrained species due to pore size contraction.
[0146] Where degradable beads are provided, it may 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 comprise reducible
cross-linking groups, such as disulfide groups, it can be useful to
avoid contacting such beads with reducing agents, e.g., DTT or
other disulfide cleaving reagents. In such cases, treatment to the
beads described herein will, in some cases be provided free of
reducing agents, such as DTT. Because reducing agents are often
provided in commercial enzyme preparations, reducing agent free (or
DTT free) enzyme preparations may be provided 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 may be used to treat the beads described
herein. The terms "reducing agent free" or "DTT free" preparations
can refer to a preparation having less than about 1/10th, less than
about 1/50th, or even 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
even less than about 0.0001 mM DTT. In many cases, the amount of
DTT can be undetectable.
[0147] Numerous chemical triggers may be used to trigger the
degradation of beads. Examples of these chemical changes may
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.
[0148] In some embodiments, a bead may be formed from materials
that comprise degradable chemical crosslinkers, such as BAC or
cystamine. Degradation of such degradable crosslinkers may be
accomplished through a number of mechanisms. In some examples, a
bead may be contacted with a chemical degrading agent that may
induce oxidation, reduction or other chemical changes. For example,
a chemical degrading agent may be a reducing agent, such as
dithiothreitol (DTT). Additional examples of reducing agents may
include .beta.-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane
(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP),
or combinations thereof. A reducing agent may degrade the disulfide
bonds formed between gel precursors forming the bead, and thus,
degrade the bead. In other cases, a change in pH of a solution,
such as an increase in pH, may trigger degradation of a bead. In
other cases, exposure to an aqueous solution, such as water, may
trigger hydrolytic degradation, and thus degradation of the bead.
In some cases, any combination of stimuli may trigger degradation
of a bead. For example, a change in pH may enable a chemical agent
(e.g., DTT) to become an effective reducing agent.
[0149] Beads may 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 may cause melting of a bead
such that a portion of the bead degrades. In other cases, heat may
increase the internal pressure of the bead components such that the
bead ruptures or explodes. Heat may also act upon heat-sensitive
polymers used as materials to construct beads.
[0150] Any suitable agent may degrade beads. In some embodiments,
changes in temperature or pH may be used to degrade
thermo-sensitive or pH-sensitive bonds within beads. In some
embodiments, chemical degrading agents may be used to degrade
chemical bonds within beads by oxidation, reduction or other
chemical changes. For example, a chemical degrading agent may be a
reducing agent, such as DTT, wherein DTT may degrade the disulfide
bonds formed between a crosslinker and gel precursors, thus
degrading the bead. In some embodiments, a reducing agent may be
added to degrade the bead, which may or may not cause the bead to
release its contents. Examples of reducing agents may include
dithiothreitol (DTT), .beta.-mercaptoethanol,
(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA),
tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof. The
reducing agent may be present at a concentration of about 0.1 mM,
0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present at a
concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM,
or greater than 10 mM. The reducing agent may be present at
concentration of at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM,
or less.
[0151] Any suitable number of molecular tag molecules (e.g.,
primer, barcoded oligonucleotide) can be associated with a bead
such that, upon release from the bead, the molecular tag molecules
(e.g., primer, e.g., barcoded oligonucleotide) are present in the
partition at a pre-defined concentration. Such pre-defined
concentration may 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 oligonucleotide
bearing beads.
[0152] Although FIG. 1 and FIG. 2 have been described in terms of
providing substantially singly occupied partitions, above, in
certain cases, multiply occupied partitions can be provided, e.g.,
containing two, three, four or more cells and/or microcapsules
(e.g., beads) comprising barcoded nucleic acid molecules (e.g.,
oligonucleotides) within a single partition. Accordingly, as noted
above, the flow characteristics of the biological particle or
analyte carrier and/or bead containing fluids and partitioning
fluids may be controlled to provide for such multiply occupied
partitions. In particular, the flow parameters may be controlled to
provide a given occupancy rate at greater than about 50% of the
partitions, greater than about 75%, and in some cases greater than
about 80%, 90%, 95%, or higher.
[0153] In some cases, additional microcapsules can be used to
deliver additional reagents to a partition. In such cases, it may
be advantageous to introduce different beads into a common channel
or droplet generation junction, from different bead sources (e.g.,
containing different associated reagents) through different channel
inlets into such common channel or droplet generation junction
(e.g., junction 210). In such cases, the flow and frequency of the
different beads into the channel or junction may be controlled to
provide for a certain ratio of microcapsules from each source,
while ensuring a given pairing or combination of such beads into a
partition with a given number of biological particles or analyte
carriers (e.g., one biological particle or analyte carrier and one
bead per partition).
[0154] The partitions described herein may comprise 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.
[0155] For example, in the case of droplet based partitions, the
droplets may have overall volumes that are less than about 1000 pL,
900 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, or less. Where co-partitioned with
microcapsules, it will be appreciated that the sample fluid volume,
e.g., including co-partitioned biological particles or analyte
carriers and/or beads, within the partitions may be less than about
90% of the above described volumes, less than about 80%, less than
about 70%, less than about 60%, less than about 50%, less than
about 40%, less than about 30%, less than about 20%, or less than
about 10% of the above described volumes.
[0156] As is described elsewhere herein, partitioning species may
generate a population or plurality of partitions. In such cases,
any suitable number of partitions can be generated or otherwise
provided. For example, at least about 1,000 partitions, at least
about 5,000 partitions, at least about 10,000 partitions, at least
about 50,000 partitions, at least about 100,000 partitions, at
least about 500,000 partitions, at least about 1,000,000
partitions, at least about 5,000,000 partitions at least about
10,000,000 partitions, at least about 50,000,000 partitions, at
least about 100,000,000 partitions, at least about 500,000,000
partitions, at least about 1,000,000,000 partitions, or more
partitions can be generated or otherwise provided. Moreover, the
plurality of partitions may comprise both unoccupied partitions
(e.g., empty partitions) and occupied partitions.
Reagents
[0157] In accordance with certain aspects, biological particles or
analyte carriers may be partitioned along with lysis reagents in
order to release the contents of the biological particles or
analyte carriers within the partition. In such cases, the lysis
agents can be contacted with the biological particle or analyte
carrier suspension concurrently with, or immediately prior to, the
introduction of the biological particles or analyte carriers into
the partitioning junction/droplet generation zone (e.g., junction
210), such as through an additional channel or channels upstream of
the channel junction. In accordance with other aspects,
additionally or alternatively, biological particles or analyte
carriers may be partitioned along with other reagents, as will be
described further below.
[0158] FIG. 3 shows an example of a microfluidic channel structure
300 for co-partitioning biological particles or analyte carriers
and reagents. The channel structure 300 can include channel
segments 301, 302, 304, 306 and 308. Channel segments 301 and 302
communicate at a first channel junction 309. Channel segments 302,
304, 306, and 308 communicate at a second channel junction 310.
[0159] In an example operation, the channel segment 301 may
transport an aqueous fluid 312 that includes a plurality of
biological particles or analyte carriers 314 along the channel
segment 301 into the second junction 310. As an alternative or in
addition to, channel segment 301 may transport beads (e.g., gel
beads). The beads may comprise barcode molecules.
[0160] For example, the channel segment 301 may be connected to a
reservoir comprising an aqueous suspension of biological particles
or analyte carriers 314. Upstream of, and immediately prior to
reaching, the second junction 310, the channel segment 301 may meet
the channel segment 302 at the first junction 309. The channel
segment 302 may transport a plurality of reagents 315 (e.g., lysis
agents) suspended in the aqueous fluid 312 along the channel
segment 302 into the first junction 309. For example, the channel
segment 302 may be connected to a reservoir comprising the reagents
315. After the first junction 309, the aqueous fluid 312 in the
channel segment 301 can carry both the biological particles or
analyte carriers 314 and the reagents 315 towards the second
junction 310. In some instances, the aqueous fluid 312 in the
channel segment 301 can include one or more reagents, which can be
the same or different reagents as the reagents 315. A second fluid
316 that is immiscible with the aqueous fluid 312 (e.g., oil) can
be delivered to the second junction 310 from each of channel
segments 304 and 306. Upon meeting of the aqueous fluid 312 from
the channel segment 301 and the second fluid 316 from each of
channel segments 304 and 306 at the second channel junction 310,
the aqueous fluid 312 can be partitioned as discrete droplets 318
in the second fluid 316 and flow away from the second junction 310
along channel segment 308. The channel segment 308 may deliver the
discrete droplets 318 to an outlet reservoir fluidly coupled to the
channel segment 308, where they may be harvested.
[0161] The second fluid 316 can comprise 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 318.
[0162] A discrete droplet generated may include an individual
biological particle or analyte carrier 314 and/or one or more
reagents 315. In some instances, a discrete droplet generated may
include a barcode carrying bead (not shown), such as via other
microfluidics structures described elsewhere herein. In some
instances, a discrete droplet may be unoccupied (e.g., no reagents,
no biological particles or analyte carriers).
[0163] Beneficially, when lysis reagents and biological particles
or analyte carriers are co-partitioned, the lysis reagents can
facilitate the release of the contents of the biological particles
or analyte carriers within the partition. The contents released in
a partition may remain discrete from the contents of other
partitions.
[0164] As will be appreciated, the channel segments described
herein may 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 300 may have other
geometries. For example, a microfluidic channel structure can have
more than two channel junctions. For example, a microfluidic
channel structure can have 2, 3, 4, 5 channel segments or more each
carrying the same or different types of beads, reagents, and/or
biological particles or analyte carriers that meet at a channel
junction. Fluid flow in each channel segment may be controlled to
control the partitioning of the different elements into droplets.
Fluid may be directed flow along one or more channels or reservoirs
via one or more fluid flow units. A fluid flow unit can comprise
compressors (e.g., providing positive pressure), pumps (e.g.,
providing negative pressure), actuators, and the like to control
flow of the fluid. Fluid may also or otherwise be controlled via
applied pressure differentials, centrifugal force, electrokinetic
pumping, vacuum, capillary or gravity flow, or the like.
[0165] 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 may
additionally or alternatively be co-partitioned with the biological
particles or analyte carriers to cause the release of the
biological particles or analyte carriers' contents into the
partitions. For example, in some cases, surfactant-based lysis
solutions may be used to lyse cells. In some cases, lysis solutions
may include non-ionic surfactants such as, for example, TritonX-100
and Tween 20. In some cases, lysis solutions may include ionic
surfactants such as, for example, sarcosyl and sodium dodecyl
sulfate (SDS). Electroporation, thermal, acoustic or mechanical
cellular disruption may also be used in certain cases, e.g.,
non-emulsion based partitioning such as encapsulation of biological
particles or analyte carriers that may 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 size, following cellular disruption.
[0166] Alternatively or in addition to the lysis agents
co-partitioned with the biological particles or analyte carriers
described above, other reagents can also be co-partitioned with the
biological particles or analyte carriers, including, for example,
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. In addition, in the case of encapsulated
biological particles or analyte carriers, the biological particles
or analyte carriers may be exposed to an appropriate stimulus to
release the biological particles or analyte carriers or their
contents from a co-partitioned microcapsule. For example, in some
cases, a chemical stimulus may be co-partitioned along with an
encapsulated biological particle or analyte carrier to allow for
the degradation of the microcapsule and release of the cell or its
contents into the larger partition. In some cases, this stimulus
may be the same as the stimulus described elsewhere herein for
release of nucleic acid molecules (e.g., oligonucleotides) from
their respective microcapsule (e.g., bead). In alternative aspects,
this may be a different and non-overlapping stimulus, in order to
allow an encapsulated biological particle or analyte carrier to be
released into a partition at a different time from the release of
nucleic acid molecules into the same partition.
[0167] Additional reagents may also be co-partitioned with the
biological particles or analyte carriers, such as endonucleases to
fragment a biological particle's or analyte carrier's DNA, DNA
polymerase enzymes and dNTPs used to amplify the biological
particle's or analyte carrier's nucleic acid fragments and to
attach the barcode molecular tags to the amplified fragments. Other
enzymes may be co-partitioned, including without limitation,
polymerase, transposase, ligase, proteinase K, DNAse, etc.
Additional reagents may 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 cases, template
switching can be used to increase the length of a cDNA. In some
cases, 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.,
polyC, to the cDNA in a template independent manner. Switch oligos
can include sequences complementary to the additional nucleotides,
e.g., polyG. The additional nucleotides (e.g., polyC) on the cDNA
can hybridize to the additional nucleotides (e.g., polyG) on the
switch oligo, whereby the switch oligo can be used by the reverse
transcriptase as template to further extend the cDNA. Template
switching oligonucleotides may comprise a hybridization region and
a template region. The hybridization region can comprise any
sequence capable of hybridizing to the target. In some cases, as
previously described, the hybridization region comprises a series
of G bases to complement the overhanging C bases at the 3' end of a
cDNA molecule. The series of G bases may comprise 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 comprise any sequence to be incorporated into
the cDNA. In some cases, the template region comprises at least 1
(e.g., at least 2, 3, 4, 5 or more) tag sequences and/or functional
sequences. Switch oligos may comprise deoxyribonucleic acids;
ribonucleic acids; modified nucleic acids including 2-Aminopurine,
2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,
2'-deoxyInosine, 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.
[0168] In some cases, the length of a switch oligo may be at least
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,
142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,
168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,
181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,
246, 247, 248, 249 or 250 nucleotides or longer.
[0169] In some cases, the length of a switch oligo may be at most
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,
142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,
168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,
181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,
246, 247, 248, 249 or 250 nucleotides.
[0170] Once the contents of the cells are released into their
respective partitions, the macromolecular components (e.g.,
macromolecular constituents of biological particles, or analyte
carriers such as RNA, DNA, or proteins) contained therein may be
further processed within the partitions. In accordance with the
methods and systems described herein, the macromolecular component
contents of individual biological particles or analyte carriers can
be provided with unique identifiers such that, upon
characterization of those macromolecular components they may be
attributed as having been derived from the same biological particle
or particles or analyte carrier or carriers. The ability to
attribute characteristics to individual biological particles or
analyte carrier or groups of biological particles or analyte
carriers is provided by the assignment of unique identifiers
specifically to an individual biological particle or analyte
carrier or groups of biological particles or analyte carriers.
Unique identifiers, e.g., in the form of nucleic acid barcodes can
be assigned or associated with individual biological particles or
analyte carriers or populations of biological particles or analyte
carriers, in order to tag or label the biological particle's or
analyte carrier's macromolecular components (and as a result, its
characteristics) with the unique identifiers. These unique
identifiers can then be used to attribute the biological particle's
or analyte carrier's components and characteristics to an
individual biological particle or analyte carrier or group of
biological particles or analyte carriers.
[0171] In some aspects, this is performed by co-partitioning the
individual biological particle or analyte carrier or groups of
biological particles or analyte carriers with the unique
identifiers, such as described above (with reference to FIG. 2). In
some aspects, the unique identifiers are provided in the form of
nucleic acid molecules (e.g., oligonucleotides) that comprise
nucleic acid barcode sequences that may be attached to or otherwise
associated with the nucleic acid contents of individual biological
particle or analyte carrier, or to other components of the
biological particle or analyte carrier, and particularly to
fragments of those nucleic acids. The nucleic acid molecules are
partitioned such that as between nucleic acid molecules in a given
partition, the nucleic acid barcode sequences contained therein are
the same, but as between different partitions, the nucleic acid
molecule can, and do have differing barcode sequences, or at least
represent a large number of different barcode sequences across all
of the partitions in a given analysis. In some aspects, only one
nucleic acid barcode sequence can be associated with a given
partition, although in some cases, two or more different barcode
sequences may be present.
[0172] The nucleic acid barcode sequences can include from about 6
to about 20 or more nucleotides within the sequence of the nucleic
acid molecules (e.g., oligonucleotides). The nucleic acid barcode
sequences can include from about 6 to about 20, 30, 40, 50, 60, 70,
80, 90, 100 or more nucleotides. In some cases, the length of a
barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length
of a barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some
cases, the length of a barcode sequence may be at most about 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or
shorter. These nucleotides may be completely contiguous, i.e., in a
single stretch of adjacent nucleotides, or they may be separated
into two or more separate subsequences that are separated by 1 or
more nucleotides. In some cases, separated barcode subsequences can
be from about 4 to about 16 nucleotides in length. In some cases,
the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16 nucleotides or longer. In some cases, the barcode
subsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16 nucleotides or longer. In some cases, the barcode
subsequence may be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16 nucleotides or shorter.
[0173] The co-partitioned nucleic acid molecules can also comprise
other functional sequences useful in the processing of the nucleic
acids from the co-partitioned biological particles or analyte
carriers. These sequences include, e.g., targeted or
random/universal amplification primer sequences for amplifying the
genomic DNA from the individual biological particles or analyte
carriers within the partitions while attaching the associated
barcode sequences, sequencing primers or primer recognition sites,
hybridization or probing sequences, e.g., for identification of
presence of the sequences or for pulling down barcoded nucleic
acids, or any of a number of other potential functional sequences.
Other mechanisms of co-partitioning oligonucleotides may also be
employed, including, e.g., coalescence of two or more droplets,
where one droplet contains oligonucleotides, or microdispensing of
oligonucleotides into partitions, e.g., droplets within
microfluidic systems.
[0174] In an example, microcapsules, such as beads, are provided
that each include large numbers of the above described barcoded
nucleic acid molecules (e.g., barcoded oligonucleotides) releasably
attached to the beads, where all of the nucleic acid molecules
attached to a particular bead will include the same nucleic acid
barcode sequence, but where a large number of diverse barcode
sequences are represented across the population of beads used. In
some embodiments, hydrogel beads, e.g., comprising polyacrylamide
polymer matrices, are used as a solid support and delivery vehicle
for the nucleic acid molecules into the partitions, as they are
capable of carrying large numbers of nucleic acid molecules, and
may be configured to release those nucleic acid molecules upon
exposure to a particular stimulus, as described elsewhere herein.
In some cases, the population of beads provides a diverse barcode
sequence library that includes at least about 1,000 different
barcode sequences, at least about 5,000 different barcode
sequences, at least about 10,000 different barcode sequences, at
least about 50,000 different barcode sequences, at least about
100,000 different barcode sequences, at least about 1,000,000
different barcode sequences, at least about 5,000,000 different
barcode sequences, or at least about 10,000,000 different barcode
sequences, or more. Additionally, each bead can be provided with
large numbers of nucleic acid (e.g., oligonucleotide) molecules
attached. In particular, the number of molecules of nucleic acid
molecules including the barcode sequence on an individual bead can
be at least about 1,000 nucleic acid molecules, at least about
5,000 nucleic acid molecules, at least about 10,000 nucleic acid
molecules, at least about 50,000 nucleic acid molecules, at least
about 100,000 nucleic acid molecules, at least about 500,000
nucleic acids, at least about 1,000,000 nucleic acid molecules, at
least about 5,000,000 nucleic acid molecules, at least about
10,000,000 nucleic acid molecules, at least about 50,000,000
nucleic acid molecules, at least about 100,000,000 nucleic acid
molecules, at least about 250,000,000 nucleic acid molecules and in
some cases at least about 1 billion nucleic acid molecules, or
more. Nucleic acid molecules of a given bead can include identical
(or common) barcode sequences, different barcode sequences, or a
combination of both. Nucleic acid molecules of a given bead can
include multiple sets of nucleic acid molecules. Nucleic acid
molecules of a given set can include identical barcode sequences.
The identical barcode sequences can be different from barcode
sequences of nucleic acid molecules of another set.
[0175] Moreover, when the population of beads is partitioned, the
resulting population of partitions can also include a diverse
barcode library that includes at least about 1,000 different
barcode sequences, at least about 5,000 different barcode
sequences, at least about 10,000 different barcode sequences, at
least at least about 50,000 different barcode sequences, at least
about 100,000 different barcode sequences, at least about 1,000,000
different barcode sequences, at least about 5,000,000 different
barcode sequences, or at least about 10,000,000 different barcode
sequences. Additionally, each partition of the population can
include at least about 1,000 nucleic acid molecules, at least about
5,000 nucleic acid molecules, at least about 10,000 nucleic acid
molecules, at least about 50,000 nucleic acid molecules, at least
about 100,000 nucleic acid molecules, at least about 500,000
nucleic acids, at least about 1,000,000 nucleic acid molecules, at
least about 5,000,000 nucleic acid molecules, at least about
10,000,000 nucleic acid molecules, at least about 50,000,000
nucleic acid molecules, at least about 100,000,000 nucleic acid
molecules, at least about 250,000,000 nucleic acid molecules and in
some cases at least about 1 billion nucleic acid molecules.
[0176] In some cases, multiple different barcodes are incorporated
within a given partition, either attached to a single or multiple
beads within the partition. For example, in some cases, a mixed,
but known set of barcode sequences may provide greater assurance of
identification in the subsequent processing, e.g., by providing a
stronger address or attribution of the barcodes to a given
partition, as a duplicate or independent confirmation of the output
from a given partition.
[0177] The nucleic acid molecules (e.g., oligonucleotides) are
releasable from the beads upon the application of a particular
stimulus to the beads. In some cases, the stimulus may be a
photo-stimulus, e.g., through cleavage of a photo-labile linkage
that releases the nucleic acid molecules. In other cases, a thermal
stimulus may be used, where elevation of the temperature of the
beads environment will result in cleavage of a linkage or other
release of the nucleic acid molecules form the beads. In still
other cases, a chemical stimulus can be used that cleaves a linkage
of the nucleic acid molecules to the beads, or otherwise results in
release of the nucleic acid molecules from the beads. In one case,
such compositions include the polyacrylamide matrices described
above for encapsulation of biological particles or analyte
carriers, and may be degraded for release of the attached nucleic
acid molecules through exposure to a reducing agent, such as
DTT.
[0178] In some aspects, provided are systems and methods for
controlled partitioning. Droplet size may be controlled by
adjusting certain geometric features in channel architecture (e.g.,
microfluidics channel architecture). For example, an expansion
angle, width, and/or length of a channel may be adjusted to control
droplet size.
[0179] FIG. 4 shows an example of a microfluidic channel structure
for the controlled partitioning of beads into discrete droplets. A
channel structure 400 can include a channel segment 402
communicating at a channel junction 406 (or intersection) with a
reservoir 404. The reservoir 404 can be a chamber. Any reference to
"reservoir," as used herein, can also refer to a "chamber." In
operation, an aqueous fluid 408 that includes suspended beads 412
may be transported along the channel segment 402 into the junction
406 to meet a second fluid 410 that is immiscible with the aqueous
fluid 408 in the reservoir 404 to create droplets 416, 418 of the
aqueous fluid 408 flowing into the reservoir 404. At the junction
406 where the aqueous fluid 408 and the second fluid 410 meet,
droplets can form based on factors such as the hydrodynamic forces
at the junction 406, flow rates of the two fluids 408, 410, fluid
properties, and certain geometric parameters (e.g., w, h.sub.0,
.alpha., etc.) of the channel structure 400. A plurality of
droplets can be collected in the reservoir 404 by continuously
injecting the aqueous fluid 408 from the channel segment 402
through the junction 406.
[0180] A discrete droplet generated may include a bead (e.g., as in
occupied droplets 416). Alternatively, a discrete droplet generated
may include more than one bead. Alternatively, a discrete droplet
generated may not include any beads (e.g., as in unoccupied droplet
418). In some instances, a discrete droplet generated may contain
one or more biological particles or analyte carriers, as described
elsewhere herein. In some instances, a discrete droplet generated
may comprise one or more reagents, as described elsewhere
herein.
[0181] In some instances, the aqueous fluid 408 can have a
substantially uniform concentration or frequency of beads 412. The
beads 412 can be introduced into the channel segment 402 from a
separate channel (not shown in FIG. 4). The frequency of beads 412
in the channel segment 402 may be controlled by controlling the
frequency in which the beads 412 are introduced into the channel
segment 402 and/or the relative flow rates of the fluids in the
channel segment 402 and the separate channel. In some instances,
the beads can be introduced into the channel segment 402 from a
plurality of different channels, and the frequency controlled
accordingly.
[0182] In some instances, the aqueous fluid 408 in the channel
segment 402 can comprise biological particles or analyte carriers
(e.g., described with reference to FIGS. 1 and 2). In some
instances, the aqueous fluid 408 can have a substantially uniform
concentration or frequency of biological particles or analyte
carriers. As with the beads, the biological particles or analyte
carriers can be introduced into the channel segment 402 from a
separate channel. The frequency or concentration of the biological
particles or analyte carriers in the aqueous fluid 408 in the
channel segment 402 may be controlled by controlling the frequency
in which the biological particles or analyte carriers are
introduced into the channel segment 402 and/or the relative flow
rates of the fluids in the channel segment 402 and the separate
channel. In some instances, the biological particles or analyte
carriers can be introduced into the channel segment 402 from a
plurality of different channels, and the frequency controlled
accordingly. In some instances, a first separate channel can
introduce beads and a second separate channel can introduce
biological particles or analyte carriers into the channel segment
402. The first separate channel introducing the beads may be
upstream or downstream of the second separate channel introducing
the biological particles or analyte carriers.
[0183] The second fluid 410 can comprise 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.
[0184] In some instances, the second fluid 410 may not be subjected
to and/or directed to any flow in or out of the reservoir 404. For
example, the second fluid 410 may be substantially stationary in
the reservoir 404. In some instances, the second fluid 410 may be
subjected to flow within the reservoir 404, but not in or out of
the reservoir 404, such as via application of pressure to the
reservoir 404 and/or as affected by the incoming flow of the
aqueous fluid 408 at the junction 406. Alternatively, the second
fluid 410 may be subjected and/or directed to flow in or out of the
reservoir 404. For example, the reservoir 404 can be a channel
directing the second fluid 410 from upstream to downstream,
transporting the generated droplets.
[0185] The channel structure 400 at or near the junction 406 may
have certain geometric features that at least partly determine the
sizes of the droplets formed by the channel structure 400. The
channel segment 402 can have a height, h.sub.0 and width, w, at or
near the junction 406. By way of example, the channel segment 402
can comprise a rectangular cross-section that leads to a reservoir
404 having a wider cross-section (such as in width or diameter).
Alternatively, the cross-section of the channel segment 402 can be
other shapes, such as a circular shape, trapezoidal shape,
polygonal shape, or any other shapes. The top and bottom walls of
the reservoir 404 at or near the junction 406 can be inclined at an
expansion angle, .alpha.. The expansion angle, .alpha., allows the
tongue (portion of the aqueous fluid 408 leaving channel segment
402 at junction 406 and entering the reservoir 404 before droplet
formation) to increase in depth and facilitate decrease in
curvature of the intermediately formed droplet. Droplet size may
decrease with increasing expansion angle. The resulting droplet
radius, R.sub.d, may be predicted by the following equation for the
aforementioned geometric parameters of h.sub.0, w, and .alpha.:
R d .apprxeq. 0.44 ( 1 + 2.2 tan .alpha. w h 0 ) h 0 tan .alpha.
##EQU00001##
[0186] By way of example, for a channel structure with w=21 .mu.m,
h=21 .mu.m, and .alpha.=3.degree., the predicted droplet size is
121 .mu.m. In another example, for a channel structure with w=25
.mu.m, h=25 .mu.m, and .alpha.=5.degree., the predicted droplet
size is 123 .mu.m. In another example, for a channel structure with
w=28 .mu.m, h=28 .mu.m, and .alpha.=7.degree., the predicted
droplet size is 124 .mu.m.
[0187] In some instances, the expansion angle, .alpha., 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. In some instances,
the width, w, can be between a range of from about 100 micrometers
(.mu.m) to about 500 .mu.m. In some instances, the width, w, can be
between a range of from about 10 .mu.m to about 200 .mu.m.
Alternatively, the width can be less than about 10 .mu.m.
Alternatively, the width can be greater than about 500 .mu.m. In
some instances, the flow rate of the aqueous fluid 408 entering the
junction 406 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 408 entering the junction 406 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 408
entering the junction 406 can be less than about 0.01 .mu.L/min.
Alternatively, the flow rate of the aqueous fluid 408 entering the
junction 406 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 408 entering the junction
406.
[0188] In some instances, at least about 50% of the droplets
generated can have uniform size. In some instances, at least about
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
greater of the droplets generated can have uniform size.
Alternatively, less than about 50% of the droplets generated can
have uniform size.
[0189] The throughput of droplet generation can be increased by
increasing the points of generation, such as increasing the number
of junctions (e.g., junction 406) between aqueous fluid 408 channel
segments (e.g., channel segment 402) and the reservoir 404.
Alternatively or in addition, the throughput of droplet generation
can be increased by increasing the flow rate of the aqueous fluid
408 in the channel segment 402.
[0190] FIG. 5 shows an example of a microfluidic channel structure
for increased droplet generation throughput. A microfluidic channel
structure 500 can comprise a plurality of channel segments 502 and
a reservoir 504. Each of the plurality of channel segments 502 may
be in fluid communication with the reservoir 504. The channel
structure 500 can comprise a plurality of channel junctions 506
between the plurality of channel segments 502 and the reservoir
504. Each channel junction can be a point of droplet generation.
The channel segment 402 from the channel structure 400 in FIG. 4
and any description to the components thereof may correspond to a
given channel segment of the plurality of channel segments 502 in
channel structure 500 and any description to the corresponding
components thereof. The reservoir 404 from the channel structure
400 and any description to the components thereof may correspond to
the reservoir 504 from the channel structure 500 and any
description to the corresponding components thereof.
[0191] Each channel segment of the plurality of channel segments
502 may comprise an aqueous fluid 508 that includes suspended beads
512. The reservoir 504 may comprise a second fluid 510 that is
immiscible with the aqueous fluid 508. In some instances, the
second fluid 510 may not be subjected to and/or directed to any
flow in or out of the reservoir 504. For example, the second fluid
510 may be substantially stationary in the reservoir 504. In some
instances, the second fluid 510 may be subjected to flow within the
reservoir 504, but not in or out of the reservoir 504, such as via
application of pressure to the reservoir 504 and/or as affected by
the incoming flow of the aqueous fluid 508 at the junctions.
Alternatively, the second fluid 510 may be subjected and/or
directed to flow in or out of the reservoir 504. For example, the
reservoir 504 can be a channel directing the second fluid 510 from
upstream to downstream, transporting the generated droplets.
[0192] In operation, the aqueous fluid 508 that includes suspended
beads 512 may be transported along the plurality of channel
segments 502 into the plurality of junctions 506 to meet the second
fluid 510 in the reservoir 504 to create droplets 516, 518. A
droplet may form from each channel segment at each corresponding
junction with the reservoir 504. At the junction where the aqueous
fluid 508 and the second fluid 510 meet, droplets can form based on
factors such as the hydrodynamic forces at the junction, flow rates
of the two fluids 508, 510, fluid properties, and certain geometric
parameters (e.g., w, h.sub.0, .alpha., etc.) of the channel
structure 500, as described elsewhere herein. A plurality of
droplets can be collected in the reservoir 504 by continuously
injecting the aqueous fluid 508 from the plurality of channel
segments 502 through the plurality of junctions 506. Throughput may
significantly increase with the parallel channel configuration of
channel structure 500. For example, a channel structure having five
inlet channel segments comprising the aqueous fluid 508 may
generate droplets five times as frequently than a channel structure
having one inlet channel segment, provided that the fluid flow rate
in the channel segments are substantially the same. The fluid flow
rate in the different inlet channel segments may or may not be
substantially the same. A channel structure may have as many
parallel channel segments as is practical and allowed for the size
of the reservoir. For example, the channel structure may have at
least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 150, 500, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1000, 1500, 5000 or more parallel or substantially parallel
channel segments.
[0193] The geometric parameters, w, h.sub.a and .alpha., may or may
not be uniform for each of the channel segments in the plurality of
channel segments 502. For example, each channel segment may have
the same or different widths at or near its respective channel
junction with the reservoir 504. For example, each channel segment
may have the same or different height at or near its respective
channel junction with the reservoir 504. In another example, the
reservoir 504 may have the same or different expansion angle at the
different channel junctions with the plurality of channel segments
502. When the geometric parameters are uniform, beneficially,
droplet size may also be controlled to be uniform even with the
increased throughput. In some instances, the geometric parameters
for the plurality of channel segments 502 may be varied accordingly
to provide a different distribution of droplet sizes.
[0194] In some instances, at least about 50% of the droplets
generated can have uniform size. In some instances, at least about
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
greater of the droplets generated can have uniform size.
Alternatively, less than about 50% of the droplets generated can
have uniform size.
[0195] FIG. 6 shows another example of a microfluidic channel
structure for increased droplet generation throughput. A
microfluidic channel structure 600 can comprise a plurality of
channel segments 602 arranged generally circularly around the
perimeter of a reservoir 604. Each of the plurality of channel
segments 602 may be in fluid communication with the reservoir 604.
The channel structure 600 can comprise a plurality of channel
junctions 606 between the plurality of channel segments 602 and the
reservoir 604. Each channel junction can be a point of droplet
generation. The channel segment 402 from the channel structure 400
in FIG. 2 and any description to the components thereof may
correspond to a given channel segment of the plurality of channel
segments 602 in channel structure 600 and any description to the
corresponding components thereof. The reservoir 404 from the
channel structure 400 and any description to the components thereof
may correspond to the reservoir 604 from the channel structure 600
and any description to the corresponding components thereof.
[0196] Each channel segment of the plurality of channel segments
602 may comprise an aqueous fluid 608 that includes suspended beads
612. The reservoir 604 may comprise a second fluid 610 that is
immiscible with the aqueous fluid 608. In some instances, the
second fluid 610 may not be subjected to and/or directed to any
flow in or out of the reservoir 604. For example, the second fluid
610 may be substantially stationary in the reservoir 604. In some
instances, the second fluid 610 may be subjected to flow within the
reservoir 604, but not in or out of the reservoir 604, such as via
application of pressure to the reservoir 604 and/or as affected by
the incoming flow of the aqueous fluid 608 at the junctions.
Alternatively, the second fluid 610 may be subjected and/or
directed to flow in or out of the reservoir 604. For example, the
reservoir 604 can be a channel directing the second fluid 610 from
upstream to downstream, transporting the generated droplets.
[0197] In operation, the aqueous fluid 608 that includes suspended
beads 612 may be transported along the plurality of channel
segments 602 into the plurality of junctions 606 to meet the second
fluid 610 in the reservoir 604 to create a plurality of droplets
616. A droplet may form from each channel segment at each
corresponding junction with the reservoir 604. At the junction
where the aqueous fluid 608 and the second fluid 610 meet, droplets
can form based on factors such as the hydrodynamic forces at the
junction, flow rates of the two fluids 608, 610, fluid properties,
and certain geometric parameters (e.g., widths and heights of the
channel segments 602, expansion angle of the reservoir 604, etc.)
of the channel structure 600, as described elsewhere herein. A
plurality of droplets can be collected in the reservoir 604 by
continuously injecting the aqueous fluid 608 from the plurality of
channel segments 602 through the plurality of junctions 606.
Throughput may significantly increase with the substantially
parallel channel configuration of the channel structure 600. A
channel structure may have as many substantially parallel channel
segments as is practical and allowed for by the size of the
reservoir. For example, the channel structure may have at least
about 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, 1500, 5000 or more parallel or substantially parallel channel
segments. The plurality of channel segments may be substantially
evenly spaced apart, for example, around an edge or perimeter of
the reservoir. Alternatively, the spacing of the plurality of
channel segments may be uneven.
[0198] The reservoir 604 may have an expansion angle, a (not shown
in FIG. 6) at or near each channel junction. Each channel segment
of the plurality of channel segments 602 may have a width, w, and a
height, h.sub.0, at or near the channel junction. The geometric
parameters, w, h.sub.a and .alpha., may or may not be uniform for
each of the channel segments in the plurality of channel segments
602. For example, each channel segment may have the same or
different widths at or near its respective channel junction with
the reservoir 604. For example, each channel segment may have the
same or different height at or near its respective channel junction
with the reservoir 604.
[0199] The reservoir 604 may have the same or different expansion
angle at the different channel junctions with the plurality of
channel segments 602. For example, a circular reservoir (as shown
in FIG. 6) may have a conical, dome-like, or hemispherical ceiling
(e.g., top wall) to provide the same or substantially same
expansion angle for each channel segments 602 at or near the
plurality of channel junctions 606. When the geometric parameters
are uniform, beneficially, resulting droplet size may be controlled
to be uniform even with the increased throughput. In some
instances, the geometric parameters for the plurality of channel
segments 602 may be varied accordingly to generate a different
distribution of droplet sizes.
[0200] In some instances, at least about 50% of the droplets
generated can have uniform size. In some instances, at least about
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
greater of the droplets generated can have uniform size.
Alternatively, less than about 50% of the droplets generated can
have uniform size. The beads and/or biological particle or analyte
carrier injected into the droplets may or may not have uniform
size.
[0201] FIG. 7A shows a cross-section view of another example of a
microfluidic channel structure with a geometric feature for
controlled partitioning. A channel structure 700 can include a
channel segment 702 communicating at a channel junction 706 (or
intersection) with a reservoir 704. In some instances, the channel
structure 700 and one or more of its components can correspond to
the channel structure 100 and one or more of its components. FIG.
7B shows a perspective view of the channel structure 700 of FIG.
7A.
[0202] An aqueous fluid 712 comprising a plurality of particles 716
may be transported along the channel segment 702 into the junction
706 to meet a second fluid 714 (e.g., oil, etc.) that is immiscible
with the aqueous fluid 712 in the reservoir 704 to create droplets
720 of the aqueous fluid 712 flowing into the reservoir 704. At the
junction 706 where the aqueous fluid 712 and the second fluid 714
meet, droplets can form based on factors such as the hydrodynamic
forces at the junction 706, relative flow rates of the two fluids
712, 714, fluid properties, and certain geometric parameters (e.g.,
.DELTA.h, etc.) of the channel structure 700. A plurality of
droplets can be collected in the reservoir 704 by continuously
injecting the aqueous fluid 712 from the channel segment 702 at the
junction 706.
[0203] A discrete droplet generated may comprise one or more
particles of the plurality of particles 716. As described elsewhere
herein, a particle may be any particle, such as a bead, cell bead,
gel bead, biological particle or analyte carrier, macromolecular
constituents of biological particle or analyte carrier, or other
particles. Alternatively, a discrete droplet generated may not
include any particles.
[0204] In some instances, the aqueous fluid 712 can have a
substantially uniform concentration or frequency of particles 716.
As described elsewhere herein (e.g., with reference to FIG. 4), the
particles 716 (e.g., beads) can be introduced into the channel
segment 702 from a separate channel (not shown in FIG. 7). The
frequency of particles 716 in the channel segment 702 may be
controlled by controlling the frequency in which the particles 716
are introduced into the channel segment 702 and/or the relative
flow rates of the fluids in the channel segment 702 and the
separate channel. In some instances, the particles 716 can be
introduced into the channel segment 702 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 or
analyte carriers into the channel segment 702. The first separate
channel introducing the beads may be upstream or downstream of the
second separate channel introducing the biological particles or
analyte carriers.
[0205] In some instances, the second fluid 714 may not be subjected
to and/or directed to any flow in or out of the reservoir 704. For
example, the second fluid 714 may be substantially stationary in
the reservoir 704. In some instances, the second fluid 714 may be
subjected to flow within the reservoir 704, but not in or out of
the reservoir 704, such as via application of pressure to the
reservoir 704 and/or as affected by the incoming flow of the
aqueous fluid 712 at the junction 706. Alternatively, the second
fluid 714 may be subjected and/or directed to flow in or out of the
reservoir 704. For example, the reservoir 704 can be a channel
directing the second fluid 714 from upstream to downstream,
transporting the generated droplets.
[0206] The channel structure 700 at or near the junction 706 may
have certain geometric features that at least partly determine the
sizes and/or shapes of the droplets formed by the channel structure
700. The channel segment 702 can have a first cross-section height,
h.sub.1, and the reservoir 704 can have a second cross-section
height, h.sub.2. The first cross-section height, h.sub.1, and the
second cross-section height, h.sub.2, may be different, such that
at the junction 706, there is a height difference of zlh. The
second cross-section height, h.sub.2, may be greater than the first
cross-section height, h.sub.1. In some instances, the reservoir may
thereafter gradually increase in cross-section height, for example,
the more distant it is from the junction 706. In some instances,
the cross-section height of the reservoir may increase in
accordance with expansion angle, .beta., at or near the junction
706. The height difference, .DELTA.h, and/or expansion angle,
.beta., can allow the tongue (portion of the aqueous fluid 712
leaving channel segment 702 at junction 706 and entering the
reservoir 704 before droplet formation) to increase in depth and
facilitate decrease in curvature of the intermediately formed
droplet. For example, droplet size may decrease with increasing
height difference and/or increasing expansion angle.
[0207] 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,
.beta., 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.
[0208] In some instances, the flow rate of the aqueous fluid 712
entering the junction 706 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 712 entering the junction 706 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 712
entering the junction 706 can be less than about 0.01 .mu.L/min.
Alternatively, the flow rate of the aqueous fluid 712 entering the
junction 706 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 712 entering the junction
706. The second fluid 714 may be stationary, or substantially
stationary, in the reservoir 704. Alternatively, the second fluid
714 may be flowing, such as at the above flow rates described for
the aqueous fluid 712.
[0209] In some instances, at least about 50% of the droplets
generated can have uniform size. In some instances, at least about
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
greater of the droplets generated can have uniform size.
Alternatively, less than about 50% of the droplets generated can
have uniform size.
[0210] While FIGS. 7A and 7B illustrate the height difference,
.DELTA.h, being abrupt at the junction 706 (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 706, 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 FIGS. 7A and 7B illustrate the expanding
reservoir cross-section height as linear (e.g., constant expansion
angle, .beta.), 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.
[0211] The channel networks, e.g., as described above or elsewhere
herein, can be fluidly coupled to appropriate fluidic components.
For example, the inlet channel segments are fluidly coupled to
appropriate sources of the materials they are to deliver to a
channel junction. These sources may include any of a variety of
different fluidic components, from simple reservoirs defined in or
connected to a body structure of a microfluidic device, to fluid
conduits that deliver fluids from off-device sources, manifolds,
fluid flow units (e.g., actuators, pumps, compressors) or the like.
Likewise, the outlet channel segment (e.g., channel segment 208,
reservoir 604, etc.) may be fluidly coupled to a receiving vessel
or conduit for the partitioned cells for subsequent processing.
Again, this may be a reservoir defined in the body of a
microfluidic device, or it may be a fluidic conduit for delivering
the partitioned cells to a subsequent process operation, instrument
or component.
[0212] The methods and systems described herein may be used to
greatly increase the efficiency of cell applications and/or other
applications receiving droplet-based input. For example, following
the sorting of occupied cells and/or appropriately-sized cells,
subsequent operations that can be performed can include generation
of amplification products, purification (e.g., via solid phase
reversible immobilization (SPRI)), further processing (e.g.,
shearing, ligation of functional sequences, and subsequent
amplification (e.g., via PCR)). These operations may occur in bulk
(e.g., outside the partition). In the case where a partition is a
droplet in an emulsion, the emulsion can be broken and the contents
of the droplet pooled for additional operations. Additional
reagents that may be co-partitioned along with the barcode bearing
bead may include oligonucleotides to block ribosomal RNA (rRNA) and
nucleases to digest genomic DNA from cells. Alternatively, rRNA
removal agents may be applied during additional processing
operations. The configuration of the constructs generated by such a
method can help minimize (or avoid) sequencing of the poly-T
sequence during sequencing and/or sequence the 5' end of a
polynucleotide sequence. The amplification products, for example,
first amplification products and/or second amplification products,
may be subject to sequencing for sequence analysis. In some cases,
amplification may be performed using the Partial Hairpin
Amplification for Sequencing (PHASE) method.
[0213] A variety of applications require the evaluation of the
presence and quantification of different biological particle or
analyte carrier or organism types within a population of biological
particles or analyte carriers, including, for example, microbiome
analysis and characterization, environmental testing, food safety
testing, epidemiological analysis, e.g., in tracing contamination
or the like.
Computer Systems
[0214] The present disclosure provides computer systems that are
programmed to implement methods of the disclosure. FIG. 9 shows an
example computer system 901 that can be programmed or otherwise
configured to, for example, process and/or analyze a metabolite,
control addition of reagents to reaction mixtures, control
partition generation, control of reagent addition to partitions,
provide conditions sufficient to conduct reactions, obtain and
process sequencing data, output sequencing results to a user,
provide an interface for user input to control devices coupled to
the computer processor. The computer system 901 can regulate
various aspects of the present disclosure, such as, for example,
regulating fluid flow, delivery of reagents, partition generation,
modulate reactions conditions, etc.. The computer system 901 can be
an electronic device of a user or a computer system that is
remotely located with respect to the electronic device. The
electronic device can be a mobile electronic device.
[0215] The computer system 901 includes a central processing unit
(CPU, also "processor" and "computer processor" herein) 905, which
can be a single core or multi core processor, or a plurality of
processors for parallel processing. The computer system 901 also
includes memory or memory location 910 (e.g., random-access memory,
read-only memory, flash memory), electronic storage unit 915 (e.g.,
hard disk), communication interface 920 (e.g., network adapter) for
communicating with one or more other systems, and peripheral
devices 925, such as cache, other memory, data storage and/or
electronic display adapters. The memory 910, storage unit 915,
interface 920 and peripheral devices 925 are in communication with
the CPU 905 through a communication bus (solid lines), such as a
motherboard. The storage unit 915 can be a data storage unit (or
data repository) for storing data. The computer system 901 can be
operatively coupled to a computer network ("network") 930 with the
aid of the communication interface 920. The network 930 can be the
Internet, an internet and/or extranet, or an intranet and/or
extranet that is in communication with the Internet. The network
930 in some cases is a telecommunication and/or data network. The
network 930 can include one or more computer servers, which can
enable distributed computing, such as cloud computing. The network
930, in some cases with the aid of the computer system 901, can
implement a peer-to-peer network, which may enable devices coupled
to the computer system 901 to behave as a client or a server.
[0216] The CPU 905 can execute a sequence of machine-readable
instructions, which can be embodied in a program or software. The
instructions may be stored in a memory location, such as the memory
910. The instructions can be directed to the CPU 905, which can
subsequently program or otherwise configure the CPU 905 to
implement methods of the present disclosure. Examples of operations
performed by the CPU 905 can include fetch, decode, execute, and
writeback.
[0217] The CPU 905 can be part of a circuit, such as an integrated
circuit. One or more other components of the system 901 can be
included in the circuit. In some cases, the circuit is an
application specific integrated circuit (ASIC).
[0218] The storage unit 915 can store files, such as drivers,
libraries and saved programs. The storage unit 915 can store user
data, e.g., user preferences and user programs. The computer system
901 in some cases can include one or more additional data storage
units that are external to the computer system 901, such as located
on a remote server that is in communication with the computer
system 901 through an intranet or the Internet.
[0219] The computer system 901 can communicate with one or more
remote computer systems through the network 930. For instance, the
computer system 901 can communicate with a remote computer system
of a user (e.g., operator). Examples of remote computer systems
include personal computers (e.g., portable PC), slate or tablet
PC's (e.g., Apple.RTM. iPad, Samsung.RTM. Galaxy Tab), telephones,
Smart phones (e.g., Apple.RTM. iPhone, Android-enabled device,
Blackberry.RTM.), or personal digital assistants. The user can
access the computer system 901 via the network 930.
[0220] Methods as described herein can be implemented by way of
machine (e.g., computer processor) executable code stored on an
electronic storage location of the computer system 901, such as,
for example, on the memory 910 or electronic storage unit 915. The
machine executable or machine readable code can be provided in the
form of software. During use, the code can be executed by the
processor 905. In some cases, the code can be retrieved from the
storage unit 915 and stored on the memory 910 for ready access by
the processor 905. In some situations, the electronic storage unit
915 can be precluded, and machine-executable instructions are
stored on memory 910.
[0221] The code can be pre-compiled and configured for use with a
machine having a processor adapted to execute the code, or can be
compiled during runtime. The code can be supplied in a programming
language that can be selected to enable the code to execute in a
pre-compiled or as-compiled fashion.
[0222] Aspects of the systems and methods provided herein, such as
the computer system 901, can be embodied in programming. Various
aspects of the technology may be thought of as "products" or
"articles of manufacture" typically in the form of machine (or
processor) executable code and/or associated data that is carried
on or embodied in a type of machine readable medium.
Machine-executable code can be stored on an electronic storage
unit, such as memory (e.g., read-only memory, random-access memory,
flash memory) or a hard disk. "Storage" type media can include any
or all of the tangible memory of the computers, processors or the
like, or associated modules thereof, such as various semiconductor
memories, tape drives, disk drives and the like, which may provide
non-transitory storage at any time for the software programming.
All or portions of the software may at times be communicated
through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the
software from one computer or processor into another, for example,
from a management server or host computer into the computer
platform of an application server. Thus, another type of media that
may bear the software elements includes optical, electrical and
electromagnetic waves, such as used across physical interfaces
between local devices, through wired and optical landline networks
and over various air-links. The physical elements that carry such
waves, such as wired or wireless links, optical links or the like,
also may be considered as media bearing the software. As used
herein, unless restricted to non-transitory, tangible "storage"
media, terms such as computer or machine "readable medium" refer to
any medium that participates in providing instructions to a
processor for execution.
[0223] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, a tangible storage medium, a carrier wave medium or
physical transmission medium. Non-volatile storage media include,
for example, optical or magnetic disks, such as any of the storage
devices in any computer(s) or the like, such as may be used to
implement the databases, etc. shown in the drawings. Volatile
storage media include dynamic memory, such as main memory of such a
computer platform. Tangible transmission media include coaxial
cables; copper wire and fiber optics, including the wires that
comprise a bus within a computer system. Carrier-wave transmission
media may take the form of electric or electromagnetic signals, or
acoustic or light waves such as those generated during radio
frequency (RF) and infrared (IR) data communications. Common forms
of computer-readable media therefore include for example: a floppy
disk, a flexible disk, hard disk, magnetic tape, any other magnetic
medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch
cards paper tape, any other physical storage medium with patterns
of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other
memory chip or cartridge, a carrier wave transporting data or
instructions, cables or links transporting such a carrier wave, or
any other medium from which a computer may read programming code
and/or data. Many of these forms of computer readable media may be
involved in carrying one or more sequences of one or more
instructions to a processor for execution.
[0224] The computer system 901 can include or be in communication
with an electronic display 935 that comprises a user interface (UI)
940 for providing, for example, monitoring of sample preparation,
monitoring of reactions and/or reaction conditions, monitoring of
sequencing, results of sequencing, and permitting user inputs for
sample preparation, reactions, sequencing and/or sequencing
analysis. Examples of UIs include, without limitation, a graphical
user interface (GUI) and web-based user interface.
[0225] Methods and systems of the present disclosure can be
implemented by way of one or more algorithms. An algorithm can be
implemented by way of software upon execution by the central
processing unit 905. The algorithm can, for example, implement
sample preparation protocols, reaction protocols, sequencing
protocols, data analysis protocols and system or device operation
protocols.
[0226] Devices, systems, compositions and methods of the present
disclosure may be used for various applications, such as, for
example, 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 cell. For example, a
biological particle or analyte carrier (e.g., a cell or cell bead)
is partitioned in a partition (e.g., droplet), and multiple
analytes from the biological particle or analyte carrier are
processed for subsequent processing. The multiple analytes may be
from the cell. This may enable, for example, simultaneous
proteomic, transcriptomic and genomic analysis of the cell.
EXAMPLES
Example 1: GEM Generation and Barcoding
[0227] A suspension of cells is prepared, where the viability, as
measured by trypan blue staining, is greater than 80%. The
suspension of cells can be optionally filtered through a 40 .mu.m
strainer prior to loading onto a microfluidic chip. A master mix is
then prepared which contains the reverse transcriptase (RT) reagent
mix, RT primer, DTT, the RT enzyme mix and a volume of cells
according to an expected cell recovery number, up to a volume of
100 .mu.L. The master mix may contain riboswitches which may bind
to a particular metabolite from a cell and expose a capture
sequence on the riboswitch. 90 .mu.L of the master mix is loaded
into a first channel of the microfluidic chip. 40 .mu.L of gel
beads comprising barcode sequences (e.g., each bead comprising a
single barcode sequence) is loaded into a second channel of this
chip. 270 .mu.L of partitioning oil is loaded into a third channel
of the chip. To generate a gel bead in emulsions (GEMs) comprising
a cell and a barcoded gel bead, a specially designed gasket is
attached to the chip and then the loaded chip is transferred to a
controller. The instrument will recognize the chip and the user can
initialize the generation of GEMs by pressing a button. A given GEM
may contain one or more sequences for analysis, including, for
example, RNA from a cell in the GEM (e.g., mRNA) and/or a
riboswitch sequence (e.g., a riboswitch capture sequence) in the
GEM. After the instrument has generated the GEMs, 100 .mu.l of GEMs
are aspirated from the recovery wells and are dispensed carefully
into PCR tube(s).
[0228] A gel bead in emulsion-reverse transcriptase (GEM-RT)
reaction can then be performed