U.S. patent application number 16/715119 was filed with the patent office on 2020-04-16 for methods and systems for sorting droplets and beads.
The applicant listed for this patent is 10X Genomics, Inc.. Invention is credited to Rajiv BHARADWAJ, Anthony MAKAREWICZ, Michael SCHNALL-LEVIN, Steven SHORT.
Application Number | 20200115703 16/715119 |
Document ID | / |
Family ID | 64270560 |
Filed Date | 2020-04-16 |
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United States Patent
Application |
20200115703 |
Kind Code |
A1 |
BHARADWAJ; Rajiv ; et
al. |
April 16, 2020 |
METHODS AND SYSTEMS FOR SORTING DROPLETS AND BEADS
Abstract
Methods and systems for sorting droplets are provided. In some
cases, occupied droplets may be sorted from unoccupied droplets. In
some cases, singularly occupied droplets may be sorted from
unoccupied droplets and multiply occupied droplets. Methods and
systems for sorting cell beads are provided. In some cases, cell
beads may be sorted from particles unoccupied with cell
derivatives. In some cases, singularly occupied cell beads may be
sorted from unoccupied particles and multiply occupied cell beads.
Methods and systems for selectively polymerizing droplets based on
occupancy and size of the droplets are provided.
Inventors: |
BHARADWAJ; Rajiv;
(Pleasanton, CA) ; SCHNALL-LEVIN; Michael; (San
Francisco, CA) ; MAKAREWICZ; Anthony; (Livermore,
CA) ; SHORT; Steven; (Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
10X Genomics, Inc. |
Pleasanton |
CA |
US |
|
|
Family ID: |
64270560 |
Appl. No.: |
16/715119 |
Filed: |
December 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16031880 |
Jul 10, 2018 |
10544413 |
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16715119 |
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PCT/US2018/033280 |
May 17, 2018 |
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16031880 |
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62508219 |
May 18, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/502715 20130101;
B01L 2300/0867 20130101; C12Q 2565/629 20130101; G01N 15/1056
20130101; G01N 2015/149 20130101; B01L 2400/086 20130101; B01L
2200/0652 20130101; C12N 15/1075 20130101; G01N 15/1484 20130101;
C12Q 2563/179 20130101; G01N 2015/1081 20130101; C12N 15/1006
20130101; B01L 3/502784 20130101; G01N 15/1404 20130101; G01N
21/6428 20130101; C12Q 1/6806 20130101; C12Q 1/6834 20130101; C12Q
2563/149 20130101; B01L 2400/0415 20130101; G01N 15/1031 20130101;
B01L 2400/043 20130101; C12N 15/1065 20130101; G01N 2015/1006
20130101; B01L 3/502761 20130101; B01L 2200/0673 20130101; C12N
15/1075 20130101; C12Q 2535/122 20130101; C12Q 2563/149 20130101;
C12Q 2563/179 20130101; C12Q 2565/629 20130101; C12Q 1/6806
20130101; C12Q 2531/113 20130101; C12Q 2535/122 20130101; C12Q
2563/113 20130101; C12Q 2563/159 20130101; C12Q 2565/629 20130101;
C12Q 1/6806 20130101; C12Q 2531/113 20130101; C12Q 2535/122
20130101; C12Q 2563/143 20130101; C12Q 2563/149 20130101; C12Q
2563/159 20130101; C12Q 2565/629 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; B01L 3/00 20060101 B01L003/00; G01N 15/14 20060101
G01N015/14; G01N 15/10 20060101 G01N015/10; C12Q 1/6806 20060101
C12Q001/6806; G01N 21/64 20060101 G01N021/64 |
Claims
1. A method for sorting droplets, comprising: (a) bringing a first
phase in contact with a second phase immiscible with said first
phase, to generate a plurality of droplets, wherein said plurality
of droplets comprises (i) a first subset of droplets each
including, and not more than, one biological particle, and (ii) a
second subset of droplets each either having more than one
biological particle or not having any biological particle, wherein
said biological particle is a cell, a derivative of a cell, or a
constituent of a cell; (b) directing said plurality of droplets
along a first channel towards an intersection of said first channel
with at least a second channel and a third channel; and (c)
separating at least a portion of said first subset of said
plurality of droplets from at least a portion of said second subset
of said plurality of droplets, wherein upon separation, said at
least said portion of said first subset of said plurality of
droplets flows along said second channel and said at least said
portion of said second subset of said plurality of droplets flows
along said third channel.
2. The method of claim 1, wherein said first subset of said
plurality of droplets includes particles having coupled thereto
molecules comprising barcode sequences.
3. The method of claim 2, wherein said particles are gel beads.
4. The method of claim 1, further comprising detecting individual
droplets of said first subset of said plurality of droplets, and
subjecting said individual droplets to a stimulus to facilitate
polymerization in said biological particles upon detecting said
individual droplets.
5. The method of claim 4 wherein said stimulus is applied prior to
said intersection.
6. The method of claim 4, wherein said stimulus is applied
subsequent to said intersection.
7. The method of claim 4, wherein said stimulus is an optical
stimulus or chemical stimulus.
8. The method of claim 1, wherein said biological particles are
cells enclosed within or comprising a gel or polymer matrix.
9. The method of claim 1, wherein each of said plurality of
droplets comprises field-attractable particles, and wherein (c)
comprises subjecting said plurality of droplets to an electric or
magnetic field under conditions sufficient to separate said at
least said portion of said first subset from said at least said
portion of said second subset.
10. The method of claim 9, wherein a concentration of said
field-attractable particles in droplets of said second subset not
having any biological particle is substantially uniform.
11. The method of claim 9, wherein each droplet of said first
subset comprises (i) less field attractable particles than each
droplet of said second subset not having any biological particle,
and (ii) more field attractable particles than each droplet of said
second subset having more than one biological particle.
12. The method of claim 11, wherein forces induced by said electric
or magnetic field on droplets of said second subset not having any
biological particle are greater than forces induced on said first
subset, and wherein forces induced on said first subset are greater
than forces induced on droplets of said second subset having more
than one biological particle.
13. The method of claim 9, wherein said field-attractable particles
are magnetic-field attractable particles.
14. The method of claim 9, wherein said conditions of said electric
or magnetic field sufficient to separate said at least said portion
of said first subset and said at least said portion of said second
subset are determined based at least in part on a ratio between
sizes of said plurality of droplets and sizes of said biological
particles in said first subset of said plurality of droplets.
15. The method of claim 1, further comprising, subsequent to (c),
subjecting nucleic acid molecules derived from said biological
particles in said first subset to nucleic acid sequencing.
16. The method of claim 15, further comprising, subsequent to (c),
subjecting said first subset of said plurality of droplets to
conditions sufficient to yield extension products of said nucleic
acid molecules from said biological particles in said first subset,
and subjecting said extension products or derivatives thereof to
nucleic acid sequencing.
17. The method of claim 1, wherein (c) comprises subjecting said
plurality of droplets to a pressure pulse under conditions
sufficient to separate said at least said portion of said first
subset from said at least said portion of said second subset.
18. The method of claim 17, wherein forces induced by said pressure
pulse on droplets of said second subset not having any biological
particle are greater than forces induced on said first subset, and
wherein forces induced on said first subset are greater than forces
induced on droplets of said second subset having more than one
biological particle.
19. The method of claim 1, wherein upon separation, said at least
said portion of said first subset and droplets of said second
subset having more than one biological particle flow along said
second channel and droplets of said second subset not having any
biological particle flow along said third channel.
20. The method of claim 19, further comprising (i) directing said
at least said portion of said first subset and said droplets of
said second subset having more than one biological particle along
said second channel towards a second intersection of said second
channel with at least a fourth channel and a fifth channel, and
(ii) separating said at least said portion of said first subset
from at least a portion of said droplets of said second subset
having more than one biological particle, wherein upon separation,
said at least said portion of said first subset flows along said
fourth channel and said at least said portion of said droplets of
said second subset having more than one biological particle flows
along said fifth channel.
21. A method for sorting particles, comprising: (a) providing a
plurality of particles comprising (i) a first set of particles
having a plurality of cells or material therefrom, and (ii) a
second set of particles each not having any cell or material
therefrom; and (b) sorting said plurality of particles, thereby
isolating at least a portion of said first set of particles from at
least a portion of said second set of particles.
22. The method of claim 21, wherein said first set of particles
comprises (i) a first subset of particles each including, but not
more than, one cell of said plurality of cells, or material from
said cell, and (ii) a second subset of particles each including
more than one cell of said plurality of cells, or material from
said more than one cell.
23. The method of claim 22, further comprising isolating at least a
portion of said first subset of particles from at least a portion
of said second subset of particles.
24. The method of claim 21, wherein (b) comprises subjecting said
plurality of particles to a magnetic or electric field to isolate
said at least said portion of said first subset of particles from
said at least said portion of said second subset of particles.
25. The method of claim 24, wherein each particle of said plurality
of particles comprises field-attractable particles.
26. The method of claim 21, wherein (b) comprises subjecting said
plurality of particles to a pressure pulse to isolate said at least
said portion of said first subset of particles from said at least
said portion of said second subset of particles.
27. The method of claim 21, wherein each of said plurality of
particles comprises a gel or polymer matrix.
28. The method of claim 27, further comprising, prior to (a),
generating said plurality of particles by (i) encapsulating each of
said plurality of cells or material therefrom in a gel or polymer
matrix, or (ii) generating said gel or polymer matrix in each of
said plurality of cells.
29. The method of claim 21, further comprising, subsequent to (b),
subjecting nucleic acid molecules derived from said first subset to
nucleic acid sequencing.
30. The method of claim 21, wherein a particle of said first set of
particles comprises a cell of said plurality of cells, or material
derived from said cell.
Description
CROSS-REFERENCE
[0001] This application is a continuation of International
Application No. PCT/US2018/033280, filed May 17, 2018, which claims
the benefit of U.S. Provisional Patent Application No. 62/508,219,
filed May 18, 2017, 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 using various reaction
environments, such as partitions. Partitions may be wells or
droplets. Droplets or wells may enable 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] A plurality of droplets can be generated such that one or
more droplets include cells and/or particles. The cells and/or
particles can be of interest for use in various (e.g., single cell)
applications, such as nucleic acid amplification and/or sequencing
applications.
SUMMARY
[0005] As recognized herein, when a plurality of droplets is
generated, some droplets may not include any particles, such as
cells and beads. A particle may be a bead, such as a gel bead
and/or a cell bead. A particle may be a biological particle, such
as a cell or cell derivative. A particle, such as a gel bead, may
have a molecular barcode coupled thereto. Thus, recognized herein
is a need to sort the plurality of droplets into a first subset of
droplets that include particles and a second subset of droplets
that do not. In some instances, when a plurality of cell beads is
generated, some particles generated with the plurality of cell
beads may not include any cells (e.g., non-cell bead). Recognized
herein is a need to isolate the plurality of cell beads, such as by
sorting a plurality of particles into a first subset of particles
that include cells (e.g., cell beads) and a second subset of
particles that do not.
[0006] In some aspects, the systems and methods for sorting
described herein may yield an output comprising mostly singularly
occupied droplets (containing a single particle of interest). For
example, at least about 90%, 95%, 96%, 97%, 98%, 99%, or more of a
plurality of droplets may be singularly occupied droplets. Droplets
may be sorted, such as by (i) introducing field-attractable
particles (e.g., magnetic particles) into the droplets and
subjecting the droplets to a field (e.g., magnetic field), (ii)
subjecting the droplets to a pressure pulse and separating the
droplets based on hydrodynamic forces, and/or (iii) directing the
droplets to interface physical structures (e.g., having apertures)
in a flow path of the droplets and separating the droplets based on
mechanical properties (e.g., deformability) of the droplets.
[0007] In some aspects, the systems and methods for sorting
described herein may yield an output comprising mostly singularly
occupied cell beads (containing a single cell). For example, at
least about 90%, 95%, 96%, 97%, 98%, 99%, or more of a population
of beads (or particles) may be singularly occupied cell beads. Cell
beads may be isolated (or sorted), such as by (i) generating cell
beads with field-attractable particles (e.g., magnetic particles),
such as by polymerizing the droplets containing the
field-attractable particles, and subjecting the cell beads to a
field (e.g., magnetic field), (ii) subjecting the cell beads to a
pressure pulse and separating the cell beads via hydrodynamic
forces, and/or (iii) directing the cell beads to interface physical
structures (e.g., having apertures) in a flow path of the cell
beads and separating the cell beads based on mechanical properties
(e.g., deformability) of the cell beads. In some cases, already
sorted droplets, which are mostly singularly occupied droplets
(e.g., containing a single cell), may be polymerized to generate
cell beads that are mostly singularly occupied. In some cases, a
plurality of droplets may be selectively polymerized, such that
mostly (or only) singularly occupied droplets are polymerized to
generate cell beads that are mostly singularly occupied.
[0008] Provided herein are methods and systems for sorting droplets
that can isolate droplets that include biological particles (e.g.,
a cell) and/or other particles (e.g., gel beads, cell beads, etc.)
from droplets that do not include biological particles and/or other
particles. The methods and systems may isolate droplets that are
singularly occupied from droplets that are non-singularly occupied,
such as from unoccupied droplets or multiply occupied droplets. In
another aspect, provided herein are methods and systems that can
isolate particles that include cells (e.g., cell beads) from
particles that do not include cells. The methods and systems may
isolate cell beads that are singularly occupied from particles that
are non-singularly occupied, such as from unoccupied particles or
multiply occupied cell beads. The isolated droplets (that include
biological particles and/or other particles) and/or isolated cell
beads (that include cells) can be subject to further applications,
such as nucleic acid amplification and/or sequencing. Beneficially,
such pre-sorting may increase efficiency of downstream applications
by significantly saving time and resources (e.g., valuable
reagents).
[0009] The methods and systems generally operate by generating a
plurality of droplets such that each of the plurality of droplets
comprises field-attractable particles. A given droplet in the
plurality of droplets may or may not include one or more particles
(e.g., biological particles, beads, etc.). Thus, the plurality of
droplets comprising field attractable particles can comprise a
first subset of droplets that include one or more particles and a
second subset of droplets that do not include any particles. A
given droplet in the first subset of droplets that include one or
more particles can comprise a sufficiently discrepant number or
concentration of field-attractable particles than a given droplet
in the second subset of droplets that do not include any particles
such that when the plurality of droplets is subject to an electric
or magnetic field, the first subset of droplets and the second
subset of droplets are separated from each other. In some cases,
when the plurality of droplets is subjected to an electric or
magnetic field, singularly occupied droplets may be separated from
unoccupied droplets and otherwise multiply occupied droplets.
[0010] In some instances, a plurality of particles may be generated
with field-attractable particles, such as by polymerizing the
plurality of droplets comprising the field-attractable particles. A
given particle may or may not include a cell. Thus, the plurality
of particles comprising field attractable particles may comprise a
first subset of particles (e.g., cell beads) that include cells and
a second subset of particles that does not include cells. A given
cell bead in the first subset of particles can comprise a
sufficiently discrepant number or concentration of
field-attractable particles than a given particle in the second
subset of particles, such that when the plurality of particles is
subject to an electric or magnetic field, the first subset of
particles and the second subset of particles are separated from
each other. In some cases, when the plurality of particles is
subjected to an electric or magnetic field, singularly occupied
cell beads may be separated from unoccupied particles and otherwise
multiply occupied cell beads.
[0011] In some instances, a plurality of droplets can be generated
without field-attractable particles. A given droplet in the
plurality of droplets may or may not include one or more particles.
Thus, the plurality of droplets can comprise a first subset of
droplets that include one or more particles and a second subset of
droplets that do not include any particles. The plurality of
droplets can be subject to a pressure pulse and the first subset of
droplets and the second subset of droplets can be separated from
each other via hydrodynamic forces. In some cases, the plurality of
droplets can be subject to an electric field, and the first subset
and the second subset of droplets can be separated via
dielectrophoresis. In some cases, singularly occupied droplets may
be separated from unoccupied droplets and otherwise multiply
occupied droplets.
[0012] In some instances, a plurality of particles can be generated
without field-attractable particles. A given particle in the
plurality particles may or may not include one or more cells. Thus,
the plurality of particles can comprise a first subset of particles
(e.g., cell beads) that include one or more cells and a second
subset of particles that do not include any cells. The plurality of
particles can be subject to a pressure pulse and the first subset
of particles and the second subset of particles can be separated
from each other via hydrodynamic forces. In some cases, the
plurality of particles can be subject to an electric field, and the
first subset and the second subset can be separated via
dielectrophoresis. In some cases, singularly occupied cell beads
may be separated from unoccupied particles and otherwise multiply
occupied cell beads.
[0013] In some instances, a plurality of droplets comprising a
first subset of droplets that include one or more particles and a
second subset of droplets that do not include any particles can be
sorted via a passive mechanism based on mechanical properties of
the droplets, such as the respective deformability properties of
the droplets. When the plurality of droplets is directed to pass
through one or more apertures, each aperture having a size smaller
than a minimum dimension of a droplet, only deforming droplets may
pass through the apertures and non-deforming droplets may be
trapped on the apertures. Unoccupied droplets may have higher
deformability and/or lower surface tension properties compared to
occupied droplets, thus allowing occupied droplets to be trapped on
one or more apertures, and allowing unoccupied droplets to pass
through the one or more apertures, thereby separating the first
subset and second subset of droplets from the plurality of
droplets.
[0014] In some instances, a plurality of particles comprising a
first subset of particles (e.g., cell beads) that include one or
more cells and a second subset of particles that do not include any
particles can be sorted via a passive mechanism based on mechanical
properties of the particles, such as the respective deformability
properties (or rigidity) of the particles. When the plurality of
particles is directed to pass through one or more apertures, each
aperture having a size smaller than a minimum dimension of a
particle, only deforming particles may pass through the apertures
and non-deforming particles may be trapped on the apertures.
Unoccupied particles (e.g., not having cells or their derivatives)
may have higher deformability and/or lower surface tension
properties compared to cell beads, thus allowing cell beads to be
trapped on one or more apertures, and allowing unoccupied particles
to pass through the one or more apertures, thereby separating the
first subset and second subset of particles from the plurality of
particles.
[0015] In an aspect, provided is a method for sorting droplets,
comprising: (a) bringing a first phase in contact with a second
phase to generate a plurality of droplets, wherein the first phase
and second phase are immiscible, wherein the plurality of droplets
comprises field-attractable particles and wherein (i) a first
subset of the plurality of droplets includes biological particles
or particles having coupled thereto molecular barcodes, and (ii) a
second subset of the plurality of droplets does not include the
biological particles; (b) directing the plurality of droplets along
a first channel towards an intersection of the first channel with a
second channel and a third channel; and (c) subjecting the
plurality of droplets comprising the field-attractable particles to
an electric or magnetic field under conditions sufficient to
separate at least a portion of the first subset of the plurality of
droplets from at least a portion of the second subset of the
plurality of droplets, wherein upon separation, the at least the
portion of the first subset of the plurality of droplets flows
along the second channel and the at least the portion of the second
subset of the plurality of droplets flows along the third
channel.
[0016] In some embodiments, the second subset of the plurality of
droplets does not include the particles having coupled thereto
molecular barcodes.
[0017] In some embodiments, a concentration of the
field-attractable particles in the second subset of the plurality
of droplets is substantially uniform.
[0018] In some embodiments, each droplet of the first subset of the
plurality of droplets comprises less field attractable particles
than each droplet of the second subset of the plurality of
droplets. In some embodiments, wherein the electric or magnetic
field induces forces on the second subset of the plurality of
droplets that is greater than forces induced on the first subset of
the plurality of droplets.
[0019] In some embodiments, the field-attractable particles are
magnetic-field attractable particles. In some embodiments, the
field-attractable particles are paramagnetic particles.
[0020] In some embodiments, the field-attractable particles are
electric-field attractable particles. In some embodiments, the
field-attractable particles are conductive particles.
[0021] In some embodiments, the first subset of the plurality of
droplets includes biological particles and the particles having
coupled thereto molecular barcodes. In some embodiments, the
particles having coupled thereto molecular barcodes are beads. In
some embodiments, the beads are gel beads.
[0022] In some embodiments, the method further comprises,
subsequent to (c), subjecting nucleic acid molecules derived from
the biological particles in the first subset to nucleic acid
sequencing. In some embodiments, the method further comprises,
subsequent to (c), subjecting the first subset of the plurality of
droplets to nucleic acid amplification conditions to yield
amplification products of the nucleic acid molecules from the
biological particles in the first subset. In some embodiments, the
method further comprises subjecting the amplification products to
nucleic acid sequencing.
[0023] In some embodiments, the conditions of the electric or
magnetic field sufficient to separate the at least the portion of
the first subset of the plurality of droplets and the at least the
portion of the second subset of the plurality of droplets are
determined based at least in part on a ratio between sizes of the
plurality of droplets and sizes of the biological particles and/or
particles having coupled thereto molecular barcodes in the first
subset of the plurality of droplets.
[0024] In some embodiments, the plurality of droplets is directed
along the first channel using a pressure pulse.
[0025] In some embodiments, the molecular barcodes are releasably
coupled to the particles.
[0026] In some embodiments, the method further comprises subjecting
individual droplets of the first subset of the plurality of
droplets to a stimulus to facilitate polymerization in the
biological particles. In some embodiments, the stimulus is an
optical stimulus. In some embodiments, the optical stimulus a laser
or ultraviolet light. In some embodiments, the stimulus is a
chemical stimulus. In some embodiments, the stimulus is applied
prior to the intersection. In some embodiments, the stimulus is
applied along the first channel. In some embodiments, the stimulus
is applied along the second channel. In some embodiments, the
method further comprises (i) detecting the individual droplets and
(ii) subjecting the individual droplets to the stimulus upon
detecting the individual droplets.
[0027] In some embodiments, the biological particles are cells
enclosed within or comprising a gel or polymer matrix.
[0028] In some embodiments, the first subset comprises a third
subset of droplets each comprising a single biological particle and
a fourth subset of droplets each comprising multiple biological
particles, the method further comprising: directing the first
subset of the plurality of droplets along the second channel
towards a second intersection of the second channel with a fourth
channel and a fifth channel, and subjecting the first subset to an
electric or magnetic field under conditions sufficient to separate
at least a portion of the third subset from at least a portion of
the fourth subset, wherein upon separation, the at least the
portion of the third subset of droplets flows along a fourth
channel and the at least the portion of the fourth subset of
droplets flows along a fifth channel.
[0029] In another aspect, provided is a system for sorting
droplets, comprising: a fluid flow path comprising a first channel,
a second channel and a third channel; a fluid flow unit that is
configured to subject a plurality of droplets to flow along the
first channel, wherein the plurality of droplets is generated upon
bringing a first phase in contact with a second phase, wherein the
first phase and second phase are immiscible, wherein the plurality
of droplets comprises field-attractable particles, and wherein (i)
a first subset of the plurality of droplets includes biological
particles or particles having coupled thereto molecular barcodes,
and (ii) a second subset of the plurality of droplets does not
include the biological particles; a field application unit that is
configured to apply an electric or magnetic field; and a controller
operatively coupled to the fluid flow unit and the field
application unit, wherein the controller is programmed to (i)
direct the fluid flow unit to subject the plurality of droplets to
flow along the first channel to an intersection of the first
channel with the second channel and the third channel, and (ii)
direct the field application unit to subject the plurality of
droplets comprising the field-attractable particles to the electric
or magnetic field under conditions sufficient to separate at least
a portion of the first subset of the plurality of droplets from at
least a portion of the second subset of the plurality of droplets,
wherein upon separation, the at least the portion of the first
subset of the plurality of droplets flows along the second channel
and the at least the portion of the second subset of the plurality
of droplets flows along the third channel.
[0030] In some embodiments, the second subset of the plurality of
droplets does not include the particles having coupled thereto
molecular barcodes.
[0031] In some embodiments, the field application unit is
configured to apply the electric field. In some embodiments, the
field application unit is configured to apply the magnetic field.
In some embodiments, the field application unit is configured to
apply the electric field and magnetic field.
[0032] In some embodiments, the field-attractable particles are
magnetic-field attractable particles. In some embodiments, the
field-attractable particles are paramagnetic particles.
[0033] In some embodiments, the field-attractable particles are
electric-field attractable particles. In some embodiments, the
field-attractable particles are conductive particles.
[0034] In some embodiments, the fluid flow unit includes at least
one pump that is configured to provide negative pressure. In some
embodiments, the fluid flow unit includes at least one compressor
that is configured to provide positive pressure.
[0035] In some embodiments, the fluid flow unit is configured to
apply a pressure pulse to direct the plurality of droplets along
the first channel.
[0036] In some embodiments, the fluid flow unit is configured to
apply a pressure pulse to direct the first or second subset of the
plurality of droplets along the second channel or third channel,
respectively.
[0037] In some embodiments, the controller is programmed to direct
the fluid flow unit to subject the first subset of the plurality of
droplets to a pressure pulse at the intersection to subject the
first subset of the plurality of droplets to flow along the second
channel.
[0038] In some embodiments, the fluid flow unit includes an
actuator that is configured to subject the plurality of droplets to
flow.
[0039] In some embodiments, the controller is programmed to
determine the conditions of the electric or magnetic field
sufficient to separate the at least the portion of the first subset
of the plurality of droplets and the at least the portion of the
second subset of the plurality of droplets based at least in part
on a ratio between sizes of the plurality of droplets and/or sizes
of the biological particles or particles having coupled thereto
molecular barcodes in the first subset of the plurality of
droplets.
[0040] In some embodiments, each droplet of the first subset of the
plurality of droplets comprises less field attractable particles
than each droplet of the second subset of the plurality of
droplets. In some embodiments, wherein the electric or magnetic
field induces forces on the second subset of the plurality of
droplets that is greater than forces induced on the first subset of
the plurality of droplets.
[0041] In some embodiments, the biological particles are cells
enclosed within or comprising a gel or polymer matrix.
[0042] In another aspect, provided is a non-transitory
computer-readable medium comprising machine-executable code that,
upon execution by one or more computer processors, implements a
method for sorting droplets, comprising: (a) bringing a first phase
in contact with a second phase to generate a plurality of droplets,
wherein the first phase and second phase are immiscible, wherein
the plurality of droplets comprises field-attractable particles,
and wherein (i) a first subset of the plurality of droplets
includes biological particles or particles having coupled thereto
molecular barcodes, and (ii) a second subset of the plurality of
droplets does not include the biological particles; (b) directing
the plurality of droplets along a first channel towards an
intersection of the first channel with a second channel and a third
channel; and (c) subjecting the plurality of droplets comprising
the field-attractable particles to an electric or magnetic field
under conditions sufficient to separate at least a portion of the
first subset of the plurality of droplets from at least a portion
of the second subset of the plurality of droplets, wherein upon
separation, the at least the portion of the first subset of the
plurality of droplets flows along the second channel and the at
least the portion of the second subset of the plurality of droplets
flows along the third channel.
[0043] In another aspect, provided is a method for sorting
droplets, comprising: (a) bringing a first phase in contact with a
second phase to generate a plurality of droplets, wherein the first
phase and second phase are immiscible, and wherein (i) a first
subset of the plurality of droplets includes biological particles
or particles, which particles comprise molecular barcodes coupled
thereto, and (ii) a second subset of the plurality of droplets does
not include the biological particles; (b) directing the plurality
of droplets along a first channel towards an intersection of the
first channel with a second channel and a third channel; and (c) at
the intersection, subjecting the plurality of droplets to a
pressure pulse under conditions sufficient to separate at least a
portion of the first subset of the plurality of droplets from at
least a portion of the second subset of the plurality of droplets,
wherein upon separation, the at least the portion of the first
subset of the plurality of droplets flows along the second channel
and the at least the portion of the second subset of the plurality
of droplets flows along the third channel.
[0044] In some embodiments, the second subset of the plurality of
droplets does not include the particles having coupled thereto
molecular barcodes.
[0045] In some embodiments, the pressure pulse induces forces on
the second subset of the plurality of droplets that is greater than
forces induced on the first subset of the plurality of
droplets.
[0046] In some embodiments, the first subset of the plurality of
droplets includes biological particles and the particles having
coupled thereto molecular barcodes. In some embodiments, the
particles having coupled thereto molecular barcodes are beads. In
some embodiments, the beads are gel beads.
[0047] In some embodiments, the method further comprises,
subsequent to (c), subjecting nucleic acid molecules derived from
the biological particles in the first subset to nucleic acid
sequencing. In some embodiments, the method further comprises,
subsequent to (c), subjecting the first subset of the plurality of
droplets to nucleic acid amplification conditions to yield
amplification products of the nucleic acid molecules from the
biological particles in the first subset. In some embodiments, the
method further comprises subjecting the amplification products to
nucleic acid sequencing.
[0048] In some embodiments, the molecular barcodes are releasably
coupled to the particles.
[0049] In some embodiments, the method further comprises subjecting
individual droplets of the first subset of the plurality of
droplets to a stimulus to facilitate polymerization in the
biological particles. In some embodiments, the method further
comprises (i) detecting the individual droplets and (ii) subjecting
the individual droplets to the stimulus upon detecting the
individual droplets.
[0050] In some embodiments, the biological particles are cells
enclosed within or comprising a gel or polymer matrix.
[0051] In another aspect, provided is a system for sorting
droplets, comprising: a fluid flow path comprising a first channel,
a second channel and a third channel; a fluid flow unit that is
configured to subject a plurality of droplets to flow along the
first channel, wherein the plurality of droplets is generated upon
bringing a first phase in contact with a second phase, wherein the
first phase and second phase are immiscible, and wherein (i) a
first subset of the plurality of droplets includes biological
particles or particles having coupled thereto molecular barcodes,
and (ii) a second subset of the plurality of droplets does not
include the biological particles; a pressure application unit that
is configured to apply a pressure pulse; and a controller
operatively coupled to the fluid flow unit and the pressure
application unit, wherein the controller is programmed to (i)
direct the fluid flow unit to subject the plurality of droplets to
flow along the first channel to an intersection of the first
channel with the second channel and the third channel, and (ii)
direct the pressure application unit to subject the plurality of
droplets to the pressure pulse under conditions sufficient to
separate at least a portion of the first subset of the plurality of
droplets from at least a portion of the second subset of the
plurality of droplets, wherein upon separation, the at least the
portion of the first subset of the plurality of droplets flows
along the second channel and the at least the portion of the second
subset of the plurality of droplets flows along the third
channel.
[0052] In another aspect, provided is a non-transitory
computer-readable medium comprising machine-executable code that,
upon execution by one or more computer processors, implements a
method for sorting droplets, comprising: (a) bringing a first phase
in contact with a second phase to generate a plurality of droplets,
wherein the first phase and second phase are immiscible, and
wherein (i) a first subset of the plurality of droplets includes
biological particles or particles having coupled thereto molecular
barcodes, and (ii) a second subset of the plurality of droplets
does not include the biological particles; (b) directing the
plurality of droplets along a first channel towards an intersection
of the first channel with a second channel and a third channel; and
(c) subjecting the plurality of droplets to a pressure pulse under
conditions sufficient to separate at least a portion of the first
subset of the plurality of droplets from at least a portion of the
second subset of the plurality of droplets, wherein upon
separation, the at least the portion of the first subset of the
plurality of droplets flows along the second channel and the at
least the portion of the second subset of the plurality of droplets
flows along the third channel.
[0053] In another aspect, provided is a method for droplet
processing, comprising: (a) bringing a first phase in contact with
a second phase to generate a plurality of droplets, wherein the
first phase and second phase are immiscible, wherein (i) a first
subset of the plurality of droplets includes biological particles,
and (ii) a second subset of the plurality of droplets does not
include the biological particles; (b) directing the plurality of
droplets along a first channel towards an intersection of the first
channel with a second channel and a third channel; (c) prior to the
intersection, selectively subjecting individual droplets of the
first subset of the plurality of droplets to a stimulus to
facilitate polymerization in the biological particles; and (d)
separating at least a portion of the first subset of the plurality
of droplets from at least a portion of the second subset of the
plurality of droplets at the intersection, wherein upon separation,
the at least the portion of the first subset of the plurality of
droplets flows along the second channel and the at least the
portion of the second subset of the plurality of droplets flows
along the third channel.
[0054] In some embodiments, the first subset of the plurality of
droplets include particles having coupled thereto molecular
barcodes.
[0055] In some embodiments, the second subset of the plurality of
droplets does not include the particles having coupled thereto
molecular barcodes.
[0056] In some embodiments, the method further comprises (i)
detecting the individual droplets and (ii) selectively subjecting
the individual droplets to the stimulus upon detecting the
individual droplets.
[0057] In another aspect, provided is a method for sorting
droplets, comprising: (a) bringing a first phase in contact with a
second phase to generate a plurality of droplets, wherein the first
phase and second phase are immiscible, wherein the plurality of
droplets comprises field-attractable particles and wherein the
plurality of droplets comprises (i) a first subset of droplets each
including, and not more than, one biological particle, and (ii) a
second subset of droplets each either not including any biological
particle or including more than one biological particle; (b)
directing the plurality of droplets along a first channel towards
an intersection of the first channel with a second channel and a
third channel; and (c) subjecting the plurality of droplets
comprising the field-attractable particles to an electric or
magnetic field under conditions sufficient to separate at least a
portion of the first subset of the plurality of droplets from at
least a portion of the second subset of the plurality of droplets,
wherein upon separation, the at least the portion of the first
subset of the plurality of droplets flows along the second channel
and the at least the portion of the second subset of the plurality
of droplets flows along the third channel.
[0058] In some embodiments, the first subset of the plurality of
droplets include particles having coupled thereto molecular
barcodes. In some embodiments, the particles having coupled thereto
molecular barcodes are beads. In some embodiments, the beads are
gel beads.
[0059] In some embodiments, a concentration of the
field-attractable particles in droplets of the second subset which
do not include any biological particle is substantially
uniform.
[0060] In some embodiments, each droplet of the first subset of the
plurality of droplets comprises (i) less field attractable
particles than each droplet of the second subset of the plurality
of droplets which do not include any biological particle, and (ii)
more field attractable particles than each droplet of the second
subset of the plurality of droplets which includes more than one
biological particle. In some embodiments, forces induced by the
electric or magnetic field on droplets of the second subset which
do not include any biological particle is greater than forces
induced on the first subset, which forces induced on the first
subset are greater than forces induced on droplets of the second
subset which includes more than one biological particle.
[0061] In some embodiments, the field-attractable particles are
magnetic-field attractable particles. In some embodiments, the
field-attractable particles are paramagnetic particles.
[0062] In some embodiments, the field-attractable particles are
electric-field attractable particles. In some embodiments, the
field-attractable particles are conductive particles.
[0063] In some embodiments, the method further comprises,
subsequent to (c), subjecting nucleic acid molecules derived from
the biological particles in the first subset to nucleic acid
sequencing.
[0064] In some embodiments, the method further comprises,
subsequent to (c), subjecting the first subset of the plurality of
droplets to nucleic acid amplification conditions to yield
amplification products of the nucleic acid molecules from the
biological particles in the first subset. In some embodiments, the
method further comprises subjecting the amplification products to
nucleic acid sequencing.
[0065] In some embodiments, the conditions of the electric or
magnetic field sufficient to separate the at least the portion of
the first subset of the plurality of droplets and the at least the
portion of the second subset of the plurality of droplets are
determined based at least in part on a ratio between sizes of the
plurality of droplets and sizes of the biological particles in the
first subset of the plurality of droplets.
[0066] In some embodiments, the plurality of droplets is directed
along the first channel using a pressure pulse.
[0067] In some embodiments, the method further comprises subjecting
individual droplets of the first subset of the plurality of
droplets to a stimulus to facilitate polymerization in the
biological particles. In some embodiments, the stimulus is applied
prior to the intersection. In some embodiments, the method further
comprises (i) detecting the individual droplets and (ii) subjecting
the individual droplets to the stimulus upon detecting the
individual droplets.
[0068] In some embodiments, the biological particles are cells
enclosed within or comprising a gel or polymer matrix.
[0069] In another aspect, provided is a method for sorting
droplets, comprising: (a) bringing a first phase in contact with a
second phase to generate a plurality of droplets, wherein the first
phase and second phase are immiscible, wherein the plurality of
droplets comprises (i) a first subset of droplets each including,
and not more than, one biological particle, and (ii) a second
subset of droplets each either not including any biological
particle or including more than one biological particle; (b)
directing the plurality of droplets along a first channel towards
an intersection of the first channel with a second channel and a
third channel; and (c) subjecting the plurality of droplets to a
pressure pulse under conditions sufficient to separate at least a
portion of the first subset of the plurality of droplets from at
least a portion of the second subset of the plurality of droplets,
wherein upon separation, the at least the portion of the first
subset of the plurality of droplets flows along the second channel
and the at least the portion of the second subset of the plurality
of droplets flows along the third channel.
[0070] In some embodiments, the first subset of the plurality of
droplets include particles having coupled thereto molecular
barcodes. In some embodiments, the particles having coupled thereto
molecular barcodes are beads. In some embodiments, the beads are
gel beads.
[0071] In some embodiments, forces induced by the pressure pulse on
droplets of the second subset which do not include any biological
particle is greater than forces induced on the first subset, which
forces induced on the first subset are greater than forces induced
on droplets of the second subset which includes more than one
biological particle.
[0072] In some embodiments, the method further comprises,
subsequent to (c), subjecting nucleic acid molecules derived from
the biological particles in the first subset to nucleic acid
sequencing. In some embodiments, the method further comprises,
subsequent to (c), subjecting the first subset of the plurality of
droplets to nucleic acid amplification conditions to yield
amplification products of the nucleic acid molecules from the
biological particles in the first subset. In some embodiments, the
method further comprises subjecting the amplification products to
nucleic acid sequencing.
[0073] In some embodiments, the method further comprises subjecting
individual droplets of the first subset of the plurality of
droplets to a stimulus to facilitate polymerization in the
biological particles. In some embodiments, the method further
comprises (i) detecting the individual droplets and (ii) subjecting
the individual droplets to the stimulus upon detecting the
individual droplets.
[0074] In some embodiments, the biological particles are cells
enclosed within or comprising a gel or polymer matrix.
[0075] In another aspect, provided is a method for sorting
particles, comprising: (a) providing a plurality of particles,
wherein the plurality of particles comprises (i) a first subset of
particles each including a biological particle from or contents of
a plurality of cells and (ii) a second subset of particles each not
including a biological particle from or contents of the plurality
of cells; and (b) sorting the plurality of particles, thereby
isolating at least a portion of the first subset of particles from
at least a portion of the second subset of particles.
[0076] In some embodiments, the first subset of particles comprises
a third subset of particles each including, but not more than, one
biological particle from the plurality of cells and a fourth subset
of particles each including more than one biological particle from
the plurality of cells. In some embodiments, the method further
comprises sorting the first subset of particles, thereby isolating
at least a portion of the third subset of particles from at least a
portion of the fifth subset of particles.
[0077] In some embodiments, (b) comprises subjecting the plurality
of particles to a magnetic or electric field. In some embodiments,
each particle of the plurality of particles comprises
field-attractable particles.
[0078] In some embodiments, wherein (b) comprises subjecting the
plurality of particles to a pressure pulse.
[0079] In another aspect, provided is a method for sorting
particles, comprising: (a) providing a plurality of particles
generated from a plurality of cells, wherein the plurality of
particles comprises (i) a first subset of particles each including,
but not more than, one biological particle from or contents of a
single cell from the plurality of cells and (ii) a second subset of
particles each either not including a biological particle from or
contents of the plurality of cells or including more than one
biological particle from or contents of the plurality of cells; and
(b) sorting the plurality of particles, thereby isolating at least
a portion of the first subset of particles from at least a portion
of the second subset of particles.
[0080] In some embodiments, (b) comprises subjecting the plurality
of particles to a magnetic or electric field. In some embodiments,
each particle of the plurality of particles comprises
field-attractable particles.
[0081] In some embodiments, (b) comprises subjecting the plurality
of particles to a pressure pulse.
[0082] In another aspect, provided is a method for processing
droplets. The method can comprise: providing a plurality of gel
beads in a first phase, wherein the plurality of gel beads comprise
(i) molecular barcodes and (ii) field-attractable particles; and
subjecting the plurality of gel beads comprising the
field-attractable particles to an electric or magnetic field under
conditions sufficient to separate the plurality of gel beads from
at least 50% of the first phase, thereby providing the plurality of
gel beads in a second phase that is immiscible with respect to the
first phase.
[0083] In some embodiments, the plurality of gel beads can be
separated from at least 60% of the first phase. In some
embodiments, the plurality of gel beads can be separated from at
least 80% of the first phase. In some embodiments, the plurality of
gel beads can be separated from at least 90% of the first
phase.
[0084] In some embodiments, the first phase can be an oil phase. In
some embodiments, the second phase can be an aqueous phase.
[0085] In another aspect, provided is a method for sorting gel
beads, comprising: processing a plurality of droplets to generate a
plurality of gel beads, wherein the plurality of droplets comprises
field-attractable particles and wherein (i) a first subset of the
plurality of gel beads includes biological particles or particles
having coupled thereto molecular barcodes, and (ii) a second subset
of the plurality of gel beads does not include the biological
particles, directing the plurality of gel beads along a first
channel towards an intersection of the first channel with a second
channel and a third channel; and subjecting the plurality of gel
beads to an electric or magnetic field under conditions sufficient
to separate the first subset of the plurality of gel beads from the
second subset of the plurality of gel beads, wherein upon
separation, the first subset of the plurality of gel beads flows
along the second channel and the second subset of the plurality of
gel beads flows along the third channel.
[0086] In some embodiments, the processing comprises polymerizing
the plurality of droplets.
[0087] In another aspect, provided is a method for sorting gel
beads, comprising: processing a plurality of droplets to generate a
plurality of gel beads, wherein (i) a first subset of the plurality
of gel beads includes biological particles or particles having
coupled thereto molecular barcodes, and (ii) a second subset of the
plurality of gel beads does not include the biological particles,
directing the plurality of gel beads along a first channel towards
an intersection of the first channel with a second channel and a
third channel; and at the intersection, subjecting the plurality of
gel beads to a pressure pulse under conditions sufficient to
separate the first subset of the plurality of gel beads from the
second subset of the plurality of gel beads, wherein upon
separation, the first subset of the plurality of gel beads flows
along the second channel and the second subset of the plurality of
gel beads flows along the third channel.
[0088] 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
[0089] 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
[0090] 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:
[0091] FIG. 1 shows an example of a microfluidic channel structure
for partitioning individual biological particles.
[0092] FIG. 2A shows an example of a microfluidic channel structure
for separating occupied droplets from unoccupied droplets.
[0093] FIG. 2B shows an example of a multi-stage microfluidic
channel structure for separating singularly occupied droplets.
[0094] FIG. 3 shows another example of a microfluidic channel
structure for separating occupied droplets from unoccupied
droplets.
[0095] FIG. 4 shows an example of a microfluidic channel structure
for selective polymerization of partitions based on occupancy.
[0096] FIG. 5 shows another example of a microfluidic channel
structure for selective polymerization of partitions based on
occupancy.
[0097] FIG. 6 shows an example of a microfluidic channel structure
for selective polymerization of partitions based on droplet
size.
[0098] FIG. 7 shows a flowchart for a method of sorting occupied
droplets and unoccupied droplets.
[0099] FIG. 8 shows a flowchart for another method of sorting
occupied droplets and unoccupied droplets.
[0100] FIG. 9 shows a flowchart for a method of selectively
polymerizing occupied droplets.
[0101] FIG. 10 shows a flowchart for a method of selectively
polymerizing appropriately sized droplets.
[0102] FIG. 11 shows an example of a microfluidic channel structure
for separating occupied droplets from unoccupied droplets.
[0103] FIG. 12 shows an example of a microfluidic channel structure
for delivering barcode carrying beads to droplets.
[0104] FIG. 13 shows an example of a microfluidic channel structure
for co-partitioning biological particles and reagents.
[0105] FIG. 14 shows an example of a microfluidic channel structure
for the controlled partitioning of beads into discrete
droplets.
[0106] FIG. 15 shows an example of a microfluidic channel structure
for increased droplet generation throughput.
[0107] FIG. 16 shows another example of a microfluidic channel
structure for increased droplet generation throughput.
[0108] FIG. 17A shows a cross-section view of another example of a
microfluidic channel structure with a geometric feature for
controlled partitioning. FIG. 17B shows a perspective view of the
channel structure of FIG. 17A.
[0109] FIG. 18 shows an example computer control system that is
programmed or otherwise configured to implement methods provided
herein.
[0110] FIG. 19 illustrates an example of a barcode carrying
bead.
DETAILED DESCRIPTION
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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).
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] The term "biological particle," as used herein, generally
refers to a discrete biological system derived from a biological
sample. The biological particle may be a macromolecule. The
biological particle may be a small molecule. The biological
particle may be a virus. The biological particle may be a cell or
derivative of a cell. The biological particle may be an organelle.
The biological particle may be a rare cell from a population of
cells. The biological particle 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
particle may be a constituent of a cell. The biological particle
may be or may include DNA, RNA, organelles, proteins, or any
combination thereof. The biological particle 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 may be obtained from a tissue of a
subject. The biological particle may be a hardened cell. Such
hardened cell may or may not include a cell wall or cell membrane.
The biological particle 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.
[0122] The term "macromolecular constituent," as used herein,
generally refers to a macromolecule contained within or from a
biological particle. The macromolecular constituent may comprise a
nucleic acid. In some cases, the biological particle 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.
[0123] 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.
[0124] 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.
[0125] The efficiency of many single cell applications can increase
by improving cell throughput. For example, this can be achieved by
sorting a plurality of droplets that may or may not contain cells
and/or particles therein to collect only the droplets that contain
the cells and/or particles therein. The plurality of droplets may
be sorted to isolate singularly occupied droplets from
non-singularly occupied droplets (e.g., unoccupied, multiply
occupied, etc.). In another example, higher efficiency can be
achieved by isolating a plurality of cell beads from a plurality of
particles that may or may not contain cells therein. The plurality
of particles may be sorted to isolate singularly occupied cell
beads (e.g., particles containing cells or their derivatives) from
non-singularly occupied cell beads (e.g., unoccupied particles,
multiply occupied cell beads, etc.). The isolated population of
droplets that contain (e.g., singularly contain) the cells and/or
particles therein, and/or cell beads that contain (e.g., singularly
contain) the cells therein, can then be subject to further
applications, such as nucleic acid amplification and/or sequencing
applications.
[0126] Provided are methods and systems for sorting droplets. The
methods and systems generally operate by generating a plurality of
droplets such that each of the plurality of droplets comprises
field-attractable particles. A given droplet in the plurality of
droplets may or may not include therein one or more cells and/or
other particles (e.g., cell beads, gel beads, etc.). In some cases,
the other particles (e.g., gel beads) may have molecular barcodes
coupled thereto. Thus, the plurality of droplets comprising field
attractable particles can comprise a first subset of droplets that
include one or more cells and/or other particles and a second
subset of droplets that do not include any cells and/or other
particles. A given droplet in the first subset of droplets that
includes one or more cells and/or other particles can comprise a
sufficiently discrepant number or concentration of
field-attractable particles than a given droplet in the second
subset of droplets that does not include any cells and/or other
particles such that when the plurality of droplets is subject to an
electric or magnetic field, the first subset of droplets and the
second subset of droplets are separated from each other. In some
cases, when the plurality of droplets is subjected to an electric
or magnetic field, singularly occupied droplets may be separated
from unoccupied droplets and otherwise multiply occupied
droplets.
[0127] In some instances, a plurality of droplets can be generated
with or without field-attractable particles. A given droplet in the
plurality of droplets may or may not include one or more cells
and/or particles. Thus, the plurality of droplets can comprise a
first subset of droplets that include one or more cells and/or
particles and a second subset of droplets that do not include any
cells and/or particles. The plurality of droplets can be subject to
a pressure pulse and the first subset of droplets and the second
subset of droplets can be separated from each other via
hydrodynamic forces. In some cases, singularly occupied droplets
may be separated from unoccupied droplets and otherwise multiply
occupied droplets.
[0128] In an aspect, the methods and systems described herein
provide for the compartmentalization, depositing, or partitioning
of macromolecular constituent contents of individual biological
particles from a sample material containing biological particles
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. The
partition can be a well. The partition can be a bead, such as a gel
bead and/or a cell bead. A partition may or may not contain
biological particles and/or macromolecular constituents thereof. In
accordance with some embodiments, each partition may contain at
least some field attractable particles. The amount and/or
concentration of field attractable particles in each partition can
vary depending on whether the partition contains biological
particles (or other particles, such as beads). In accordance with
some other embodiments, a partition may not contain field
attractable particles.
[0129] In some instances, unique identifiers, such as barcodes, may
be previously, subsequently or concurrently delivered to the
partitions that hold the compartmentalized or partitioned
biological particle, in order to allow for the later attribution of
the characteristics of the individual biological particle to the
particular partition. Barcodes may be delivered, for example on an
oligonucleotide, to a partition via any suitable mechanism.
Barcoded oligonucleotides can be delivered to a partition via a
microcapsule. In some cases, barcoded oligonucleotides can be
initially associated with the microcapsule and then released from
the microcapsule upon application of a stimulus which allows the
oligonucleotides to dissociate or to be released from the
microcapsule.
[0130] A microcapsule, in some instances, can comprise a bead. In
some cases, 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.
[0131] In some instances, the bead may contain molecular precursors
(e.g., monomers or polymers), which may form a polymer network via
polymerization of the 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.
[0132] A bead may comprise natural and/or synthetic materials. For
example, a polymer can be a natural polymer or a synthetic polymer.
In some cases, a bead can comprise 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.
[0133] In some cases, a chemical cross-linker may be a precursor
used to cross-link monomers during polymerization of the monomers
and/or may be used to attach oligonucleotides (e.g., barcoded
oligonucleotides) to the bead. In some cases, polymers may be
further polymerized with a cross-linker species or other type of
monomer to generate a further polymeric network. Non-limiting
examples of chemical cross-linkers (also referred to as a
"crosslinker" or a "crosslinker agent" herein) include cystamine,
gluteraldehyde, dimethyl suberimidate, N-Hydroxysuccinimide
crosslinker BS3, formaldehyde, carbodiimide (EDC), SMCC,
Sulfo-SMCC, vinylsilane, N,N'diallyltartardiamide (DATD),
N,N'-Bis(acryloyl)cystamine (BAC), or homologs thereof. In some
cases, the crosslinker used in the present disclosure contains
cystamine.
[0134] Crosslinking may be permanent or reversible, depending upon
the particular crosslinker used. Reversible crosslinking 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.
[0135] 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
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.
[0136] 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.
[0137] In some cases, the bead may comprise covalent or ionic bonds
between polymeric precursors (e.g., monomers, oligomers, linear
polymers), oligonucleotides, primers, and other entities. In some
cases, the covalent bonds can be carbon-carbon bonds or thioether
bonds.
[0138] In some cases, a bead may comprise an acrydite moiety, which
in certain aspects may be used to attach one or more
oligonucleotides (e.g., barcode sequence, 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 an
oligonucleotide (e.g., barcode sequence, 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.
[0139] Functionalization of beads for attachment of
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.
[0140] 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.
[0141] 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.
[0142] FIG. 19 illustrates an example of a barcode carrying bead. A
nucleic acid molecule 1902, such as an oligonucleotide, can be
coupled to a bead 1904 by a releasable linkage 1906, such as, for
example, a disulfide linker. The same bead 1904 may be coupled
(e.g., via releasable linkage) to one or more other nucleic acid
molecules 1918, 1920. The nucleic acid molecule 1902 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 1902 may comprise a functional sequence 1908 that may
be used in subsequent processing. For example, the functional
sequence 1908 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 1902 may comprise a barcode sequence 1910 for use in
barcoding the sample (e.g., DNA, RNA, protein, etc.). In some
cases, the barcode sequence 1910 can be bead-specific such that the
barcode sequence 1910 is common to all nucleic acid molecules
(e.g., including nucleic acid molecule 1902) coupled to the same
bead 1904. Alternatively or in addition, the barcode sequence 1910
can be partition-specific such that the barcode sequence 1910 is
common to all nucleic acid molecules coupled to one or more beads
that are partitioned into the same partition. The nucleic acid
molecule 1902 may comprise a specific priming sequence 1912, 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 1902 may comprise an anchoring sequence 1914
to ensure that the specific priming sequence 1912 hybridizes at the
sequence end (e.g., of the mRNA). For example, the anchoring
sequence 1914 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.
[0143] The nucleic acid molecule 1902 may comprise a unique
molecular identifying sequence 1916 (e.g., unique molecular
identifier (UMI)). In some cases, the unique molecular identifying
sequence 1916 may comprise from about 5 to about 8 nucleotides.
Alternatively, the unique molecular identifying sequence 1916 may
compress less than about 5 or more than about 8 nucleotides. The
unique molecular identifying sequence 1916 may be a unique sequence
that varies across individual nucleic acid molecules (e.g., 1902,
1918, 1920, etc.) coupled to a single bead (e.g., bead 1904). In
some cases, the unique molecular identifying sequence 1916 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. 19 shows three nucleic acid molecules 1902, 1918,
1920 coupled to the surface of the bead 1904, 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., 1908, 1910, 1912, etc.) and variable or unique
sequence segments (e.g., 1916) between different individual nucleic
acid molecules coupled to the same bead.
[0144] In operation, a biological particle (e.g., cell, DNA, RNA,
etc.) can be co-partitioned along with a barcode bearing bead 1904.
The barcoded nucleic acid molecules 1902, 1918, 1920 can be
released from the bead 1904 in the partition. By way of example, in
the context of analyzing sample RNA, the poly-T segment (e.g.,
1912) of one of the released nucleic acid molecules (e.g., 1902)
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 1908, 1910,
1916 of the nucleic acid molecule 1902. Because the nucleic acid
molecule 1902 comprises an anchoring sequence 1914, 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 1910. However, the
transcripts made from the different mRNA molecules within a given
partition may vary at the unique molecular identifying sequence
1912 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 (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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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 any
swelling method as is known to one having skill in the art. 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
any de-swelling method as is known to one having skill in the art.
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.
[0150] In some cases, an acrydite moiety linked to precursor,
another species linked to a precursor, or a precursor itself
comprises a labile bond, such as chemically, thermally, or
photo-sensitive bonds e.g., disulfide bonds, UV sensitive bonds, 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.
[0151] 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,
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.
[0152] 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.
[0153] 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)).
[0154] Species that do not participate in polymerization may also
be encapsulated in beads during bead generation (e.g., during
polymerization of precursors). 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, oligonucleotides, reagents for a nucleic acid
amplification reaction (e.g., primers, polymerases, dNTPs,
co-factors (e.g., ionic co-factors)) including those described
herein, reagents for enzymatic reactions (e.g., enzymes,
co-factors, substrates), or reagents for a nucleic acid
modification reactions such as polymerization, ligation, or
digestion. 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.
[0155] Beads may be of uniform size or heterogeneous size. In some
cases, the diameter of a bead may be at least about 1 micrometers
(.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 1 .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.
[0156] In certain aspects, beads can be provided as a population or
plurality of beads having a relatively monodisperse size
distribution. Where it may be desirable to provide relatively
consistent amounts of reagents within partitions, 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.
[0157] Beads 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.
[0158] In addition to, or as an alternative to the cleavable
linkages between the beads and the associated molecules, such as
barcode containing oligonucleotides, described above, 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 oligonucleotide, e.g., barcoded oligonucleotide) may
result in release of the species from the bead.
[0159] 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.
[0160] A degradable bead may be useful in more quickly releasing an
attached species (e.g., an oligonucleotide, 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.
[0161] 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) 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 breaks 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.
[0162] 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.
[0163] Where degradable beads are provided, it may be desirable to
avoid exposing such beads to the stimulus or stimuli that cause
such degradation prior to the desired time, in order to 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 will be
desirable 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, it may
be desirable to provide reducing agent free (or DTT free) enzyme
preparations in treating the beads described herein. Examples of
such enzymes include, e.g., polymerase enzyme preparations, reverse
transcriptase enzyme preparations, ligase enzyme preparations, as
well as many other enzyme preparations that 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] The methods, compositions, devices, and kits of this
disclosure may be used with any suitable agent to 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.
[0168] 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.
[0169] The compartments or 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 comprise droplets of aqueous fluid
within a non-aqueous continuous phase, e.g., an oil phase. The
partitions can comprise droplets of a first phase within a second
phase, wherein the first and second phases are immiscible. 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 detail in, e.g., U.S. Patent Application
Publication No. 2010/0105112, which is entirely incorporated herein
by reference for all purposes.
[0170] In the case of droplets in an emulsion, allocating
individual biological particles to discrete partitions may
generally be accomplished by introducing a flowing stream of
biological 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. By providing the aqueous stream at a certain
concentration of biological particles, the occupancy of the
resulting partitions (e.g., number of biological particles per
partition) can be controlled. Where single biological particle
partitions are desired, the relative flow rates of the immiscible
fluids can be selected such that, on average, the partitions
contain less than one biological particle per partition, in order
to ensure that those partitions that are occupied, are primarily
singularly occupied. In some embodiments, the relative flow rates
of the fluids can be selected such that a majority of partitions
are occupied, e.g., allowing for only a small percentage of
unoccupied partitions. The flows and channel architectures can be
controlled as to ensure a desired number of singularly occupied
partitions, less than a certain level of unoccupied partitions
and/or less than a certain level of multiply occupied
partitions.
[0171] The systems and methods described herein can be operated
such that a majority of occupied partitions include no more than
one biological particle per occupied partition. In some cases, the
partitioning process is conducted such that fewer than 25% of the
occupied partitions contain more than one biological particle, and
in many cases, fewer than 20% of the occupied partitions have more
than one biological particle. In some cases, fewer than 10% or even
fewer than 5% of the occupied partitions include more than one
biological particle per partition.
[0172] In some cases, it is desirable to avoid the creation of
excessive numbers of empty partitions. For example, from a cost
perspective and/or efficiency perspective, it may desirable to
minimize the number of empty partitions. However, while this may be
accomplished by providing sufficient numbers of biological
particles into the partitioning zone, the Poissonian distribution
may expectedly increase the number of partitions that may include
multiple biological particles. As such, 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. In some cases, the flow of one or more of the cells, or
other fluids directed into the partitioning zone can be conducted
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 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 creates
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.
[0173] After the partitions are generated, comprising in part
singularly occupied partitions, in part multiply occupied
partitions, and/or in part unoccupied partitions, the occupied
partitions can be sorted from the unoccupied partitions. In some
cases, singularly occupied partitions may be isolated from
non-singularly occupied partitions (e.g., multiply occupied
partitions and unoccupied partitions). Such sorting can be achieved
by including field attractable particles during the generation of
droplets in an emulsion. For example, a flowing stream of aqueous
fluid containing biological particles and field attractable
particles can be introduced into a flowing stream of a non-aqueous
fluid, such that droplets are generated at the junction of the two
streams.
[0174] As will be appreciated, the above-described occupancy rates
are also applicable to partitions that include both biological
particles and additional reagents, including, but not limited to,
microcapsules carrying barcoded oligonucleotides. 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 oligonucleotides
and a biological particle.
[0175] Although described in terms of providing substantially
singularly occupied partitions, above, in certain cases, it is
desirable to provide multiply occupied partitions, e.g., containing
two, three, four or more cells and/or microcapsules (e.g., beads)
comprising barcoded oligonucleotides within a single partition.
Accordingly, as noted above, the flow characteristics of the
biological particle 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 desired 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.
[0176] In some cases, additional microcapsules are 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, i.e.,
containing different associated reagents, through different channel
inlets into such common channel or droplet generation junction. In
such cases, the flow and frequency of the different beads into the
channel or junction may be controlled to provide for the desired
ratio of microcapsules from each source, while ensuring the desired
pairing or combination of such beads into a partition with the
desired number of biological particles.
[0177] The partitions described herein may comprise small volumes,
e.g., 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.
[0178] 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, 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% the above described
volumes.
[0179] 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 to generate the
plurality of partitions. For example, in a method described herein,
a plurality of partitions may be generated that comprises 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. Moreover, the plurality of partitions may
comprise both unoccupied partitions (e.g., empty partitions) and
occupied partitions.
[0180] Microfluidic channel networks can be utilized to generate
partitions as described herein. Alternative mechanisms may also be
employed in the partitioning of individual biological particles,
including porous membranes through which aqueous mixtures of cells
are extruded into non-aqueous fluids.
[0181] FIG. 1 shows an example of a microfluidic channel structure
for partitioning individual biological particles. As described
elsewhere herein, in some cases, the majority of occupied
partitions can include no more than one biological particle per
occupied partition and, in some cases, some of the generated
partitions can be unoccupied (of any biological particle). In some
cases, though, some of the occupied partitions may include more
than one biological particle. 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, and
in many cases, fewer than about 20% of the occupied partitions have
more than one biological particle, while in some cases, fewer than
about 10% or even fewer than about 5% of the occupied partitions
include more than one biological particle per partition.
[0182] As shown in FIG. 1, the channel structure 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 (e.g., cells) 114 and suspended
field-attractable particles 115, 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. A discrete
droplet generated may include an individual biological particle 114
and field-attractable particles 115 (such as droplets 118). A
discrete droplet generated may include more than one individual
biological particle 114 and field-attractable particles 115 (not
shown in FIG. 1). A discrete droplet may contain field-attractable
particles 115 but no biological particle 114 (such as droplet
120).
[0183] The field-attractable particles 115 may be paramagnetic
particles. In some cases, the field-attractable particles may be
superparamagnetic particles. For example, the field-attractable
particles can comprise polystyrene magnetic particles (e.g.,
polystyrene core particle coated with at least a layer of magnetite
(e.g., iron oxide) and polystyrene), amino magnetic particles,
carboxyl magnetic particles, dimethylamino magnetic particles,
hydroxyethyl magnetic particles, and/or a combination of the above.
A paramagnetic particle can comprise a polymer matrix of amine
silane, glucuronic acid, bromoacetyl, chitosan,
carboxymethyldextran, citric acid, starch, DEAE-starch,
phosphate-starch, dextran, dextran-sulfate, lipid, oleic acid,
diphosphate, polyaspartic acid, polyacrylamide, polyacrylic acid,
polydimethylamine, polyethylene glycole alpha-methoxy-omega-amine,
polyethylene glycol alpha-,omega-diphosphate, poly(maleic
acid-co-olefin), polystyrenesulfonate, polyvinyl alcohol,
poly(4-vinylpyridine), poly-diallyldimethylamin, uncoated
magnetite, and/or other matrices. A paramagnetic particle may have
micrometer or nanometer size. For example, a paramagnetic particle
can have a maximum dimension (e.g., width, length, height,
diameter, etc.) of at most about 20 micrometers (.mu.m), 10 .mu.m,
9 .mu.m, 8 .mu.m, 7 .mu.m, 6 .mu.m, 5 .mu.m, 4 .mu.m, 3 .mu.m, 2
.mu.m, 1 .mu.m, 0.5 .mu.m, or less. The paramagnetic particle can
have a maximum dimension of at most about 500 nanometer (nm), 400
nm, 300 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9
nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, or less.
Alternatively, a paramagnetic particle can have a maximum dimension
that is greater than about 20 .mu.m. The paramagnetic particles may
be responsive when exposed to magnetic fields. In some cases, the
paramagnetic particles can be smooth surface particles, wherein a
thick polymer layer coats the magnetite (e.g., iron oxide) layer.
The smooth surface can shield the magnetite from interfering with
enzyme activities or other undesirable effects with other particles
or cells caused by exposure to the magnetite. In some cases, the
paramagnetic particles can be cross-linked particles, wherein the
particles are coated with cross-linked polymer on the surfaces of
the iron oxide crystals. The cross-linked polymer can render the
paramagnetic particle resistant to common organic solvents, such as
acetone, acetonitrile, dimethlyformanide (DMF) and chloroform. The
magnetite content on each paramagnetic particle can be adjusted
(e.g., to have higher or lower percentage) to be more responsive or
less responsive to the same magnetic field. In some cases, the
field-attractable particles can be diamagnetic particles or
ferromagnetic particles.
[0184] In some cases, the field-attractable particles 115 may be
conductive particles. A conductive particle may have micrometer or
nanometer size. For example, a conductive particle can have a
maximum dimension (e.g., width, length, height, diameter, etc.) of
at most about 20 micrometers (.mu.m), 10 .mu.m, 9 .mu.m, 8 .mu.m, 7
.mu.m, 6 .mu.m, 5 .mu.m, 4 .mu.m, 3 .mu.m, 2 .mu.m, 1 .mu.m, 0.5
.mu.m, or less. The conductive particle can have a maximum
dimension of at most about 500 nanometer (nm), 400 nm, 300 nm, 200
nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6
nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, or less. Alternatively, a
conductive particle can have a maximum dimension that is greater
than about 20 .mu.m. A conductive particle can have a substantially
spherical shape. Alternatively, a conductive particle can have a
different shape. The conductive particles may be responsive when
exposed to electric fields. The conductive (e.g., metal) content on
each conductive particle can be adjusted (e.g., to have higher or
lower percentage) to be more responsive or less responsive to the
same electric field. In some cases, the field-attractable particles
115 can comprise both paramagnetic and conductive particles.
[0185] The first aqueous fluid 112 can have a substantially uniform
concentration of field-attractable particles 115 as the first
aqueous fluid 112 is introduced into junction 110. For example, the
concentration of the field-attractable particles 115 in the first
aqueous fluid 112 in the channel segment 102 at time x can be
substantially uniform with the concentration of field-attractable
particles 115 in the first aqueous fluid 112 in the channel segment
102 at time x+.delta. (where x and .delta. are positive). In some
instances, the concentration of field-attractable particles 115 can
be substantially uniform in only the volume of the first aqueous
fluid 112, that is, the volume not including the volume of each of
the biological particles 114 suspended in the first aqueous fluid
112.
[0186] This second fluid 116 can comprise an oil, such as a
fluorinated oil, that includes a fluorosurfactant for stabilizing
the resulting droplets, e.g., inhibiting subsequent coalescence of
the resulting droplets. 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.
[0187] The generated droplets may comprise two subsets of droplets:
(1) occupied droplets 118, containing one or more biological
particles 114, and (2) unoccupied droplets 120, not containing any
biological particles 114. Each droplet generated, occupied or
unoccupied, may contain some number and/or concentration of
field-attractable particles 115. In some instances, the
concentration of the field-attractable particles 115 in each of the
unoccupied droplets 120 can be substantially uniform, wherein the
concentration is a number of field-attractable particles per total
droplet volume (and not just the volume of the first aqueous fluid
112 in the droplet). In some instances, the concentration of the
field-attractable particles 115 in each of the occupied droplets
118 can be substantially uniform, wherein the concentration is a
number of field-attractable particles per total droplet volume. In
other instances, as can be easily appreciated, the concentration of
the field-attractable particles 115 in each of the occupied
droplets 118 can vary with size and/or the number of biological
particles 114 contained in the droplet. In any case, the
concentration of field-attractable particles in any of the
unoccupied droplets 120 can be greater than the concentration of
field-attractable particles in any of the occupied droplets 118 to
account for the volume occupied by the biological particle 114 in
the occupied droplets 118. In most cases, the concentration of
field-attractable particles in a droplet from the unoccupied
droplets 120 can be greater than the concentration of
field-attractable particles in a droplet from the occupied droplets
118 to account for the volume occupied by the biological particle
114 in the occupied droplets 118.
[0188] For example, assuming that (i) the droplet is spherical and
has the radius R.sub.D, (ii) a biological particle is spherical and
has the radius R.sub.+, and (iii) the concentration of
field-attractable particles in the volume of aqueous fluid is
substantially uniform, the ratio of a number of field-attractable
particles in a singularly occupied droplet (N.sub.+) to a number of
field-attractable particles in an unoccupied droplet (N) will
be:
N + N - = 1 - ( R + R D ) 3 ##EQU00001##
[0189] As can be appreciated, the above ratio may change with
deviations from the above assumptions. For example, an occupied
droplet containing three biological particles can have a ratio of
about:
1 - 3 ( R + R D ) 3 . ##EQU00002##
[0190] In another aspect, in addition to or as an alternative to
droplet based partitioning, biological particles may be
encapsulated within a microcapsule, such as a cell bead, that
comprises an outer shell or layer or porous matrix in which is
entrained one or more individual biological particles or small
groups of biological particles, and may include other reagents.
Encapsulation of biological particles may be performed by a variety
of processes. Such processes combine an aqueous fluid containing
the biological particles and also containing the field-attractable
particles to be analyzed 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 include, e.g., thermal stimuli
(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), or the like.
[0191] Preparation of microcapsules comprising biological particles
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 small
groups of biological particles. Likewise, membrane based
encapsulation systems may be used to generate microcapsules
comprising encapsulated biological particles 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, such as to generate a plurality of particles, each particle
comprising field-attractable particles. The plurality of particles
may comprise a first subset of particles occupied by biological
particles (e.g., cell beads) and a second subset of particles
unoccupied by biological particles. In particular, and with
reference to FIG. 1, the aqueous fluid comprising (i) the
biological particles 114, (ii) the field-attractable particles 115,
and (ii) the polymer precursor material (not shown) is flowed into
channel junction 110, where it is partitioned into droplets 118 or
120 comprising or not comprising the individual biological
particles 114, respectively, but always comprising the
field-attractable particles 115, through the flow of non-aqueous
fluid 116. In the case of encapsulation methods, non-aqueous fluid
116 may also include an initiator to cause polymerization and/or
crosslinking of the polymer precursor to form the microcapsule that
includes the entrained biological particles. 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.
[0192] For example, in the case where the polymer precursor
material comprises a linear polymer material, e.g., 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 in channel segments 104 and 106, which
initiates the copolymerization of the acrylamide and BAC into a
cross-linked polymer network or, hydrogel.
[0193] Upon contact of the second fluid stream 116 with the first
fluid stream 112 at junction 110 in the formation of droplets, the
TEMED may diffuse from the second fluid 116 into the aqueous first
fluid 112 comprising the linear polyacrylamide, which will activate
the crosslinking of the polyacrylamide within the droplets,
resulting in the formation of the gel, e.g., hydrogel,
microcapsules (e.g., droplet 118, 120), 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+, 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, or the like. In some cases,
encapsulated biological particles can be selectively releasable
from the microcapsule, e.g., through passage of time, or upon
application of a particular stimulus, that degrades the
microcapsule sufficiently to allow the cell, or its contents to be
released from the microcapsule, e.g., into a partition, such as a
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, e.g., U.S. Patent Application
Publication No. 2014/0378345, which is entirely incorporated herein
by reference for all purposes).
[0194] Encapsulated biological particles (e.g., cell beads) can
provide certain potential advantages of being storable, and more
portable than droplet based partitioned biological particles.
Furthermore, in some cases, it may be desirable to allow biological
particles to be analyzed to incubate for a select period of time,
in order to characterize changes in such biological particles over
time, either in the presence or absence of different stimuli. In
such cases, encapsulation of individual biological particles may
allow for longer incubation than partitioning in emulsion droplets,
although in some cases, droplet partitioned biological particles
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 may constitute the
partitioning of the biological particles into which other reagents
are co-partitioned. Alternatively, encapsulated biological
particles may be readily deposited into other partitions, e.g.,
droplets, as described above.
[0195] In accordance with certain aspects, the biological particles
may be partitioned along with lysis reagents in order to release
the contents of the biological particles within the partition. In
such cases, the lysis agents can be contacted with the biological
particle suspension concurrently with, or immediately prior to the
introduction of the biological particles into the partitioning
junction/droplet generation zone, e.g., through an additional
channel or channels upstream of channel junction 110. 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 to cause the release of the biological particles's
contents into the partitions. For example, in some cases,
surfactant based lysis solutions may be used to lyse cells,
although these may be less desirable for emulsion based systems
where the surfactants can interfere with stable emulsions. 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 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 desired size, following cellular disruption.
[0196] In addition to the lysis agents co-partitioned with the
biological particles described above, other reagents can also be
co-partitioned with the biological particles, 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, the biological particles may be exposed to an
appropriate stimulus to release the biological particles 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 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
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 to
be released into a partition at a different time from the release
of oligonucleotides into the same partition.
[0197] Additional reagents may also be co-partitioned with the
biological particles, such as endonucleases to fragment a
biological particle's DNA, DNA polymerase enzymes and dNTPs used to
amplify the biological particle's nucleic acid fragments and to
attach the barcode molecular tags to the amplified fragments.
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.
[0198] In some cases, the length of a switch oligo may be 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, 250 nucleotides or longer.
[0199] In some cases, the length of a switch oligo may be at least
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.
[0200] In some cases, the length of a switch oligo may be at most
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.
[0201] Once the contents of the cells are released into their
respective partitions, the macromolecular components (e.g.,
macromolecular constituents of biological particles, 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 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. The ability
to attribute characteristics to individual biological particles or
groups of biological particles is provided by the assignment of
unique identifiers specifically to an individual biological
particle or groups of biological particles. Unique identifiers,
e.g., in the form of nucleic acid barcodes, can be assigned or
associated with individual biological particles or populations of
biological particle, in order to tag or label the biological
particle'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
components and characteristics to an individual biological particle
or group of biological particles. In some aspects, this is
performed by co-partitioning the individual biological particle or
groups of biological particles with the unique identifiers. In some
aspects, the unique identifiers are provided in the form of
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 to other components
of the biological particle, and particularly to fragments of those
nucleic acids. The oligonucleotides are partitioned such that as
between oligonucleotides in a given partition, the nucleic acid
barcode sequences contained therein are the same, but as between
different partitions, the oligonucleotides 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.
[0202] The nucleic acid barcode sequences can include from 6 to
about 20 or more nucleotides within the sequence of the
oligonucleotides. In some cases, the length of a barcode sequence
may be 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 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 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 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 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 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
[0203] The co-partitioned oligonucleotides can also comprise other
functional sequences useful in the processing of the nucleic acids
from the co-partitioned biological particles. These sequences
include, e.g., targeted or random/universal amplification primer
sequences for amplifying the genomic DNA from the individual
biological particles 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.
[0204] In an example, microcapsules, such as beads (e.g., see FIG.
19), are provided that each includes large numbers of the above
described barcoded oligonucleotides releasably attached to the
beads, where all of the oligonucleotides 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 oligonucleotides into
the partitions, as they are capable of carrying large numbers of
oligonucleotide molecules, and may be configured to release those
oligonucleotides upon exposure to a particular stimulus, as
described elsewhere herein. In some cases, the population of beads
will provide 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 oligonucleotide molecules
attached. In particular, the number of molecules of
oligonucleotides including the barcode sequence on an individual
bead can be at least about 1,000 oligonucleotide molecules, at
least about 5,000 oligonucleotide molecules, at least about 10,000
oligonucleotide molecules, at least about 50,000 oligonucleotide
molecules, at least about 100,000 oligonucleotide molecules, at
least about 500,000 oligonucleotides, at least about 1,000,000
oligonucleotide molecules, at least about 5,000,000 oligonucleotide
molecules, at least about 10,000,000 oligonucleotide molecules, at
least about 50,000,000 oligonucleotide molecules, at least about
100,000,000 oligonucleotide molecules, and in some cases at least
about 1 billion oligonucleotide molecules, or more.
[0205] 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 oligonucleotide molecules, at least
about 5,000 oligonucleotide molecules, at least about 10,000
oligonucleotide molecules, at least about 50,000 oligonucleotide
molecules, at least about 100,000 oligonucleotide molecules, at
least about 500,000 oligonucleotides, at least about 1,000,000
oligonucleotide molecules, at least about 5,000,000 oligonucleotide
molecules, at least about 10,000,000 oligonucleotide molecules, at
least about 50,000,000 oligonucleotide molecules, at least about
100,000,000 oligonucleotide molecules, and in some cases at least
about 1 billion oligonucleotide molecules.
[0206] In some cases, it may be desirable to incorporate multiple
different barcodes within a given partition, either attached to a
single or multiple beads within the partition. For example, in some
cases, a mixed, but known barcode sequences set 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.
[0207] The 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 oligonucleotides. 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 oligonucleotides form the beads. In
still other cases, a chemical stimulus is used that cleaves a
linkage of the oligonucleotides to the beads, or otherwise results
in release of the oligonucleotides from the beads. In one case,
such compositions include the polyacrylamide matrices described
above for encapsulation of biological particles, and may be
degraded for release of the attached oligonucleotides through
exposure to a reducing agent, such as DTT.
[0208] For example, in FIG. 1, concurrent to the stream of the
first aqueous fluid 112 flowing through channel 102 towards the
junction 110, a second aqueous stream comprising barcode carrying
beads suspended in a third fluid can be flowed through another
channel (not shown in FIG. 1) towards the junction 110. The third
fluid can be the same fluid material as the first aqueous fluid
112. A non-aqueous partitioning fluid 116 is introduced into
channel junction 110 from each of side channels 104 and 106, and
the combined streams are flowed into outlet channel 108. Within
channel junction 110, the two combined aqueous streams from channel
segments 102 and the other channel carrying the third fluid are
combined, and partitioned into droplets 118, 120. The occupied
droplets may contain either one or more biological particles,
either one or more barcode carrying beads, or both at least a
biological particle and at least a barcode carrying bead. The
unoccupied droplets may contain neither biological particles nor
barcode carrying beads. However, all droplets, both occupied and
unoccupied droplets, can comprise at least some concentration of
field-attractable particles 115. As noted previously, by
controlling the flow characteristics of each of the fluids
combining at channel junction 110, as well as controlling the
geometry of the channel junction, partitioning can be optimized to
achieve a desired occupancy level of beads, biological particles,
or both, within the partitions that are generated.
[0209] In some cases, assuming that (i) the droplet is spherical
and has the radius R.sub.D, (ii) a biological particle is spherical
and has the radius R.sub.+, (iii) a barcode carrying bead is
spherical and has the radius R.sub.B and (iv) the concentration of
field-attractable particles in the volume of aqueous fluid is
substantially uniform, the ratio of a number of field-attractable
particles in a singularly occupied droplet (N.sub.+,B) (containing
one of each of a biological particle and a barcode carrying bead)
to a number of field-attractable particles in an unoccupied droplet
(N.sub.-) will be:
N + , B N - = 1 - ( R + R D ) 3 - ( R B R D ) 3 ##EQU00003##
[0210] FIG. 2A shows an example of a microfluidic channel structure
for separating occupied droplets from unoccupied droplets. As
described elsewhere herein, when droplets are generated, there may
be a first subset population of occupied droplets containing one or
more biological particles and a second subset population of
unoccupied droplets not containing any biological particles. In
some cases, the droplets may additionally contain one or more
barcode carrying beads. For example, a droplet may have only a
biological particle, a droplet may have only a barcode carrying
bead, a droplet may have both a biological particle and a barcode
carrying bead, or a droplet may have neither biological particles
nor barcode carrying beads. In some cases, the majority of occupied
partitions can include no more than one biological particle per
occupied partition and, in some cases, some of the generated
partitions can be unoccupied (of any biological particle). In some
cases, though, some of the occupied partitions may include more
than one biological particle. In some cases, the partitioning
process may be controlled such that fewer than 25% of the occupied
partitions contain more than one biological particle, and in many
cases, fewer than 20% of the occupied partitions have more than one
biological particle, while in some cases, fewer than 10% or even
fewer than 5% of the occupied partitions include more than one
biological particle per partition.
[0211] As shown in FIG. 2A, the channel structure can include
channel segments 202, 204, and 206 meeting at a channel
intersection 211. In some instances, the outflow channel 108 of the
emulsion carrying the generated droplets in FIG. 1 can be upstream
of the channel segment 202, such that the generated droplets are
directed to flow to the channel intersection 211 for subsequent
sorting. A controller 220 can be operatively coupled to a fluid
flow unit 218, to facilitate flow of fluid in the channel
structure, and a field application unit 216, to apply one or more
fields to the channel structure.
[0212] In operation, a plurality of discrete droplets, each
comprising a first aqueous fluid 210 can flow as emulsions in a
second fluid 208, wherein the second fluid 208 is immiscible to the
first aqueous fluid 210. The droplets being transported along
channel segment 202 into intersection 211 can comprise a first
subset of droplets 214 that are each occupied with at least a
biological particle and/or a barcode carrying bead and a second
subset of droplets 212 that are each unoccupied. Every droplet,
including occupied and unoccupied droplets, can comprise some
concentration of field-attractable particles. As described above, a
given unoccupied droplet can have a higher concentration of
field-attractable particles than a given occupied droplet to
account for the volume occupied by a biological particle and/or a
barcode bead in an occupied droplet.
[0213] After sorting at or near the intersection 211, the first
subset of droplets 214 can be directed to flow along channel
segment 206 and away from the intersection 211, and the second
subset of droplets 212 can be directed to flow along channel
segment 204 and away from the intersection 211.
[0214] The fluid flow unit 218 can be configured to subject the
second fluid 208 containing a plurality of droplets, including both
occupied droplets and unoccupied droplets, to flow along the
channel 202 towards the intersection 211. The fluid flow unit 218
can be configured to subject the second fluid 208 containing a
plurality of droplets, wherein a majority of the droplets is
unoccupied droplets, to flow along the channel 204 away from the
intersection 211. The fluid flow unit 218 can be configured to
subject the second fluid 208 containing a plurality of droplets,
wherein a majority of the droplets is occupied droplets, to flow
along the channel 206 away from the intersection 211.
Alternatively, the fluid flow unit 218 can be configured to subject
the second fluid 208 containing a plurality of droplets, wherein a
majority of the droplets is unoccupied droplets, to flow along the
channel 206 away from the intersection 211, and configured to
subject the second fluid 208 containing a plurality of droplets,
wherein a majority of the droplets is occupied droplets, to flow
along the channel 204 away from the intersection 211. The fluid
flow unit 218 can be operatively coupled to the controller 220. For
example, the fluid flow unit 218 may receive instructions from the
controller 220 regarding fluid pressure and/or velocity.
[0215] In some instances, the fluid flow unit 218 may comprise a
compressor to provide positive pressure at an upstream location to
direct the fluid from the upstream location to flow to a downstream
location. In some instances, the fluid flow unit 218 may comprise a
pump to provide negative pressure at a downstream location to
direct the fluid from an upstream location to flow to the
downstream location. In some instances, the fluid flow unit 218 may
comprise both a compressor and a pump, each at different locations.
In some instances, the fluid flow unit 218 may comprise different
devices at different locations. The fluid flow unit 218 may
comprise an actuator. While FIG. 2A depicts one fluid flow unit
218, it may be appreciated that there may be a plurality of fluid
flow units 218, each in communication with the controller 220
and/or with each other. For example, there can be a separate fluid
flow unit to direct the fluid in channel 202 towards the
intersection 211, a separate fluid flow unit to direct the fluid in
channel 204 away from the intersection 211, and a separate fluid
flow unit to direct the fluid in channel 206 away from the
intersection 211.
[0216] The field application unit 216 can be configured to apply a
force field to the channel structure. In some instances, the field
application unit 216 can be configured to apply a force field at or
near the intersection 211 such that the second subset of droplets
(unoccupied droplets) are generally directed along the channel
segment 204 and away from the intersection 211, and the first
subset of droplets (occupied droplets) are generally directed along
the channel segment 206 and away from the intersection 211, thereby
isolating the two subsets of droplets.
[0217] For example, the field application unit 216 can apply a
magnetic field at or near the intersection 211. The field
application unit 216 can be a magnet and/or a circuit (e.g.,
current carrying device) configured to generate a magnetic field.
On account of each droplet containing field-attractable particles
(e.g., paramagnetic particles), each droplet may be attracted
(e.g., due to paramagnetic particles) or repelled (e.g., due to
diamagnetic particles) to or away, respectively, from the magnetic
field. The degree of attraction (or repulsion) can be proportional
to a number (and/or a concentration) of field-attractable particles
in each droplet. That is, the magnetic force acting on a droplet,
from the same magnetic field, can be proportional to a number
(and/or a concentration) of field-attractable particles in the
droplet. As previously described above, assuming that (i) the
droplet is spherical and has the radius R.sub.D, (ii) a biological
particle is spherical and has the radius R.sub.+, and (iii) the
concentration of field-attractable particles in the volume of
aqueous fluid is substantially uniform, the ratio of a number of
field-attractable particles in a singularly occupied droplet
(N.sub.+) (wherein the occupied droplet contains a single
biological particle) to a number of field-attractable particles in
an unoccupied droplet (N.sub.-) will be, and thus the ratio of a
magnetic force acting on a singularly occupied droplet (F.sub.M+)
to a magnetic force acting on an unoccupied droplet (F.sub.M-) will
be:
N + N - = F M + F M - = 1 - ( R + R D ) 3 ##EQU00004##
[0218] That is, there may be a stronger (differential) force acting
on a given unoccupied droplet than a given occupied droplet. As can
be appreciated, the above ratio may change with deviations from the
above assumptions (e.g., non-spherical biological particle,
non-spherical droplet, non-uniform concentration of
field-attractable particles in volume of aqueous fluid, etc.).
[0219] In another example, the field application unit 216 can apply
an electric field at or near the intersection 211. On account of
each droplet containing field-attractable particles (e.g.,
conductive particles), each droplet may be attracted or repelled to
or away, respectively, from the electric field. The degree of
attraction (or repulsion) can be proportional to a number (and/or a
concentration) of field-attractable particles in each droplet. That
is, the electric force acting on a droplet, from the same electric
field, can be proportional to a number (and/or a concentration) of
field-attractable particles in the droplet. As previously described
above, assuming that (i) the droplet is spherical and has the
radius R.sub.D, (ii) a biological particle is spherical and has the
radius R.sub.+, and (iii) the concentration of field-attractable
particles in the volume of aqueous fluid is substantially uniform,
the ratio of a number of field-attractable particles in a
singularly occupied droplet (N.sub.+) (wherein the occupied droplet
contains a single biological particle) to a number of
field-attractable particles in an unoccupied droplet (N.sub.-) will
be, and thus the ratio of an electric force acting on a singularly
occupied droplet (F.sub.E+) to an electric force acting on an
unoccupied droplet (F.sub.E-) will be:
N + N - = F E + F E - = 1 - ( R + R D ) 3 ##EQU00005##
[0220] As can be appreciated, the above ratio may change with
deviations from the above assumptions (e.g., non-spherical
biological particle, non-spherical droplet, non-uniform
concentration of field-attractable particles in volume of aqueous
fluid, etc.). In some instances, the fluid flow unit 218 can apply
both an electric field and a magnetic field.
[0221] The field application unit 216 can be operatively coupled to
the controller 220. For example, the field application unit 216 may
receive instructions from the controller 220 regarding force field
strength, orientation, frequency, and/or other variables. While
FIG. 2A depicts one field application unit 216, it may be
appreciated that there may be a plurality of field application
units 216, each in communication with the controller 220, other
controllers, and/or with each other. For example, there can be a
plurality of field application units, each located at a different
location. The controller 220 may instruct the field application
unit 216 to apply a force field sufficiently strong and in a
sufficiently targeted direction towards the mixed (occupied and
unoccupied) droplets such as to direct the unoccupied droplets in
one channel and direct the occupied droplets to another channel. In
an example, the field application unit 216 can be placed in a
location closer to a first channel (e.g., channel 204) than a
second channel (e.g., channel 206) to direct the unoccupied
droplets (which are subject to a stronger force from the same
field) to the first channel, assuming that the field is strongest
when closest to the field application unit 216. The stronger a
force from the field acts on a droplet, the more likely that the
droplet will deviate from an initial flow direction (e.g.,
direction of flow in channel 202) into another channel having
another direction. In some instances, the field application unit
may be located at least in part downstream, from the intersection
211, of a channel intended to isolate unoccupied droplets (e.g.,
channel 204).
[0222] For example, a force field applied can be strong enough to
direct the unoccupied droplets to flow to a first channel but weak
enough to direct (or leave be) the occupied droplets to flow to a
second channel. In some instances, a magnetic field applied by the
field application unit 216 can have a magnetic flux density range
from at least about 10.sup.-5 Teslas (T) to about 1 T.
Alternatively, the magnetic flux density can be less than or equal
to about 10.sup.-5 T and/or greater than or equal to about 1 T. In
some instances, an electric field applied by the field application
unit 216 can have an electric field strength of at least about 1
volt per meter (V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10 V/m, or more.
Alternatively, the electric field strength can be less than about
10 V/m, 5 V/m, 4 V/m, 3 V/m, 2 V/m, 1 V/m, or less.
[0223] FIG. 2B shows an example of a multi-stage microfluidic
channel structure for separating singularly occupied droplets. As
described elsewhere herein, when droplets are generated, there may
be a first subset population of occupied droplets containing a
single biological particle (e.g., cell), a second subset population
of unoccupied droplets not containing any biological particles, and
a third subset population of occupied droplets containing multiple
(e.g., two or more) biological particles. In some cases, the
droplets may additionally contain one or more barcode carrying
beads. For example, a droplet may have only biological particle(s),
a droplet may have only barcode carrying bead(s), a droplet may
have both biological particle(s) and barcode carrying bead(s), or a
droplet may have neither biological particle(s) nor barcode
carrying bead(s). The generated droplets may be sorted to isolate
the first subset population, second subset population, and third
subset population in multiple stages.
[0224] As shown in FIG. 2B, the channel structure can include
channel segments 222, 224, 226, 244, and 246. Channel segments 222,
224, and 226 may meet at channel intersection 231. Channel segments
226, 244, and 246 may meet at channel intersection 233. In some
instances, the outflow channel 108 of the emulsion carrying the
generated droplets in FIG. 1 can be upstream of the channel segment
222, such that the generated droplets are directed to flow to the
channel intersection 231 for subsequent sorting. The generated
droplets may comprise a first subset population of singularly
occupied droplets (e.g., 230A), a second subset population of
unoccupied droplets (e.g., 230B), and a third subset population of
multiply occupied droplets (e.g., 230C). A first controller 240 can
be operatively coupled to a fluid flow unit 238, to facilitate flow
of fluid in the channel structure, and a first field application
unit 236, to apply one or more fields to the channel structure. A
second controller 241 can be operatively coupled to a second field
application unit 237, to apply one or more fields to the channel
structure.
[0225] In operation, a plurality of discrete droplets can flow as
emulsions in a fluid 228. The droplets being transported along
channel segment 222 into intersection 231 can comprise the first
subset population of singularly occupied droplets (e.g., 230A), the
second subset population of unoccupied droplets (e.g., 230B), and
the third subset population of multiply occupied droplets (e.g.,
230C). Every droplet, including singularly occupied, multiply
occupied, and unoccupied droplets, can comprise some concentration
of field-attractable particles. As described above, a given
unoccupied droplet can have a higher concentration of
field-attractable particles than a given occupied droplet to
account for the volume occupied by a biological particle and/or a
barcode bead in an occupied droplet. Within occupied droplets, a
given singly occupied droplet can have a higher concentration of
field-attractable particles than a given multiply occupied droplet
to account for the differential volume occupied by the different
numbers of biological particles (and/or barcode beads) in the
droplet.
[0226] After a first stage of sorting at or near the intersection
231, the first and third subsets of droplets (e.g., 230A, C) can be
directed to flow along channel segment 226 and away from the
intersection 231, and the second subset of droplets 230B can be
directed to flow along channel segment 224 and away from the
intersection 231.
[0227] The fluid flow unit 238 can be configured to subject the
fluid 228 containing a plurality of droplets including the occupied
and/or unoccupied droplets (e.g., as emulsion suspensions) to or
from intersections 231, 233 along channel segments 222, 224, 226,
244, and 246. The fluid flow unit 238 can be operatively coupled to
the first controller 240. For example, the fluid flow unit 238 may
receive instructions from the first controller 240 regarding fluid
pressure and/or velocity.
[0228] In some instances, the fluid flow unit 238 may comprise a
compressor to provide positive pressure at an upstream location to
direct the fluid from the upstream location to flow to a downstream
location. In some instances, the fluid flow unit 238 may comprise a
pump to provide negative pressure at a downstream location to
direct the fluid from an upstream location to flow to the
downstream location. In some instances, the fluid flow unit 238 may
comprise both a compressor and a pump, each at different locations.
In some instances, the fluid flow unit 238 may comprise different
devices at different locations. The fluid flow unit 238 may
comprise an actuator. While FIG. 2B depicts one fluid flow unit
238, it may be appreciated that there may be a plurality of fluid
flow units 238, each in communication with the first controller
240, other controllers (e.g., second controller 241), and/or with
each other. For example, there can be a separate fluid flow unit to
direct the fluid in channel 222 towards the intersection 231, a
separate fluid flow unit to direct the fluid in channel 224 away
from the intersection 231, and a separate fluid flow unit to direct
the fluid in channel 226 away from the intersection 231.
[0229] The first field application unit 236 can be configured to
apply a force field to the channel structure. In some instances,
the field application unit 236 can be configured to apply a force
field at or near the intersection 231 such that the second subset
of droplets (unoccupied droplets) are generally directed along the
channel segment 224 and away from the intersection 231, and the
first and third subset of droplets (occupied droplets) are
generally directed along the channel segment 226 and away from the
intersection 231, thereby separating the subsets of droplets. For
example, as described elsewhere herein, the first field application
unit 236 may apply a magnetic field at or near the intersection
231. On account of each droplet containing field-attractable
particles (e.g., paramagnetic particles), each droplet may be
attracted (e.g., due to paramagnetic particles) or repelled (e.g.,
due to diamagnetic particles) to or away, respectively, from the
magnetic field. The degree of attraction (or repulsion) can be
proportional to a number (and/or a concentration) of
field-attractable particles in each droplet. That is, the magnetic
force acting on a droplet, from the same magnetic field, can be
proportional to a number (and/or a concentration) of
field-attractable particles in the droplet. As described above,
assuming that (i) the droplet is spherical and has the radius
R.sub.D, (ii) a biological particle is spherical and has the radius
R.sub.+, and (iii) the concentration of field-attractable particles
in the volume of aqueous fluid is substantially uniform, the ratio
of a number of field-attractable particles in a singularly occupied
droplet (N.sub.+) (wherein the occupied droplet contains a single
biological particle) to a number of field-attractable particles in
an unoccupied droplet (N.sub.-) will be, and thus the ratio of a
magnetic force acting on a singularly occupied droplet (F.sub.M+)
to a magnetic force acting on an unoccupied droplet (F.sub.M-) will
be:
N + N - = F M + F M - = 1 - ( R + R D ) 3 ##EQU00006##
Similarly, the ratio of a number of field-attractable particles in
a doubly occupied droplet (N.sub.2+) (wherein the occupied droplet
contains two biological particles) to a number of field-attractable
particles in an unoccupied droplet (N.sub.-) will be, and thus the
ratio of a magnetic force acting on a doubly occupied droplet
(F.sub.M,2+) to a magnetic force acting on an unoccupied droplet
(F.sub.M-) will be:
N 2 + N - = F M , 2 + F M - = 1 - 2 ( R + R D ) 3 ##EQU00007##
[0230] That is, there may be a stronger (differential) force acting
on a given unoccupied droplet than a given occupied droplet.
Between occupied droplets, there may be a stronger (differential)
force acting on a given singularly occupied droplet than a given
doubly occupied droplet. Similarly, the more occupied a droplet is
(with more biological particles), the less it will be affected by
the magnetic field. As can be appreciated, the above ratios may
change with deviations from the above assumptions (e.g.,
non-spherical biological particle, non-spherical droplet,
non-uniform concentration of field-attractable particles in volume
of aqueous fluid, etc.).
[0231] In another example, as described elsewhere herein, the first
field application unit 236 can apply an electric field at or near
the intersection 231. On account of each droplet containing
field-attractable particles (e.g., conductive particles), each
droplet may be attracted or repelled to or away, respectively, from
the electric field. The degree of attraction (or repulsion) can be
proportional to a number (and/or a concentration) of
field-attractable particles in each droplet. That is, the electric
force acting on a droplet, from the same electric field, can be
proportional to a number (and/or a concentration) of
field-attractable particles in the droplet.
[0232] The first field application unit 236 can be operatively
coupled to the first controller 240. For example, the first field
application unit 236 may receive instructions from the first
controller 240 regarding force field strength, orientation,
frequency, and/or other variables. The first controller 240 may
instruct the first field application unit 236 to apply a force
field sufficiently strong and in a sufficiently targeted direction
towards the mixed (occupied and unoccupied) droplets such as to
direct the unoccupied droplets in one channel and direct the
occupied droplets to another channel. In an example, the first
field application unit 236 can be placed in a location closer to a
first channel (e.g., channel 224) than a second channel (e.g.,
channel 226) to direct the unoccupied droplets (which are subject
to a stronger force from the same field) to the first channel,
assuming that the field is strongest when closest to the first
field application unit 236. The stronger a force from the field
acts on a droplet, the more likely that the droplet will deviate
from an initial flow direction (e.g., direction of flow in channel
222) into another channel having another direction. In some
instances, the field application unit may be located at least in
part downstream, from the intersection 231, of a channel intended
to isolate unoccupied droplets (e.g., channel 224).
[0233] For example, a force field applied can be strong enough to
direct the unoccupied droplets to flow to a first channel but weak
enough to direct (or leave be) the occupied droplets (whether
singularly, doubly, or otherwise multiply occupied) to flow to a
second channel. In some instances, a magnetic field applied by the
field application unit 236 can have a magnetic flux density range
from at least about 10.sup.-5 Teslas (T) to about 1 T.
Alternatively, the magnetic flux density can be less than or equal
to about 10.sup.-5 T and/or greater than or equal to about 1 T. In
some instances, an electric field applied by the field application
unit 236 can have an electric field strength of at least about 1
volt per meter (V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10 V/m, or more.
Alternatively, the electric field strength can be less than about
10 V/m, 5 V/m, 4 V/m, 3 V/m, 2 V/m, 1 V/m, or less.
[0234] The first and third sets of droplets (e.g., 230A, C)
isolated to channel 226 may be subjected to a second stage of
sorting at or near the intersection 233. After the second stage of
sorting at or near the intersection 233, the third subset of
droplets (e.g., 230C) can be directed to flow along channel segment
246 and away from the intersection 233, and the first subset of
droplets 230A can be directed to flow along channel segment 244 and
away from the intersection 233. Thus, the singularly occupied
droplets (first subset of droplets) may be isolated from both the
unoccupied droplets and the multiply occupied droplets.
[0235] The second field application unit 237 can be configured to
apply a force field to the channel structure. In some instances,
the field application unit 237 can be configured to apply a force
field at or near the intersection 233 such that the first subset of
droplets (singularly occupied droplets) are generally directed
along the channel segment 244 and away from the intersection 233,
and the third subset of droplets (multiply occupied droplets) are
generally directed along the channel segment 246 and away from the
intersection 233, thereby separating the first and third subsets of
droplets. For example, as described elsewhere herein, the second
field application unit 237 may apply a magnetic field at or near
the intersection 233. As described elsewhere herein, as between
occupied droplets, there may be a stronger (differential) force
acting on a given singularly occupied droplet than a given doubly
occupied droplet. Similarly, the more occupied a droplet is (with
more biological particles), the less it will be affected by the
magnetic field. In another example, as described elsewhere herein,
the second field application unit 237 can apply an electric field
at or near the intersection 233.
[0236] The second field application unit 237 can be operatively
coupled to the second controller 241. For example, the second field
application unit 237 may receive instructions from the second
controller 241 regarding force field strength, orientation,
frequency, and/or other variables. The second controller 241 may
instruct the second field application unit 237 to apply a force
field sufficiently strong and in a sufficiently targeted direction
towards the mixed (singularly occupied and multiply occupied)
droplets such as to direct the singularly droplets in one channel
and direct the multiply occupied droplets to another channel. In an
example, the second field application unit 237 can be placed in a
location closer to a first channel (e.g., channel 244) than a
second channel (e.g., channel 246) to direct the singularly
occupied droplets (which are subject to a stronger force from the
same field than multiply occupied droplets) to the first channel,
assuming that the field is strongest when closest to the second
field application unit 237. The stronger a force from the field
acts on a droplet, the more likely that the droplet will deviate
from an initial flow direction (e.g., direction of flow in channel
226) into another channel having another direction. In some
instances, the field application unit may be located at least in
part downstream, from the intersection 233, of a channel intended
to isolate singularly occupied droplets (e.g., channel 244).
Alternatively or in addition to, the second field application unit
236 can be operatively coupled to the first controller 240.
[0237] For example, a force field applied can be strong enough to
direct the singularly occupied droplets to flow to a first channel
but weak enough to direct (or leave be) the multiply occupied
droplets (whether singularly, doubly, or otherwise multiply
occupied) to flow to a second channel. In some instances, the force
field applied by the second field application unit 237 may be
stronger than the force field applied by the first field
application unit 236. In some instances, a magnetic field applied
by the second field application unit 237 can have a magnetic flux
density range from at least about 10.sup.-5 Teslas (T) to about 1
T. Alternatively, the magnetic flux density can be less than or
equal to about 10.sup.-5 T and/or greater than or equal to about 1
T. In some instances, an electric field applied by the field
application unit 236 can have an electric field strength of at
least about 1 volt per meter (V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10
V/m, or more. Alternatively, the electric field strength can be
less than about 10 V/m, 5 V/m, 4 V/m, 3 V/m, 2 V/m, 1 V/m, or
less.
[0238] The systems and methods described with respect to FIGS. 2A-B
may be used to isolate cell beads from particles unoccupied with
biological particles, and/or separate singularly occupied cell
beads from unoccupied and multiply occupied cell beads. As
described elsewhere herein, a plurality of particles may comprise a
first subset of particles (e.g., cell beads) occupied by biological
particles (e.g., cells) and a second subset of particles unoccupied
by biological particles. Both occupied and unoccupied particles may
comprise field-attractable particles. Occupied particles may
include singularly occupied cell beads, each containing a single
biological particle, and multiply occupied cell beads, each
containing two or more biological particles. For example, such
particles comprising the field-attractable particles may be
generated from polymerizing the plurality of droplets comprising
the field-attractable particles (e.g., in FIG. 1). In a channel
structure including channel segments 202, 204, and 206 meeting at a
channel intersection 211, the plurality of particles may be
directed to flow (e.g., as suspensions in a fluid, e.g., aqueous
fluid) to the channel intersection 211 for subsequent sorting. A
controller 220 can be operatively coupled to a fluid flow unit 218,
to facilitate flow of fluid in the channel structure, and a field
application unit 216, to apply one or more fields to the channel
structure.
[0239] In operation, a plurality of discrete particles, including
both cell beads and unoccupied particles, can be directed to flow
along channel segment 202 into intersection 211. The plurality of
particles can comprise a first subset of particles (e.g., cell
beads) that are each occupied with at least a biological particle
and a second subset of particles that are each unoccupied. Every
particle, including cell beads and unoccupied particles, can
comprise some concentration of field-attractable particles. As
described with respect to the relative concentrations of
field-attractable particles in occupied and unoccupied droplets, a
given unoccupied particle can have a higher concentration of
field-attractable particles than a given cell bead (e.g., a given
occupied particle) to account for the volume occupied by a
biological particle in a cell bead.
[0240] After sorting at or near the intersection 211, the first
subset of particles (e.g., cell beads) can be directed to flow
along channel segment 206 and away from the intersection 211, and
the second subset of particles can be directed to flow along
channel segment 204 and away from the intersection 211.
[0241] The fluid flow unit 218 can be configured to subject the
second fluid 208 containing the plurality of particles, including
both cell beads and unoccupied particles, to flow along the channel
202 towards the intersection 211. The fluid flow unit 218 can be
configured to subject the second fluid 208 containing a plurality
of particles, wherein a majority of the particles is unoccupied, to
flow along the channel 204 away from the intersection 211. The
fluid flow unit 218 can be configured to subject the second fluid
208 containing a plurality of particles, wherein a majority of the
particles is cell beads (e.g., occupied particles), to flow along
the channel 206 away from the intersection 211. Alternatively, the
fluid flow unit 218 can be configured to subject the second fluid
208 containing a plurality of particles wherein a majority of the
particles is unoccupied particles, to flow along the channel 206
away from the intersection 211, and configured to subject the
second fluid 208 containing a plurality of particles, wherein a
majority of the particles is cell beads, to flow along the channel
204 away from the intersection 211. The fluid flow unit 218 can be
operatively coupled to the controller 220. For example, the fluid
flow unit 218 may receive instructions from the controller 220
regarding fluid pressure and/or velocity.
[0242] The field application unit 216 can be configured to apply a
force field to the channel structure. In some instances, the field
application unit 216 can be configured to apply a force field at or
near the intersection 211 such that the second subset of particles
(unoccupied particles) is generally directed along the channel
segment 204 and away from the intersection 211, and the first
subset of particles (cell beads) is generally directed along the
channel segment 206 and away from the intersection 211, thereby
isolating the two subsets of particles.
[0243] For example, the field application unit 216 can apply a
magnetic field at or near the intersection 211. The field
application unit 216 can be a magnet and/or a circuit (e.g.,
current carrying device) configured to generate a magnetic field.
On account of each particle containing field-attractable particles
(e.g., paramagnetic particles), each particle may be attracted
(e.g., due to paramagnetic particles) or repelled (e.g., due to
diamagnetic particles) to or away, respectively, from the magnetic
field. The degree of attraction (or repulsion) can be proportional
to a number (and/or a concentration) of field-attractable particles
in each particle. That is, the magnetic force acting on a particle,
from the same magnetic field, can be proportional to a number
(and/or a concentration) of field-attractable particles in the
particle. For example, assuming that (i) the particle is spherical
and has the radius R.sub.CB, (ii) a biological particle is
spherical and has the radius R.sub.+, and (iii) the concentration
of field-attractable particles in the particle is substantially
uniform, the ratio of a number of field-attractable particles in a
singularly occupied cell bead (N.sub.+) (wherein the cell bead
contains a single biological particle) to a number of
field-attractable particles in an unoccupied particle (N.sub.-)
will be, and thus the ratio of a magnetic force acting on a
singularly occupied cell bead (F.sub.M+) to a magnetic force acting
on an unoccupied particle (F.sub.M-) will be:
N + N - = F M + F M - = 1 - ( R + R CB ) 3 ##EQU00008##
[0244] That is, there may be a stronger (differential) force acting
on a given unoccupied particle than a given cell bead. As can be
appreciated, the above ratio may change with deviations from the
above assumptions (e.g., non-spherical biological particle,
non-spherical particle, non-uniform concentration of
field-attractable particles in volume of particle, etc.).
[0245] In another example, the field application unit 216 can apply
an electric field at or near the intersection 211. On account of
each particle containing field-attractable particles (e.g.,
conductive particles), each particle may be attracted or repelled
to or away, respectively, from the electric field. The degree of
attraction (or repulsion) can be proportional to a number (and/or a
concentration) of field-attractable particles in each particle.
That is, the electric force acting on a particle, from the same
electric field, can be proportional to a number (and/or a
concentration) of field-attractable particles in the particle. As
previously described above, assuming that (i) the particle is
spherical and has the radius R.sub.CB, (ii) a biological particle
is spherical and has the radius R.sub.+, and (iii) the
concentration of field-attractable particles in the volume of a
particle is substantially uniform, the ratio of a number of
field-attractable particles in a singularly occupied cell bead
(N.sub.+) (wherein the cell bead contains a single biological
particle) to a number of field-attractable particles in an
unoccupied particle (N.sup.-) will be, and thus the ratio of an
electric force acting on a singularly occupied cell bead (F.sub.E+)
to an electric force acting on an unoccupied particle (F.sub.E-)
will be:
N + N - = F E + F E - = 1 - ( R + R D ) 3 ##EQU00009##
[0246] As can be appreciated, the above ratio may change with
deviations from the above assumptions (e.g., non-spherical
biological particle, non-spherical particle, non-uniform
concentration of field-attractable particles in volume of a
particle, etc.). In some instances, the fluid flow unit 218 can
apply both an electric field and a magnetic field.
[0247] The field application unit 216 can be operatively coupled to
the controller 220. For example, the field application unit 216 may
receive instructions from the controller 220 regarding force field
strength, orientation, frequency, and/or other variables. The
controller 220 may instruct the field application unit 216 to apply
a force field sufficiently strong and in a sufficiently targeted
direction towards the mixed (occupied and unoccupied) particles
such as to direct the unoccupied particles in one channel and
direct the occupied particles to another channel. In an example,
the field application unit 216 can be placed in a location closer
to a first channel (e.g., channel 204) than a second channel (e.g.,
channel 206) to direct the unoccupied particles (which are subject
to a stronger force from the same field) to the first channel,
assuming that the field is strongest when closest to the field
application unit 216. The stronger a force from the field acts on a
particle, the more likely that the particle will deviate from an
initial flow direction (e.g., direction of flow in channel 202)
into another channel having another direction. In some instances,
the field application unit may be located at least in part
downstream, from the intersection 211, of a channel intended to
isolate unoccupied particles (e.g., channel 204).
[0248] For example, a force field applied can be strong enough to
direct the unoccupied particles to flow to a first channel but weak
enough to direct (or leave be) the occupied particles to flow to a
second channel. In some instances, a magnetic field applied by the
field application unit 216 can have a magnetic flux density range
from at least about 10.sup.-5 Teslas (T) to about 1 T.
Alternatively, the magnetic flux density can be less than or equal
to about 10.sup.-5 T and/or greater than or equal to about 1 T. In
some instances, an electric field applied by the field application
unit 216 can have an electric field strength of at least about 1
volt per meter (V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10 V/m, or more.
Alternatively, the electric field strength can be less than about
10 V/m, 5 V/m, 4 V/m, 3 V/m, 2 V/m, 1 V/m, or less.
[0249] Similarly, singularly occupied cell beads may be sorted from
unoccupied particles and multiply occupied cell beads by using the
systems and methods described with respect to FIG. 2B but
introducing a plurality of particles (comprising a first subset of
singularly occupied cell beads, a second subset of unoccupied
particles, and a third subset of multiply occupied cell beads),
each particle comprising field attractable particles, in place of
the plurality of droplets comprising the field attractable
particles.
[0250] While FIG. 2A and FIG. 2B each depicts a channel structure
wherein a second channel (e.g., channel 204) branches off a first
channel (e.g., channel 202) at a sorting intersection (e.g.,
intersection 211) such that a third channel (channel 204) continues
in the same direction as the first channel, it can be appreciated
that the systems and methods disclosed herein may be application to
different channel structures. For example, the third channel can be
at a different angle (e.g., not) 180.degree. than the first
channel. In some examples, the channel structure may have more than
two channels branching off the first channel, wherein the field
application unit 216 is configured to separate the droplets into
unoccupied droplets, droplets containing only one biological
particle, droplets containing more than one biological particles,
droplets containing only barcode carrying beads, droplets
containing both a biological particle and barcode carrying beads,
or other variations. In some examples, the channel structure may
have more than two channels branching off the first channel,
wherein the field application unit 216 is configured to separate
the particles into unoccupied particles, particles containing only
one biological particle, particles containing more than one
biological particles, particles containing only barcode carrying
beads, particles containing both a biological particle and barcode
carrying beads, or other variations. For example, the more volume
of a droplet or a particle is occupied by one or more biological
particles and/or one or more barcode carrying beads contained
therein, the weaker can be the force acting on the droplet or the
particle by the field applied by the field application unit 216,
and thus the less the deviation in direction of flow relative to
the direction of flow in the first channel. That is, upon
application of a force field, the unoccupied droplets or particles
may be capable of deviating the most (e.g., to a channel closest to
the field application unit), a droplet or particle containing a
single biological particle (e.g., singularly occupied cell bead)
may be capable of deviating but not as much as the unoccupied
droplets or particles (e.g., to a channel second closest to the
field application unit), and a droplet or particle containing both
a biological particle and a barcode carrying bead may be capable of
deviating the least of the three types of droplets or particles
(e.g., to a channel farthest from the field application unit).
[0251] While FIGS. 2A-2B depict narrow channels allowing for the
flow of droplets and/or particles only in single file, the systems
and methods disclosed herein may be applicable to channels having a
broader width (e.g., diameter) that allows for the flow of droplets
and/or particles in more than single file.
[0252] While FIG. 2A depicts one controller 220 operatively coupled
to both the fluid flow unit 218 and the field application unit 216,
a separate controller can be coupled to the fluid flow unit 218 and
a separate controller can be coupled to the field application unit
216. The two separate controllers may or may not be in
communication with each other. In some instances, there may be a
plurality of controllers (e.g., two controllers to a fluid flow
unit 218), wherein each controller may or may not be in
communication with each other. The controller 220 may send
instructions to the fluid flow unit 218 and/or the field
application unit 216 via wired connection and/or wireless
connection (e.g., Wi-Fi, Bluetooth, NFC, etc.).
[0253] FIG. 3 shows another example of a microfluidic channel
structure for separating occupied droplets from unoccupied
droplets. As described elsewhere herein, when droplets are
generated, there may be a first subset population of occupied
droplets containing one or more biological particles and a second
subset population of unoccupied droplets not containing any
biological particles. In some cases, the droplets may additionally
contain one or more barcode carrying beads. For example, a droplet
may have only a biological particle, a droplet may have only a
barcode carrying bead, a droplet may have both a biological
particle and a barcode carrying bead, or a droplet may have neither
biological particles nor barcode carrying beads. In some cases, the
majority of occupied partitions can include no more than one
biological particle per occupied partition and, in some cases, some
of the generated partitions can be unoccupied (of any biological
particle). In some cases, though, some of the occupied partitions
may include more than one biological particle. 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, and in many cases, fewer than about 20% of the occupied
partitions have more than one biological particle, while in some
cases, fewer than about 10% or even fewer than about 5% of the
occupied partitions include more than one biological particle per
partition.
[0254] As shown in FIG. 3, the channel structure can include
channel segments 302, 304, and 306 meeting at a channel
intersection 311. In some instances, the outflow channel 108 of the
emulsion carrying the generated droplets in FIG. 1 can be upstream
of the channel segment 302, such that the generated droplets are
directed to flow to the channel intersection 211 for subsequent
sorting. A controller 322 can be operatively coupled to a fluid
flow unit 318, to facilitate flow of fluid in the channel
structure, a sensor 320, to detect at least a characteristic of a
droplet or a plurality of droplets, and a pressure application unit
316, to apply a pressure pulse to the channel structure.
[0255] In operation, a plurality of discrete droplets, each
comprising a first aqueous fluid 310 can flow as emulsions in a
second fluid 308, wherein the second fluid 308 is immiscible to the
first aqueous fluid 310. The droplets being transported along
channel segment 302 into intersection 311 can comprise a first
subset of droplets 314 that are each occupied with at least a
biological particle and/or a barcode carrying bead and a second
subset of droplets 312 that are each unoccupied. Every droplet,
including occupied and unoccupied droplets, may or may not comprise
some concentration of field-attractable particles. Although FIG. 3
depicts each droplet as comprising field-attractable particles 315,
this is not required.
[0256] After sorting at or near the intersection 311, the first
subset of droplets 314 can be directed to flow along channel
segment 306 and away from the intersection 311, and the second
subset of droplets 312 can be directed to flow along channel
segment 304 and away from the intersection 311.
[0257] The fluid flow unit 318 can be configured to subject the
second fluid 308 containing a plurality of droplets, including both
occupied droplets and unoccupied droplets, to flow along the
channel 302 towards the intersection 311. The fluid flow unit 318
can be configured to subject the second fluid 308 containing a
plurality of droplets, wherein a majority of the droplets is
unoccupied droplets, to flow along the channel 304 away from the
intersection 311. The fluid flow unit 318 can be configured to
subject the second fluid 308 containing a plurality of droplets,
wherein a majority of the droplets is occupied droplets, to flow
along the channel 306 away from the intersection 311.
Alternatively, the fluid flow unit 318 can be configured to subject
the second fluid 308 containing a plurality of droplets, wherein a
majority of the droplets is unoccupied droplets, to flow along the
channel 306 away from the intersection 311, and configured to
subject the second fluid 308 containing a plurality of droplets,
wherein a majority of the droplets is occupied droplets, to flow
along the channel 304 away from the intersection 311. The fluid
flow unit 318 can be operatively coupled to the controller 322. For
example, the fluid flow unit 318 may receive instructions from the
controller 322 regarding fluid pressure and/or velocity.
[0258] In some instances, the fluid flow unit 318 may comprise a
compressor to provide positive pressure at an upstream location to
direct the fluid from the upstream location to flow to a downstream
location. In some instances, the fluid flow unit 318 may comprise a
pump to provide negative pressure at a downstream location to
direct the fluid from an upstream location to flow to the
downstream location. In some instances, the fluid flow unit 318 may
comprise both a compressor and a pump, each at different locations.
In some instances, the fluid flow unit 318 may comprise different
devices at different locations. The fluid flow unit 318 may
comprise an actuator. While FIG. 3 depicts one fluid flow unit 318,
it may be appreciated that there may be a plurality of fluid flow
units 318, each in communication with the controller 322 and/or
with each other. For example, there can be a separate fluid flow
unit to direct the fluid in channel 302 towards the intersection
311, a separate fluid flow unit to direct the fluid in channel 304
away from the intersection 311, and a separate fluid flow unit to
direct the fluid in channel 306 away from the intersection 311.
[0259] The sensor 320 can be configured to sense at least a
characteristic of a droplet or a plurality of droplets in the first
channel segment 302. In some instances, the sensor 320 may detect
the characteristic of a droplet as the droplet passes the sensor
320. The sensor 320 may be located upstream of the intersection
311. One or more characteristics detected by the sensor 320 of a
droplet can be indicative of the type of droplet, such as whether
the droplet is occupied or unoccupied, or whether the droplet
contains a biological particle and/or a barcode carrying bead. In
some instances, the sensor 320 can be an impedance sensor
configured to measure bulk impedance when droplets pass by the
sensor 320. In some instances, a higher impedance can be measured
for occupied droplets than for unoccupied droplets (e.g., due to
mass and/or weight distribution of occupied droplet, etc.). In some
instances, the sensor 320 can be an optical sensor configured to
measure optical properties of a droplet, such as to distinguish
whether the droplet is occupied or unoccupied. The optical sensor
and/or a supporting device may be configured to emit a detection
signal configured to probe one or more droplets, including for
example an electromagnetic signal (e.g., in any wavelength) and/or
an acoustic signal. In some instances, the optical sensor and/or a
supporting device may comprise an illumination source configured to
illuminate the droplet or droplets with one or more types of
electromagnetic radiation. In some instances, the electromagnetic
radiation can include illumination in one or more of the visible
spectrum, infrared spectrum, the ultraviolet spectrum, and ionizing
radiation spectrum. In some instances, the ionizing radiation can
include x-rays. Alternatively the sensor 320 may be one or more
devices that are configured to provide one or more of optical
sensing, thermal sensing, laser imaging, infrared imaging,
capacitance sensing, mass sensing, vibration sensing across at
least a portion of the electromagnetic spectrum, and magnetic
induction sensing. The sensor 320 can be operatively coupled to the
controller 322. For example, the sensor 320 may transmit sensor
data (e.g., on one or more characteristics of a droplet or a
plurality of droplets) to the controller 322. The controller 322
may then use such data to determine whether the droplet is occupied
or unoccupied.
[0260] While FIG. 3 depicts one sensor 320, it may be appreciated
that there may be a plurality of sensors, each in communication
with the controller 322 and/or with each other. For example, there
can be a plurality of sensors upstream of the intersection 311 at
different locations, for example, detecting one or more
characteristics of a droplet or a plurality of droplets at
different angles.
[0261] The pressure application unit 316 can be configured to apply
a pressure pulse to the channel structure. In some instances, the
pressure application unit 316 can be configured to apply a pressure
pulse at or near the intersection 311 such that, via hydrodynamic
forces, the second subset of droplets (unoccupied droplets) are
generally directed along the channel segment 304 and away from the
intersection 311, and the first subset of droplets (occupied
droplets) are generally directed along the channel segment 306 and
away from the intersection 311, thereby isolating the two subsets
of droplets.
[0262] In some instances, the pressure application unit 316 may
comprise a compressor to provide positive pressure pulses at an
upstream location to direct the fluid from the upstream location to
flow to a downstream location. In some instances, the pressure
application unit 316 may comprise a pump to provide negative
pressure pulses at a downstream location to direct the fluid from
an upstream location to flow to the downstream location. In some
instances, the pressure application unit 316 may comprise both a
compressor and a pump, each at different locations. In some
instances, the pressure application unit 316 may comprise different
devices at different locations. The pressure application unit 316
may comprise an actuator. While FIG. 3 depicts one pressure
application unit 316, it may be appreciated that there may be a
plurality of pressure application units, each in communication with
the controller 322 and/or with each other. In some instances, the
pressure application unit 316 and a fluid flow unit 318 may be the
same device or same devices. In some instances, the pressure
application unit 316 may be, entirely or at least in part, external
to the microfluidic structure (e.g., microfluidic channels), as
illustrated in FIG. 3. In some instances, the pressure application
unit 316 may be, entirely or at least in part, internal to and/or
integral to the microfluidic structure. For example, a pressure
pulse may be generated by deflection of membranes. In some
instances, a pressure pulse may be generated from generation of air
bubbles, wherein expansion of the bubble may displace fluid
parcels.
[0263] Occupied droplets and unoccupied droplets may respond
differently to a pressure pulse, for example due to varying
predetermined particle and fluid characteristics. In some
instances, singularly occupied droplets and multiply occupied
droplets may respond differently to a pressure pulse. The
predetermined particle and fluid characteristics can include size
of a droplet, mass of a droplet, viscosity of droplet suspension in
the emulsion, deformability, and other characteristics. That is, a
given singularly occupied droplet, a multiply occupied droplet, and
an unoccupied droplet may respond differently when subject to
hydrodynamic forces triggered by the pressure pulses. In some
instances, the controller 322 may, based on a determination made
from data received from the sensor 320 (e.g., determination on
whether droplet is occupied or unoccupied), instruct the pressure
application unit 316 to apply different pressure pulses, for
example, by varying frequency of the pulses and/or changing
pressure differential. For example, the pressure application unit
316 may apply a first type of pressure pulse when an occupied
droplet is approaching the intersection 311 and a second type of
pressure pulse when an unoccupied droplet is approaching the
intersection 311. Alternatively, the same pressure pulse can be
applied for any type of droplet approaching the intersection 311,
and the droplet may react (or respond) differently (e.g., deviating
from a fluid flow direction at different angles) depending on
whether the droplet is occupied or unoccupied.
[0264] The pressure application unit 316 can be operatively coupled
to the controller 320. For example, the pressure application unit
316 may receive instructions from the controller 322 regarding
pressure pulse strength, frequency, and/or other variables.
[0265] The systems and methods described with respect to FIG. 3 may
be used to separate occupied particles from unoccupied particles.
As described elsewhere herein, a plurality of particles may
comprise a first subset of particles (e.g., cell beads) occupied by
biological particles (e.g., cells) and a second subset of particles
unoccupied by biological particles. In a channel structure
including channel segments 302, 304, and 306 meeting at a channel
intersection 311, the plurality of particles may be directed to
flow (e.g., as suspensions in a fluid, e.g., aqueous fluid) to the
channel intersection 311 for subsequent sorting. A controller 322
can be operatively coupled to a fluid flow unit 318, to facilitate
flow of fluid in the channel structure, a sensor 320, to detect at
least a characteristic of a particle or a plurality of particles,
and a pressure application unit 316, to apply a pressure pulse to
the channel structure.
[0266] After sorting at or near the intersection 311, the first
subset of particles (e.g., cell beads) can be directed to flow
along channel segment 306 and away from the intersection 311, and
the second subset of particles can be directed to flow along
channel segment 304 and away from the intersection 311.
[0267] The fluid flow unit 318 can be configured to subject the
second fluid 308 containing a plurality of particles, including
both cell beads and unoccupied particles, to flow along the channel
302 towards the intersection 311. The fluid flow unit 318 can be
configured to subject the second fluid 308 containing a plurality
of particles, wherein a majority of the particles is unoccupied
particles, to flow along the channel 304 away from the intersection
311. The fluid flow unit 318 can be configured to subject the
second fluid 308 containing a plurality of particles, wherein a
majority of the particles is occupied particles, to flow along the
channel 306 away from the intersection 311. Alternatively, the
fluid flow unit 318 can be configured to subject the second fluid
308 containing a plurality of particles, wherein a majority of the
particles is unoccupied particles, to flow along the channel 306
away from the intersection 311, and configured to subject the
second fluid 308 containing a plurality of particles, wherein a
majority of the particles is occupied particles, to flow along the
channel 304 away from the intersection 311. The fluid flow unit 318
can be operatively coupled to the controller 322. For example, the
fluid flow unit 318 may receive instructions from the controller
322 regarding fluid pressure and/or velocity.
[0268] The sensor 320 can be configured to sense at least a
characteristic of a particle or a plurality of particles in the
first channel segment 302. In some instances, the sensor 320 may
detect the characteristic of a particle as the particle passes the
sensor 320. The sensor 320 may be located upstream of the
intersection 311. One or more characteristics detected by the
sensor 320 of a particle can be indicative of the type of particle,
such as whether the particle is occupied or unoccupied, or whether
the particle contains a biological particle and/or a barcode
carrying bead. In some instances, the sensor 320 can be an
impedance sensor configured to measure bulk impedance when
particles pass by the sensor 320. In some instances, a higher
impedance can be measured for occupied particles than for
unoccupied particles (e.g., due to mass and/or weight distribution
of occupied particle, etc.). In some instances, the sensor 320 can
be an optical sensor configured to measure optical properties of a
particle, such as to distinguish whether the particle is occupied
or unoccupied. The optical sensor and/or a supporting device may be
configured to emit a detection signal configured to probe one or
more particles, including for example an electromagnetic signal
(e.g., in any wavelength) and/or an acoustic signal. In some
instances, the optical sensor and/or a supporting device may
comprise an illumination source configured to illuminate the
particle or particles with one or more types of electromagnetic
radiation. In some instances, the electromagnetic radiation can
include illumination in one or more of the visible spectrum,
infrared spectrum, the ultraviolet spectrum, and ionizing radiation
spectrum. In some instances, the ionizing radiation can include
x-rays. Alternatively the sensor 320 may be one or more devices
that are configured to provide one or more of optical sensing,
thermal sensing, laser imaging, infrared imaging, capacitance
sensing, mass sensing, vibration sensing across at least a portion
of the electromagnetic spectrum, and magnetic induction sensing.
The sensor 320 can be operatively coupled to the controller 322.
For example, the sensor 320 may transmit sensor data (e.g., on one
or more characteristics of a particle or a plurality of particles)
to the controller 322. The controller 322 may then use such data to
determine whether the particle is occupied or unoccupied.
[0269] While FIG. 3 depicts one sensor 320, it may be appreciated
that there may be a plurality of sensors, each in communication
with the controller 322 and/or with each other. For example, there
can be a plurality of sensors upstream of the intersection 311 at
different locations, for example, detecting one or more
characteristics of a particle or a plurality of particles at
different angles.
[0270] The pressure application unit 316 can be configured to apply
a pressure pulse to the channel structure. In some instances, the
pressure application unit 316 can be configured to apply a pressure
pulse at or near the intersection 311 such that, via hydrodynamic
forces, the second subset of particles (unoccupied particles) are
generally directed along the channel segment 304 and away from the
intersection 311, and the first subset of particles (cell beads)
are generally directed along the channel segment 306 and away from
the intersection 311, thereby isolating the two subsets of
particles.
[0271] Occupied particles and unoccupied particles may respond
differently to a pressure pulse, for example due to varying
predetermined particle and fluid characteristics. The predetermined
particle and fluid characteristics can include size of a particle,
mass of a particle, viscosity of particle suspension in the fluid,
deformability, and other characteristics. That is, a given
singularly occupied cell bead, multiply occupied cell bead, and an
unoccupied particle may respond differently when subject to
hydrodynamic forces triggered by the pressure pulses. In some
instances, the controller 322 may, based on a determination made
from data received from the sensor 320 (e.g., determination on
whether particle is occupied or unoccupied), instruct the pressure
application unit 316 to apply different pressure pulses, for
example, by varying frequency of the pulses and/or changing
pressure differential. For example, the pressure application unit
316 may apply a first type of pressure pulse when an occupied
particle is approaching the intersection 311 and a second type of
pressure pulse when an unoccupied particle is approaching the
intersection 311. Alternatively, the same pressure pulse can be
applied for any type of particle approaching the intersection 311,
and the particle may react (or respond) differently (e.g.,
deviating from a fluid flow direction at different angles)
depending on whether the particle is occupied or unoccupied. In
some instances, different pressure pulses may be applied as between
occupied and unoccupied droplets, occupied and unoccupied
particles, singularly occupied and multiply occupied droplets,
and/or singularly occupied and multiply occupied cell beads.
[0272] The pressure application unit 316 can be operatively coupled
to the controller 320. For example, the pressure application unit
316 may receive instructions from the controller 322 regarding
pressure pulse strength, frequency, and/or other variables.
[0273] While FIG. 3 depicts a channel structure wherein a second
channel (channel 304) branches off a first channel (channel 302)
such that a third channel (channel 304) continues in the same
direction as the first channel, it can be appreciated that the
systems and methods disclosed herein may be application to
different channel structures. For example, the third channel can be
at a different angle (e.g., not 180.degree.) than the first
channel. In some examples, the channel structure may have more than
two channels branching off the first channel, wherein the pressure
application unit 316 is configured to separate the droplets into
unoccupied droplets, droplets containing only one biological
particle, droplets containing more than one biological particles,
droplets containing only barcode carrying beads, droplets
containing both a biological particle and barcode carrying beads,
or other variations. The pressure application unit 316 can be
configured to separate particles into unoccupied particles,
particles containing only one biological particle, particles
containing more than one biological particle, particles containing
only barcode carrying beads, particles containing both a biological
particle and barcode carrying beads, or other variations. For
example, upon application of a pressure pulse, the unoccupied
droplets or particles may be capable of deviating the most (e.g.,
to a first channel), a droplet or particle containing a single
biological particle may be capable of deviating but not as much as
the unoccupied droplets or particles (e.g., to a second channel),
and a droplet or particle containing both a biological particle and
a barcode carrying bead may be capable of deviating the least of
the three types of droplets or particles (e.g., to a third
channel).
[0274] While FIG. 3 depicts narrow channels allowing for the flow
of droplets or particles only in single file, the systems and
methods disclosed herein may be applicable to channels having a
broader width (e.g., diameter) that allows for the flow of droplets
or particles in more than single file.
[0275] While FIG. 3 depicts one controller 322 operatively coupled
to all of the fluid flow unit 318, the sensor 320, and the pressure
application unit 316, a separate controller can be coupled to the
fluid flow unit 318, a separate controller can be coupled to the
sensor 320, and a separate controller can be coupled to the
pressure application unit 216. The three separate controllers may
or may not be in communication with each other. In some instances,
there may be a plurality of controllers (e.g., two controllers to a
fluid flow unit 318), wherein each controller may or may not be in
communication with each other. The controller 322 may send
instructions to the fluid flow unit 318, sensor 320, and/or the
pressure application unit 316 via wired connection and/or wireless
connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). The controller 322
may receive data from the fluid flow unit 318, sensor 320, and/or
the pressure application unit 316 via wired connection and/or
wireless connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In some
instances, the components can be directly or indirectly be in
communication with each other, with or without going through the
controller 322. For example, the sensor 320 may be directly coupled
to the pressure application unit 316.
[0276] The separation systems and methods disclosed herein may
achieve super Poisson loading. For example, the droplets can be
separated into two subsets such that at least about 5%, 10%, 15%,
20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,
99%, or greater of a first subset of droplets that is isolated are
occupied droplets (e.g., containing at least one biological
particle). Such occupancy may be greater than or equal to 1%, 2%,
3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
higher. Alternatively, less than about 97% of the first subset of
droplets can be occupied droplets. In some instances, at least
about 97%, 98%, 99%, or a higher percentage of a second subset of
droplets that is isolated can be unoccupied droplets (e.g., not
containing any biological particle and not containing any barcode
carrying beads). Alternatively, less than about 97% of the second
subset of droplets can be unoccupied droplets. For example, the
particles can be separated into two subsets such that at least
about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 96%, 97%, 98%, 99%, or greater of a first subset of particles
that is isolated are cell beads (e.g., containing at least one
biological particle). Such occupancy may be greater than or equal
to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or higher. Alternatively, less than about 97% of the first
subset of particles can be occupied particles (e.g., cell beads).
In some instances, at least about 97%, 98%, 99%, or a higher
percentage of a second subset of particles that is isolated can be
unoccupied particles (e.g., not containing any biological particle
and not containing any barcode carrying beads). Alternatively, less
than about 97% of the second subset of particles can be unoccupied
particles.
[0277] The channel networks, e.g., as described herein above or
further below, can be fluidly coupled to appropriate fluidic
components. For example, the inlet channel segments (e.g., channel
segment 202 in FIG. 2A, channel segments 102, 104, 106 in FIG. 1)
are fluidly coupled to appropriate sources of the materials they
are to deliver to a channel junction (e.g., intersection 211 in
FIG. 2A, junction 110 in FIG. 1). For example, channel segment 202
will be fluidly coupled to a source of an aqueous suspension of
biological particles (e.g., biological particles 114 in FIG. 1) to
be analyzed and field-attractable particles (e.g.,
field-attractable particles 115 in FIG. 1). Channel segments 104
and 106 may then be fluidly connected to one or more sources of the
non-aqueous (or other immiscible) fluid. 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 206 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.
[0278] In some instances, a plurality of droplets not containing
any field-attractable particles, may also be sorted using
dielectrophoresis. For example, occupied droplets and unoccupied
droplets can have different dielectric properties. When an electric
field is applied, such as via methods described elsewhere herein
(e.g., with reference to FIG. 2A), to a plurality of droplets
comprising both occupied droplets and unoccupied droplets, such as
at an intersection wherein a first channel branches off into a
second channel and a third channel, the occupied droplets may be
directed to flow through the first channel and unoccupied droplets
may be directed to flow through the second channel, due at least in
part to the varying interactions with (or influence of) the
electric field of the occupied droplets and the unoccupied droplets
having different dielectric properties. Such systems and methods
for dielectrophoresis may also be used to sort a plurality of
particles into a first subset of occupied particles (e.g., cell
beads) and a second subset of unoccupied particles.
[0279] The biological particle can be subjected to 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, 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. 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. In this manner, the
polymer or gel may act to allow the biological particle 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.
[0280] The polymer or gel may be functionalized to bind to targeted
analytes, such as nucleic acids, proteins, 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.
[0281] 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 may be surrounded by polyacrylamide strands
linked together by disulfide bridges. In this manner, the
biological particle 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 (e.g., a cell) or macromolecular
constituents (e.g., RNA, DNA, proteins, etc.) of biological
particles. A cell bead may include a single cell or multiple cells,
or a derivative of the single 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 and cell beads (and/or droplets or
other partitions) containing macromolecular constituents of
biological particles. In some cases, the cell bead may further
comprise one or more field-attractable particles (e.g.,
paramagnetic particles, conductive particles, etc.), such as via
the systems and methods described elsewhere herein, for
facilitating subsequent sorting and/or solvent exchange. The
field-attractable particles may be trapped in the gel matrix. In
some instances, the field-attractable particles may be trapped
evenly throughout the gel matrix. In some instances, the
field-attractable particles may be trapped throughout the gel
matrix such as to subject the whole of the cell bead evenly to a
force (e.g., magnetic, electric) field.
[0282] In another aspect, provided herein are systems and methods
for selective polymerization of partitions (or cells therein). In
some instances, the partitions may be selectively polymerized based
on occupancy. In some instances, the partitions may be selectively
polymerized based on size.
[0283] FIG. 4 shows an example of a microfluidic channel structure
for selective polymerization of partitions based on occupancy. As
described elsewhere herein, in some cases, the majority of occupied
partitions can include no more than one biological particle per
occupied partition and, in some cases, some of the generated
partitions can be unoccupied (of any biological particle). In some
cases, though, some of the occupied partitions may include more
than one biological particle. In some cases, the partitioning
process may be controlled such that fewer than 25% of the occupied
partitions contain more than one biological particle, and in many
cases, fewer than 20% of the occupied partitions have more than one
biological particle, while in some cases, fewer than 10% or even
fewer than 5% of the occupied partitions include more than one
biological particle per partition.
[0284] The emulsion mechanism of FIG. 4 can largely parallel that
of FIG. 1. As shown in FIG. 4, the channel structure can include
channel segments 401, 402, 404, 406 and 408. Channel segments 401
and 402 can communicate at a channel junction 409. Channel segments
402, 404, 406, and 408 can communicate at a channel junction 410.
In operation, a first aqueous fluid 412 can be delivered to
junction 409 from each of channel segments 402 and 401. Cells 414
can be introduced into the junction 409 via the channel segment 401
as suspensions in the first aqueous fluid 412 flowing along the
channel segment 401. The first aqueous fluid 412 may or may not
contain suspended field-attractable particles 115. As described
elsewhere herein, occupied droplets and unoccupied droplets may be
sorted via the field-attractable particles 115. A second fluid 416
that is immiscible with the aqueous fluid 412 is delivered to the
junction 410 from each of channel segments 404 and 406 to create
discrete droplets 418, 420 of the first aqueous fluid 412 flowing
into channel segment 408, and flowing away from junction 410. A
discrete droplet generated may or may not include biological
particles 414.
[0285] The second fluid 416 can comprise an oil, such as a
fluorinated oil, and a surfactant, such as a fluorosurfactant for
stabilizing the resulting droplets, e.g., inhibiting subsequent
coalescence of the resulting droplets. 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.
[0286] The generated droplets may comprise two subsets of droplets:
(1) occupied droplets 418, containing one or more biological
particles 414, and (2) unoccupied droplets 420, not containing any
biological particles 414.
[0287] A photo polymerization light source 424, such as a laser,
can be located downstream of the junction 410 to selectively
polymerize occupied droplets (e.g., polymerize the biological
particles therein). In some instances, the light source 424 can be
a lamp or light emitting diode (LED). The light source can be an
ultraviolet (UV) radiation source. The light source 424 can
generate optical pulses or an electromagnetic beam at a targeted
direction. For example, the light source 424 can be configured to
only emit electromagnetic waves when an occupied droplet is passing
by. The light source 424 can be operatively coupled to a controller
426. The light source 424 can receive instructions from the
controller 426 on whether or not a droplet passing by the light
source 424, or a droplet expected to pass by the light source at a
certain time, is occupied. While FIG. 4 depicts one light source
424, it may be appreciated that there may be a plurality of light
sources, each in communication with the controller 426 and/or with
each other. For example, a plurality of light sources may each be
located at different locations, including different
upstream/downstream locations in the fluidic channels.
Alternatively, a different polymerization application unit can be
used to subject the droplets to a stimulus, such as to trigger
polymerization via heating, cooling, electromagnetic radiation,
and/or light. The stimulus can be a chemical stimulus. In some
instances, a plurality of the same or different types of
polymerization application units can be used at different
locations.
[0288] A sensor 422 can be configured to sense the presence of a
biological particle (or cell) 414 in the fluid flow. For example,
the sensor 422 can be configured to sense the presence of a
biological particle (or cell) 414 in the channel segment 401
upstream of the junction 409. The sensor 422 may be located
upstream of the junction 409. In some instances, the sensor 422 can
be an impedance sensor configured to measure bulk impedance when
cells 414 pass by the sensor 422. In some instances, a higher
impedance can be measured when a cell 414 passes by than when only
the first aqueous fluid 412 passes by. In some instances, the
sensor 422 can be an optical sensor configured to measure optical
properties of a cell 414. The optical sensor and/or a supporting
device may be configured to emit a detection signal configured to
probe one or more droplets, including for example an
electromagnetic signal (e.g., in any wavelength) and/or an acoustic
signal. In some instances, the optical sensor and/or a supporting
device may comprise an illumination source configured to illuminate
the cell with one or more types of electromagnetic radiation. In
some instances, the electromagnetic radiation can include
illumination in one or more of the visible spectrum, infrared
spectrum, the ultraviolet spectrum, and ionizing radiation
spectrum. In some instances, the ionizing radiation can include
x-rays. The illumination can be transillumination or
epi-illumination. Alternatively, the sensor 422 may be one or more
devices that are configured to provide one or more of optical
sensing, thermal sensing, laser imaging, infrared imaging,
capacitance sensing, mass sensing, vibration sensing across at
least a portion of the electromagnetic spectrum, and magnetic
induction sensing. The sensor 422 may collect sensor data on one or
more properties (e.g., optical properties, impendence properties,
etc.) or characteristics of a cell 414.
[0289] The sensor 422 can be operatively coupled to the controller
426. For example, the sensor 426 may transmit sensor data (e.g., on
presence of one or more cells 414) to the controller 426. The
controller 426 may determine when a cell has passed by the sensor
location from the sensor data. The controller 426 may then use such
sensor data, the location of the sensor and/or the location in the
fluidic channel at which the presence of a cell 414 was detected,
the time the sensor detected a presence of the cell 414 in the
flow, fluid flow rate of the first aqueous fluid 412 in the channel
segment 402, fluid flow rate of emulsion in the channel segment
408, location of the light source 424, time it takes for the sensor
426 to detect and/or transmit data to the controller 426, and/or
time it takes for the controller 426 to send instructions to the
light source 424, to send instructions to the light source 424 on
whether or not to emit an electromagnetic wave to polymerize a
droplet. For example, assuming that the fluid flow rates in the
channel segments 402 and/or 408 are substantially constant, the
controller 426 may determine whether a droplet created (e.g., at
junction 410) at a certain time contains a cell or does not contain
a cell. Based on the fluid flow rates in the channel segments 402
and/or 408, the controller 426 may determine whether a droplet
passing by the light source 424 at a certain time (e.g., time it
takes for droplet to travel from junction 410 to the location of
the light source 424 site is the same every time) is occupied or
unoccupied.
[0290] While FIG. 4 depicts one sensor 422, it may be appreciated
that there may be a plurality of sensors, each in communication
with the controller 426 and/or with each other. For example, there
can be a plurality of sensors upstream of the junction 409 and/or
upstream of the junction 410 at different locations, for example,
detecting the presence of a cell 414.
[0291] While FIG. 4 depicts one controller 426 operatively coupled
to both the sensor 422 and the light source 424, a separate
controller can be coupled to the sensor 422 and a separate
controller can be coupled to the light source 424. The separate
controllers may or may not be in communication with each other. In
some instances, there may be a plurality of controllers (e.g., two
controllers to sensor 422), wherein each controller may or may not
be in communication with each other. The controller 426 may send
instructions to sensor 422 and/or the light source 424 via wired
connection and/or wireless connection (e.g., Wi-Fi, Bluetooth, NFC,
etc.). The controller 422 may receive data from the sensor 422
and/or the light source 424 via wired connection and/or wireless
connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In some instances,
the components can be directly or indirectly be in communication
with each other, with or without going through the controller 426.
For example, the sensor 422 may be directly coupled to the light
source 426.
[0292] FIG. 5 shows another example of a microfluidic channel
structure for selective polymerization of partitions (e.g.,
biological particles therein) based on occupancy.
[0293] The emulsion mechanism of FIG. 5 can largely parallel that
of FIGS. 1 and 4. As shown in FIG. 5, the channel structure can
include channel segments 501, 502, 504, 506 and 508. Channel
segments 501 and 502 can communicate at a channel junction 509.
Channel segments 502, 504, 506, and 508 can communicate at a
channel junction 510. In operation, a first aqueous fluid 512 can
be delivered to junction 509 from each of channel segments 502 and
501. Cells 514 can be introduced into the junction 509 via the
channel segment 501 as suspensions in the first aqueous fluid 512
flowing along the channel segment 501. The first aqueous fluid 512
may or may not contain suspended field-attractable particles 515.
As described elsewhere herein, occupied droplets and unoccupied
droplets may be subsequently sorted via the field-attractable
particles 515. A second fluid 516 that is immiscible with the
aqueous fluid 512 is delivered to the junction 510 from each of
channel segments 504 and 506 to create discrete droplets 518, 520
of the first aqueous fluid 512 flowing into channel segment 508,
and flowing away from junction 510. A discrete droplet generated
may or may not include biological particles 514. Each cell 514
introduced into the droplet can comprise fluorescent labels (such
as in accordance with the widely used Fluorescence-Activated Cell
Sorting (FACS) mechanism) or other optical labels that allow for
detection by an optical sensor.
[0294] The second fluid 516 can comprise an oil, such as a
fluorinated oil, that includes a fluorosurfactant for stabilizing
the resulting droplets, e.g., inhibiting subsequent coalescence of
the resulting droplets. 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.
[0295] The generated droplets may comprise two subsets of droplets:
(1) occupied droplets 518, containing one or more biological
particles 514, and (2) unoccupied droplets 520, not containing any
biological particles 514.
[0296] A photo polymerization light source 524, such as a laser,
can be located downstream of the junction 510 to selectively
polymerize occupied droplets. In some instances, the light source
524 can be a lamp or light emitting diode (LED). The light source
524 can generate optical pulses or an electromagnetic beam at a
targeted direction. For example, the light source 524 can be
configured to only emit electromagnetic waves when an occupied
droplet is passing by. The light source 524 can be operatively
coupled to a controller 526. The light source 524 can receive
instructions from the controller 526 on whether or not a droplet
passing by the light source 524, or a droplet expected to pass by
the light source at a certain time, is occupied. While FIG. 5
depicts one light source 524, it may be appreciated that there may
be a plurality of light sources, each in communication with the
controller 526 and/or with each other. For example, a plurality of
light sources may each be located at different locations, including
different upstream/downstream locations in the fluidic channels.
Alternatively, a different polymerization application unit can be
used to subject the droplets to a stimulus, such as to trigger
polymerization of one or more biological particles therein via
heating, cooling, electromagnetic radiation, and/or light. The
stimulus can be a chemical stimulus. In some instances, a plurality
of the same or different types of polymerization application units
can be used at different locations.
[0297] A sensor 522 can be configured to sense one or more
characteristics of a droplet indicative of whether the droplet is
occupied or unoccupied. The sensor 522 may be located downstream of
the junction 510. In some instances, the sensor 522 can be an
impedance sensor configured to measure bulk impedance when droplets
pass by the sensor 522. In some instances, a higher impedance can
be measured when an occupied droplet 518 passes by than when an
unoccupied droplet 520 passes by. In some instances, the sensor 522
can be an optical sensor configured to measure optical properties
of a droplet. The optical sensor and/or a supporting device may be
configured to emit a detection signal configured to probe one or
more droplets, including for example an electromagnetic signal
(e.g., in any wavelength) and/or an acoustic signal. In some
instances, the optical sensor and/or a supporting device may
comprise an illumination source configured to illuminate the
droplet or droplets with one or more types of electromagnetic
radiation. In some instances, the electromagnetic radiation can
include illumination in one or more of the visible spectrum,
infrared spectrum, the ultraviolet spectrum, and ionizing radiation
spectrum. In some instances, the ionizing radiation can include
x-rays. The illumination can be transillumination or
epi-illumination. Alternatively the sensor 522 may be one or more
devices that are configured to provide one or more of optical
sensing, thermal sensing, laser imaging, infrared imaging,
capacitance sensing, mass sensing, vibration sensing across at
least a portion of the electromagnetic spectrum, and magnetic
induction sensing. The sensor 522 may collect sensor data on one or
more properties (e.g., optical properties, impendence properties,
etc.) or characteristics of a droplet 414. For example, the one or
more properties or characteristics determined by the sensor 522 can
be indicative of a certain type of droplets, such as occupied
droplets, unoccupied droplets, singularly occupied droplets,
multiply occupied droplets, droplets of a certain size or size
range, etc.
[0298] The sensor 522 can be operatively coupled to the controller
526. For example, the sensor 526 may transmit sensor data (e.g.,
one or more characteristics indicative of an occupancy of a
droplet) to the controller 526. The controller 526 may then use
such data, the location of the sensor and/or the location in the
fluidic channel at which the occupied droplet 518 was detected, the
time the sensor 522 detected an occupied droplet 518, fluid flow in
the channel segment 508, location of the light source 524, time it
takes for the sensor 526 to detect and/or transmit data to the
controller 526, and/or time it takes for the controller 526 to send
instructions to the light source 524, to send instructions to the
light source 524 on whether or not to emit an electromagnetic wave
to polymerize a droplet. For example, using such data, the
controller 526 may determine whether a droplet passing by the
sensor 522 location at a certain time is occupied or unoccupied.
Based on the fluid flow rate in channel segment 508, the controller
526 may determine whether a droplet passing by the light source 524
at a certain time (e.g., assuming time it takes for droplet to
travel from the sensor 522 location to the location of the light
source 524 site is the same every time) is occupied or
unoccupied.
[0299] While FIG. 5 depicts one sensor 522, it may be appreciated
that there may be a plurality of sensors, each in communication
with the controller 526 and/or with each other. For example, there
can be a plurality of sensors upstream or downstream of the
junction 510 at different locations, for example, detecting the
occupancy of a droplet.
[0300] While FIG. 5 depicts one controller 526 operatively coupled
to both the sensor 522 and the light source 524, a separate
controller can be coupled to the sensor 522 and a separate
controller can be coupled to the light source 524. The separate
controllers may or may not be in communication with each other. In
some instances, there may be a plurality of controllers (e.g., two
controllers to sensor 522), wherein each controller may or may not
be in communication with each other. The controller 526 may send
instructions to sensor 522 and/or the light source 524 via wired
connection and/or wireless connection (e.g., Wi-Fi, Bluetooth, NFC,
etc.). The controller 522 may receive data from the sensor 522
and/or the light source 524 via wired connection and/or wireless
connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In some instances,
the components can be directly or indirectly be in communication
with each other, with or without going through the controller 526.
For example, the sensor 522 may be directly coupled to the light
source 526.
[0301] FIG. 6 shows an example of a microfluidic channel structure
for selective polymerization of partitions based on droplet
size.
[0302] The emulsion mechanism of FIG. 6 can largely parallel that
of FIGS. 1, 4, and 5. As shown in FIG. 6, the channel structure can
include channel segments 601, 602, 604, 606 and 608. Channel
segments 601 and 602 can communicate at a channel junction 609.
Channel segments 602, 604, 606, and 608 can communicate at a
channel junction 610. In operation, a first aqueous fluid 612 can
be delivered to junction 609 from each of channel segments 602 and
601. Cells 614 can be introduced into the junction 609 via the
channel segment 601 as suspensions in the first aqueous fluid 612
flowing along the channel segment 601. The first aqueous fluid 612
may or may not contain suspended field-attractable particles 615.
As described elsewhere herein, occupied droplets and unoccupied
droplets may be subsequently sorted via the field-attractable
particles 615. A second fluid 616 that is immiscible with the
aqueous fluid 612 is delivered to the junction 610 from each of
channel segments 604 and 606 to create discrete droplets 618, 620
of the first aqueous fluid 612 flowing into channel segment 608,
and flowing away from junction 610. A discrete droplet generated
may or may not include biological particles 614. Each cell 614
introduced into the droplet can comprise fluorescent labels (such
as in accordance with the widely used Fluorescence-Activated Cell
Sorting (FACS) mechanism).
[0303] In some instances, each droplet generated by the above
emulsion may be of substantially uniform size that is appropriate
(and/or acceptable) for feeding to subsequent single cell
applications. In some instances, some droplets may be of
substantially uniform size, and some droplets may have different
sizes. For example, the droplets generated can have a size
distribution where at least about 10%, 20%, 30%, 40%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or a
higher percentage of droplets generated can have a substantially
uniform size. Alternatively, less than 10% of the droplets
generated can have a size distribution where less than 10% of the
droplets generated have a substantially uniform size. The system
and methods described herein may selectively polymerize only
droplets (e.g., polymerize biological particles contained therein
the droplets) that have the appropriate (and/or acceptable) size
and/or droplets that are occupied.
[0304] The second fluid 616 can comprise an oil, such as a
fluorinated oil, that includes a fluorosurfactant for stabilizing
the resulting droplets, e.g., inhibiting subsequent coalescence of
the resulting droplets. 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.
[0305] The generated droplets may comprise two subsets of droplets:
(1) occupied droplets 618A, 618B, containing one or more biological
particles 614, and (2) unoccupied droplets 620, not containing any
biological particles 614. As described above, within the subset of
occupied droplets 618A, 618B, some droplets may have the
appropriate size (e.g., droplet 618A) and some droplets may have an
inappropriate size (e.g., droplet 618B).
[0306] A photo polymerization light source 624, such as a laser,
can be located downstream of the junction 610 to selectively
polymerize occupied droplets. In some instances, the light source
624 can be a lamp or light emitting diode (LED). The light source
624 can generate optical pulses or an electromagnetic beam at a
targeted direction. For example, the light source 624 can be
configured to only emit electromagnetic waves when an occupied
droplet is passing by. The light source 624 can be operatively
coupled to a controller 626. The light source 624 can receive
instructions from the controller 626 on whether or not a droplet
passing by the light source 624, or a droplet expected to pass by
the light source at a certain time, is occupied. While FIG. 6
depicts one light source 624, it may be appreciated that there may
be a plurality of light sources, each in communication with the
controller 626 and/or with each other. For example, a plurality of
light sources may each be located at different locations, including
different upstream/downstream locations in the fluidic channels.
Alternatively, a different polymerization application unit can be
used to subject the droplets to a stimulus, such as to trigger
polymerization via heating, cooling, electromagnetic radiation,
and/or light. The stimulus can be a chemical stimulus. In some
instances, a plurality of the same or different types of
polymerization application units can be used at different
locations.
[0307] A sensor 622 can be configured to sense one or more
characteristics of a droplet indicative of whether the droplet is
occupied or unoccupied. The sensor 622 may be located downstream of
the junction 610. In some instances, the sensor 622 can be an
impedance sensor configured to measure bulk impedance when droplets
pass by the sensor 622. In some instances, a higher impedance can
be measured when a larger droplet (e.g., droplet 618B) passes by
than when a smaller droplet (e.g., droplet 618A or droplet 620)
passes by. In some instances, a higher impedance can be measured
when an occupied droplet (e.g., droplets 618A, 618B) passes by than
when an unoccupied droplet (e.g., droplet 620) passes by. In some
instances, the sensor 622 can be an optical sensor configured to
measure optical properties of a droplet. The optical sensor and/or
a supporting device may be configured to emit a detection signal
configured to probe one or more droplets, including for example an
electromagnetic signal (e.g., in any wavelength) and/or an acoustic
signal. In some instances, the optical sensor and/or a supporting
device may comprise an illumination source configured to illuminate
the droplet or droplets with one or more types of electromagnetic
radiation. In some instances, the electromagnetic radiation can
include illumination in one or more of the visible spectrum,
infrared spectrum, the ultraviolet spectrum, and ionizing radiation
spectrum. In some instances, the ionizing radiation can include
x-rays. The illumination can be transillumination or
epi-illumination. Alternatively the sensor 622 may be one or more
devices that are configured to provide one or more of optical
sensing, thermal sensing, laser imaging, infrared imaging,
capacitance sensing, mass sensing, vibration sensing across at
least a portion of the electromagnetic spectrum, and magnetic
induction sensing. The sensor 622 may collect sensor data on one or
more properties (e.g., optical properties, impendence properties,
etc.) or characteristics of a droplet. For examples, the one or
more properties and/or other characteristics of a droplet measured
by the sensor 622 can be indicative of a size and/or an occupancy
of the droplet.
[0308] The sensor 622 can be operatively coupled to the controller
626. For example, the sensor 626 may transmit sensor data (e.g.,
size and/or occupancy of a droplet) to the controller 626. The
controller 626 may then use such data, the location of the sensor
and/or the location in the fluidic channel at which the occupancy
and/or size of a droplet was detected, the time the sensor 622
detected, fluid flow in the channel segment 608, location of the
light source 624, time it takes for the sensor 626 to detect and/or
transmit data to the controller 626, and/or time it takes for the
controller 626 to send instructions to the light source 624, to
send instructions to the light source 624 on whether or not to emit
an electromagnetic wave to polymerize a droplet. For example,
assuming that the fluid flow rates in the channel segment 608 are
substantially constant, the controller 626 may determine whether a
droplet passing by a sensor 622 location at a certain time is
occupied or unoccupied. Based on the fluid flow rate in channel
segment 608, the controller 626 may determine whether a droplet
passing by the light source 624 at a certain time (e.g., assuming
time it takes for droplet to travel from the sensor 622 location to
the location of the light source 624 site is the same every time)
is occupied or unoccupied.
[0309] While FIG. 6 depicts one sensor 622, it may be appreciated
that there may be a plurality of sensors, each in communication
with the controller 626 and/or with each other. For example, there
can be a plurality of sensors upstream or downstream of the
junction 610 at different locations, for example, detecting the
occupancy of a droplet. In some instances, a separate sensor can
detect occupancy of a droplet and a separate sensor can detect size
of the droplet. The separate sensors may or may not detect such
characteristics at the same upstream/downstream location of the
fluid channel.
[0310] While FIG. 6 depicts one controller 626 operatively coupled
to both the sensor 622 and the light source 624, a separate
controller can be coupled to the sensor 622 and a separate
controller can be coupled to the light source 624. The separate
controllers may or may not be in communication with each other. In
some instances, there may be a plurality of controllers (e.g., two
controllers to sensor 622), wherein each controller may or may not
be in communication with each other. The controller 626 may send
instructions to sensor 622 and/or the light source 624 via wired
connection and/or wireless connection (e.g., Wi-Fi, Bluetooth, NFC,
etc.). The controller 622 may receive data from the sensor 622
and/or the light source 624 via wired connection and/or wireless
connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In some instances,
the components can be directly or indirectly be in communication
with each other, with or without going through the controller 626.
For example, the sensor 622 may be directly coupled to the light
source 626.
[0311] In some instances, a plurality of droplets, wherein a first
subset comprises polymerized droplets and a second subset comprises
pre-polymerized droplets can be further sorted based on
polymerization status, such as via solvent exchange. For example,
the pre-polymerized droplets can be washed away during solvent
exchange to isolate the polymerized droplets.
[0312] The separation systems and methods for sorting and/or
selective polymerization described above and further below may
achieve super Poissonian loading. For example, the droplets can be
separated into two subsets such that at least about 5%, 10%, 15%,
20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,
99%, or greater of a first subset of droplets that is isolated are
occupied droplets (e.g., containing at least one biological
particle). Such occupancy may be greater than or equal to 1%, 2%,
3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
higher. Alternatively, less than about 97% of the first subset of
droplets can be occupied droplets. In some instances, at least
about 97%, 98%, 99%, or a higher percentage of a second subset of
droplets that is isolated are unoccupied droplets (e.g., not
containing any biological particle and not containing any barcode
carrying beads). Alternatively, less than about 97% of the second
subset of droplets can be unoccupied droplets. The separation
systems and methods described above and below may achieve super
Poissonian monodispersity. For example, the droplets can be
separated into two subsets such that at least about 5%, 10%, 15%,
20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,
99%, or greater of a first subset of droplets that is isolated are
within a given droplet size range. Such monodispersity may be
greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively, less than
about 97% of the first subset of droplets can be within a given
droplet size range.
[0313] As described elsewhere herein, a cell bead can be formed
after the polymerization process, such as via the selective
polymerization systems and methods described herein. Alternatively,
a different polymerization procedure can be used. For example, cell
beads can be formed by selectively polymerizing one or more
biological particles within droplets, such as via methods described
in relation to FIG. 4-6. In another example, a cell bead can be
formed by polymerizing one or more biological particles that are
suspended in a first solvent, such as oil. In some cases, a
plurality of biological particles (e.g., cells) may be hardened
and/or polymerized in bulk by applying a stimulus (e.g., light,
chemical, temperature, etc.) to the first solvent carrying the
plurality of biological particles (e.g., as suspensions). Upon
formation, a plurality of cell beads may be surrounded by the first
solvent, such as an oil. In order to promote integration of a cell
bead into a droplet with a gel bead, the cell bead may be placed
into an aqueous environment by a solvent exchange process. The
solvent exchange process may comprise the operations of collecting
a plurality of cell beads surrounded by oil (for instance, in an
Eppendorf tube or other collection vessel), removing excess oil
(for instance, by pipetting), adding a ligation buffer (such as a
3.times. ligation buffer), vortexing, adding a buffer (such as a
1.times.1H,1H,2H,2H-perfluoro-1-octanol (PFO) buffer), vortexing,
centrifugation, and separation. The separation operation may
comprise magnetic separation.
[0314] Each of the cell beads may comprise field-attractable
particles (e.g., paramagnetic particles). For example, cell beads
comprising field-attractable particles can be formed using the
systems and methods described elsewhere herein (e.g.,
field-attractable particles 115 in FIG. 1 are suspended in fluids
that form emulsion droplets which can be polymerized to form cell
beads). The magnetic separation may be accomplished by using a
magnetic separating apparatus to pull cell beads containing
paramagnetic particles away from unwanted remaining oil and
solvents. In some instances, the magnetic separating apparatus can
be a field application unit as described elsewhere herein (e.g.,
the same type of field application unit 216 in FIG. 2A, field
application unit 316 in FIG. 3). For instance, the magnetic
separation apparatus may be used to pull cell beads containing
paramagnetic particles away from the ligation buffer and PFO to
allow removal of the ligation buffer and PFO (for instance by
pipetting). The cell beads containing paramagnetic particles may
then be suspended in a ligation buffer and vortexed. The cell beads
containing paramagnetic particles may again be separated
magnetically and the ligation buffer may be removed. This cycle of
re-suspension, vortexing, and magnetic separation may be repeated
until the cell beads are clean. For instance, the cycle may be
repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. The
cell beads may then be placed into an aqueous medium.
[0315] Once the cell beads are in an aqueous medium, the cell beads
may be further treated. For instance, the cell beads in aqueous
solution may be filtered (for instance, using a 70 .mu.m filter) to
remove clumps and/or large cell beads from the solution. In some
cases, additional reagents may be added to and/or removed from the
aqueous medium to further process the cell beads. The cell beads
may then be combined into droplets with gel beads, as described
herein.
[0316] Also provided herein are the microfluidic devices used for
partitioning the cells as described above. Such microfluidic
devices can comprise channel networks for carrying out the
partitioning process like those set forth in FIGS. 1, 4, 5, and 6.
These microfluidic devices can comprise channel networks, such as
those described herein, for partitioning cells into separate
partitions, and co-partitioning such cells with oligonucleotide
barcode library members, e.g., disposed on beads. These channel
networks can be disposed within a solid body, e.g., a glass,
semiconductor or polymer body structure in which the channels are
defined, where those channels communicate at their termini with
reservoirs for receiving the various input fluids, and for the
ultimate deposition of the partitioned cells, etc., from the output
of the channel networks. By way of example, and with reference to
FIG. 1, a reservoir fluidly coupled to channel 102 may be provided
with an aqueous suspension of cells 114, while a reservoir coupled
to another channel (not shown) may be provided with an aqueous
suspension of beads carrying the oligonucleotides. Channel segments
106 and 108 may be provided with a non-aqueous solution, e.g., an
oil, into which the aqueous fluids are partitioned as droplets at
the channel junction 110. Finally, an outlet reservoir may be
fluidly coupled to channel 108 into which the partitioned cells and
beads can be delivered and from which they may be harvested. As
will be appreciated, while described as reservoirs, it will be
appreciated that the channel segments may be coupled to any of a
variety of different fluid sources or receiving components,
including tubing, manifolds, or fluidic components of other
systems.
[0317] Also provided are systems that control flow of these fluids
through the channel networks e.g., through applied pressure
differentials, centrifugal force, electrokinetic pumping,
compressors, capillary or gravity flow, or the like.
[0318] The systems and methods described herein may allow for the
production of one or more droplets containing a single biological
particle and/or a single bead. The systems and methods may also
allow for the production of one or more droplets containing a
single biological particle and more than one bead, one or more
droplets containing more than one biological particle and a single
bead, or one or more droplets containing more than one biological
particle and more than one bead. The systems and methods described
herein may allow for the production of one or more cell beads
containing a single biological particle and/or a single bead. The
systems and methods may also allow for selective polymerization of
occupied droplets and/or appropriately sized droplets, which
mixture of polymerized and pre-polymerized droplets may or may not
be subjected to subsequent sorting.
[0319] FIG. 7 shows a flowchart for a method of sorting occupied
droplets and unoccupied droplets, wherein an occupied droplet
contains at least a biological particle (cell) and/or a barcode
carrying bead (particle).
[0320] In an operation 701, a plurality of droplets are generated
upon bringing a first phase in contact with a second phase, wherein
the first phase and the second phase are immiscible. Each of the
plurality of droplets comprises some number and/or concentration of
field-attractable particles. A first subset of the plurality of
droplets contains therein cells and/or barcode carrying particles,
and a second subset of the plurality of droplets does not contains
therein cells and/or barcode carrying particles. A given droplet in
the first subset of the plurality of droplets may contain a single
cell or a plurality of cells. A given droplet in the first subset
of the plurality of droplets may contain a single barcode carrying
particle or a plurality of barcode carrying particles. A given
droplet of the first subset of the plurality of droplets may
contain a fewer number and/or lower concentration of
field-attractable particles than a given droplet of the second
subset of the plurality of droplets, on account of the volume
occupied by the cell and/or barcode carrying particles contained in
the occupied droplet.
[0321] In an operation 702, the plurality of droplets is directed
along the first channel towards an intersection of the first
channel. The intersection can be between the first channel, a
second channel, and a third channel. The plurality of droplets may
be directed along one or more channels in a flow of fluid (e.g.,
either the first phase or the second phase used to generate the
droplets), such as via a fluid flow unit.
[0322] In an operation 703, the plurality of droplets is subject to
a force field, such as via a field application unit, at or near the
intersection. The force field can be a magnetic field and/or an
electric field. The plurality of droplets can be subjected to the
force field under conditions sufficient to separate the first
subset of droplets from the second subset of the droplets, wherein
upon separation, the first subset of droplets flows along the
second channel, and the second subset of droplets flows along the
third channel. For example, the field-attractable particles can be
paramagnetic particles when a magnetic field is applied. The
field-attractable particles can be conductive particles when an
electric field is applied. Because a given droplet in the second
subset of the plurality of droplets has a greater number and/or
concentration of field-attractable particles than a given droplet
in the first subset of the plurality of droplets, for the same
field applied, a stronger force can act on a given droplet in the
second subset of droplets than on a given droplet in the first
subset of droplets. The force differential may separate the two
subsets, such as by influencing a greater deviation in fluid flow
path direction for a given droplet in the second subset of droplets
than for a given droplet in the first subset of droplets.
[0323] The method of FIG. 7 may isolate occupied droplets (e.g.,
first subset of droplets) with super-Poissonian loading. For
example, a plurality of droplets can be separated into two subsets
such that at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a first
subset of droplets that is isolated are occupied droplets (e.g.,
containing at least one biological particle). Such occupancy may be
greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively, less than
about 97% of droplets of a separated subset of the plurality of
droplets may be occupied droplets.
[0324] The method of FIG. 7 may be similarly used to isolate
occupied particles from a plurality of particles containing
occupied and unoccupied particles. For example, a plurality of
particles may be generated to comprise a first subset of occupied
particles (containing a biological particle and/or a barcode
carrying particle therein) and a second subset of unoccupied
particles, wherein particles of both the first subset and the
second subset comprise field-attractable particles. A given
particle of the first subset may contain a fewer number and/or
lower concentration of field-attractable particles than a given
particle of the second subset, on account of the volume occupied by
the cell and/or barcode carrying particles contained in the
occupied particle. The plurality of particles may be directed along
a first channel towards an intersection of the first channel. The
intersection can be between the first channel, a second channel,
and a third channel.
[0325] The plurality of particles can be subject to a force field,
such as via a field application unit, at or near the intersection.
The force field can be a magnetic field and/or an electric field.
The plurality of particles can be subjected to the force field
under conditions sufficient to separate the first subset from the
second subset, wherein upon separation, the first subset of
particles flows along the second channel, and the second subset of
particles flows along the third channel. For example, the
field-attractable particles can be paramagnetic particles when a
magnetic field is applied. The field-attractable particles can be
conductive particles when an electric field is applied. Because a
given particle in the second subset has a greater number and/or
concentration of field-attractable particles than a given particle
in the first subset, for the same field applied, a stronger force
can act on a given particle in the second subset than on a given
particle in the first subset. The force differential may separate
the two subsets, such as by influencing a greater deviation in
fluid flow path direction for a given particle in the second subset
than for a given particle in the first subset.
[0326] The method of FIG. 7 may isolate occupied particles (e.g.,
first subset of particles) with super-Poissonian loading. For
example, a plurality of particles can be separated into two subsets
such that at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a first
subset of particles that is isolated are occupied particles (e.g.,
containing at least one biological particle). Such occupancy may be
greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively, less than
about 97% of particles of a separated subset of the plurality of
particles may be occupied particles.
[0327] FIG. 8 shows a flowchart for a method of sorting occupied
droplets and unoccupied droplets, wherein an occupied droplet
contains at least a biological particle (cell) and/or a barcode
carrying bead (particle).
[0328] In an operation 801, a plurality of droplets are generated
upon bringing a first phase in contact with a second phase, wherein
the first phase and the second phase are immiscible. Each of the
plurality of droplets comprises may or may not comprise some number
and/or concentration of field-attractable particles. The presence
of field-attractable particles is not required. A first subset of
the plurality of droplets contains therein cells and/or barcode
carrying particles, and a second subset of the plurality of
droplets does not contain therein cells and/or barcode carrying
particles. A given droplet in the first subset of the plurality of
droplets may contain a single cell or a plurality of cells. A given
droplet in the first subset of the plurality of droplets may
contain a single barcode carrying particle or a plurality of
barcode carrying particles.
[0329] In an operation 802, the plurality of droplets is directed
along the first channel towards an intersection of the first
channel. The intersection can be between the first channel, a
second channel, and a third channel. The plurality of droplets may
be directed along one or more channels in a flow of fluid (e.g.,
either the first phase or the second phase used to generate the
droplets), such as via a fluid flow unit.
[0330] In an operation 803, the plurality of droplets is subject to
a pressure pulse, such as via a pressure application unit, at or
near the intersection. The pressure pulse can be provided as a
positive pressure pulse or a negative pressure pulse. The plurality
of droplets can be subjected to the pressure pulse under conditions
sufficient to separate the first subset of droplets from the second
subset of the droplets, wherein upon separation, the first subset
of droplets flows along the second channel, and the second subset
of droplets flows along the third channel. Because a given droplet
in the second subset of the plurality of droplets has different
particle and/or suspension characteristics in the fluid than a
given droplet in the first subset of the plurality of droplets, for
the same pressure pulse applied, a different hydrodynamic force can
act on a given droplet in the second subset of droplets than on a
given droplet in the first subset of droplets. The pressure pulse
may separate the two subsets, such as by influencing a greater
deviation in fluid flow path direction for a given droplet in the
second subset of droplets than for a given droplet in the first
subset of droplets.
[0331] In some instances, a sensor, such as an impedance sensor or
an optical sensor, may measure one or more characteristics of a
droplet at an upstream location of the intersection. The sensing
data may be indicative of the occupancy of the droplet. The sensing
data may be transmitted to a controller. The controller may use the
sensing data to determine the occupancy of the droplet and instruct
the pressure application unit to generate or not generate pressure
pulses to separate the first subset from the second subset.
[0332] The method of FIG. 8 may isolate occupied droplets (e.g.,
first subset of droplets) with super-Poissonian loading. For
example, a plurality of droplets can be separated into two subsets
such that at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a first
subset of droplets that is isolated are occupied droplets (e.g.,
containing at least one biological particle). Such occupancy may be
greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively, less than
about 97% of droplets of a separated subset of the plurality of
droplets may be occupied droplets.
[0333] The method of FIG. 8 may be similarly used to isolate cell
beads from a plurality of particles containing both cell beads and
unoccupied particles. For example, a plurality of particles may be
generated to comprise a first subset of occupied particles
(containing a biological particle and/or a barcode carrying
particle therein) and a second subset of unoccupied particles. The
presence of field-attractable particles is not required. The
plurality of particles may be directed along a first channel
towards an intersection of the first channel. The intersection can
be between the first channel, a second channel, and a third
channel. The plurality of particles may be subject to a pressure
pulse, such as via a pressure application unit, at or near the
intersection. The pressure pulse can be provided as a positive
pressure pulse or a negative pressure pulse.
[0334] The plurality of particles can be subjected to the pressure
pulse under conditions sufficient to separate the first subset of
particles (e.g., cell beads) from the second subset of the
particles (e.g., unoccupied particles), wherein upon separation,
the first subset of particles flows along the second channel, and
the second subset of particles flows along the third channel.
Because a given particle in the second subset of the plurality of
particles has different particle and/or suspension characteristics
in the fluid than a given particle in the first subset of the
plurality of particles, for the same pressure pulse applied, a
different hydrodynamic force can act on a given particle in the
second subset of particles than on a given particle in the first
subset of particles. The pressure pulse may separate the two
subsets, such as by influencing a greater deviation in fluid flow
path direction for a given particle in the second subset of
particles than for a given particle in the first subset of
particles.
[0335] In some instances, a sensor, such as an impedance sensor or
an optical sensor, may measure one or more characteristics of a
particle at an upstream location of the intersection. The sensing
data may be indicative of the occupancy of the particle. The
sensing data may be transmitted to a controller. The controller may
use the sensing data to determine the occupancy of the particle and
instruct the pressure application unit to generate or not generate
pressure pulses to separate the first subset from the second
subset.
[0336] The method of FIG. 8 may isolate cell beads (e.g., first
subset of particles) with super-Poissonian loading. For example, a
plurality of particles can be separated into two subsets such that
at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a first subset of
particles that is isolated are cell beads (e.g., containing at
least one biological particle). Such occupancy may be greater than
or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or higher. Alternatively, less than about 97% of
particles of a separated subset of the plurality of particles may
be cell beads.
[0337] FIG. 9 shows a flowchart for a method of selectively
polymerizing occupied droplets, wherein an occupied droplet
contains at least a biological particle (cell) and/or a barcode
carrying bead (particle).
[0338] In an operation 901, a plurality of droplets are generated
upon bringing a first phase in contact with a second phase, wherein
the first phase and the second phase are immiscible. Each of the
plurality of droplets comprises may or may not comprise some number
and/or concentration of field-attractable particles. The presence
of field-attractable particles is not required. A first subset of
the plurality of droplets contains therein cells and/or barcode
carrying particles, and a second subset of the plurality of
droplets does not contain therein cells and/or barcode carrying
particles. A given droplet in the first subset of the plurality of
droplets may contain a single cell or a plurality of cells. A given
droplet in the first subset of the plurality of droplets may
contain a single barcode carrying particle or a plurality of
barcode carrying particles.
[0339] In an operation 902, at an upstream location, a sensor
detects and/or measures one or more characteristics of a droplet
passing through the upstream location. The one or more
characteristics can be indicative of the occupancy of the droplet.
In some instances, the sensor can be an impedance sensor configured
to detect bulk impedance as a droplet or a plurality of droplets
passes through the upstream location. A higher impedance can be
measured for occupied droplets than for unoccupied droplets. In
some instances, the sensor can be an optical sensor configured to
detect one or more optical characteristics of the droplet as the
droplet passes through the upstream location. Alternatively, a
plurality of the same or different types of sensors can be used to
detect and/or measure one or more characteristics of the droplet
passing through the upstream location.
[0340] In an operation 903, the sensing data may transmitted to a
controller. For example, the sensor can be operatively coupled to
the controller. The controller can determine, based at least in
part on the sensor data, whether a droplet passing through a
downstream location is occupied or unoccupied. For example, the
controller may use such sensor data, the location of the sensor
and/or the location in the fluidic channel at which the occupancy
of a droplet was detected, the time the sensor detected the
occupancy of a droplet, fluid flow rate of one or more channels,
location of the light source, time it takes for the sensor to
detect and/or transmit data to the controller, and/or time it takes
for the controller to send instructions to the light source, to
send instructions to the light source on whether or not to emit an
electromagnetic wave to polymerize a droplet.
[0341] Alternatively, in some instances, the sensor may detect the
presence of a cell suspended in a fluid flow before the droplets
are generated (e.g., at the intersection), and use such sensor data
of the presence of the cell, and other information (e.g., location,
times, fluid flow rates) to determine whether a droplet at a
downstream location is occupied.
[0342] In an operation 904, the controller may transmit instruction
to a light source at a downstream location to emit electromagnetic
waves to polymerize a droplet if the droplet is occupied, and not
to emit electromagnetic waves if the droplet is unoccupied, thus
letting the unoccupied droplet pass through unpolymerized.
Alternatively, other polymerization application units can be used
in place of, or in conjunction with the light source.
[0343] The method of FIG. 9 may polymerize occupied droplets (e.g.,
first subset of droplets) with super-Poissonian distribution. The
separation systems and methods disclosed herein may achieve super
Poisson loading. For example, at least about 5%, 10%, 15%, 20%,
25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or
greater of a plurality of droplets that are polymerized can be
occupied droplets. Such occupancy may be greater than or equal to
1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or higher. Alternatively, less than about 97% of polymerized
droplets may be occupied droplets.
[0344] FIG. 10 shows a flowchart for a method of selectively
polymerizing appropriately sized droplets.
[0345] In an operation 1001, a plurality of droplets are generated
upon bringing a first phase in contact with a second phase, wherein
the first phase and the second phase are immiscible. Each of the
plurality of droplets comprises may or may not comprise some number
and/or concentration of field-attractable particles. The presence
of field-attractable particles is not required. A first subset of
the plurality of droplets contains therein cells and/or barcode
carrying particles, and a second subset of the plurality of
droplets does not contain therein cells and/or barcode carrying
particles. A given droplet in the first subset of the plurality of
droplets may contain a single cell or a plurality of cells. A given
droplet in the first subset of the plurality of droplets may
contain a single barcode carrying particle or a plurality of
barcode carrying particles.
[0346] In an operation 1002, at an upstream location, a sensor
detects and/or measures one or more characteristics of a droplet
passing through the upstream location. The one or more
characteristics can be indicative of a size of the droplet. In some
instances, the sensor can be an impedance sensor configured to
detect bulk impedance as a droplet or a plurality of droplets
passes through the upstream location. A higher impedance can be
measured for larger droplets than for smaller droplets. In some
instances, the sensor can be an optical sensor configured to detect
one or more optical characteristics of the droplet as the droplet
passes through the upstream location. Alternatively, a plurality of
the same or different types of sensors can be used to detect and/or
measure one or more characteristics of the droplet passing through
the upstream location.
[0347] In an operation 1003, the sensing data may transmitted to a
controller. For example, the sensor can be operatively coupled to
the controller. The controller can determine, based at least in
part on the sensor data, whether a droplet passing through a
downstream location is appropriately sized or inappropriately
sized. For example, the controller may use such sensor data, the
location of the sensor and/or the location in the fluidic channel
at which the size of a droplet was detected, the time the sensor
detected the size of a droplet, fluid flow rate of one or more
channels, location of the light source, time it takes for the
sensor to detect and/or transmit data to the controller, and/or
time it takes for the controller to send instructions to the light
source, to send instructions to the light source on whether or not
to emit an electromagnetic wave to polymerize a droplet.
[0348] In an operation 1004, the controller may transmit
instruction to a light source at a downstream location to emit
electromagnetic waves to polymerize a droplet if the droplet is
appropriately sized, and not to emit electromagnetic waves if the
droplet is inappropriately sized, thus letting the inappropriately
sized droplet pass through unpolymerized. Alternatively, other
polymerization application units can be used in place of, or in
conjunction with the light source.
[0349] The method of FIG. 10 may polymerize appropriately sized
droplets (e.g., first subset of droplets) with super-Poissonian
distribution. For example, at least about 5%, 10%, 15%, 20%, 25%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or
greater of a plurality of droplets that is polymerized can be
appropriately sized droplets. Alternatively, less than about 97% of
polymerized droplets may be appropriately sized droplets.
[0350] In some instances, the methods of FIG. 9 and FIG. 10 can be
combined such that one or more sensors detect one or more
characteristics of a droplet at an upstream location (or upstream
locations), wherein the one or more characteristics of the droplet
are indicative of both a size and an occupancy of the droplet, and
transmits the sensor data to a controller. The controller transmits
instructions to a light source at a downstream location to
polymerize the droplet only if the droplet is both appropriately
sized and occupied.
[0351] In another aspect, provided is a passive mechanism for
sorting occupied droplets from unoccupied droplets. The passive
mechanism may not require application of external forces (e.g.,
magnetic field, electric field, pressure pulse, etc.) on the
droplets to achieve sorting. The passive mechanism may sort
droplets based at least in part on mechanical properties of the
droplets. For example, the passive mechanism may sort droplets
based at least in part on properties such as deformability and
surface tension (e.g., surface interface energy) of the droplets.
In some instances, due to the presence of one or more biological
particles in an occupied droplet, the occupied droplet may
demonstrate lower deformability and/or higher surface tension
properties than unoccupied droplets, making occupied droplets
`harder` or `stiffer` than unoccupied droplets. Thus, when a
plurality of droplets comprising both a first subset of occupied
droplets and a second subset of unoccupied droplets is directed to
pass through an aperture which is smaller in size than a diameter
of a given droplet in the plurality of droplets, only those
droplets capable of deforming (e.g., unoccupied droplets having
higher deformability properties) may pass through the aperture,
trapping the occupied droplets, thereby sorting the occupied
droplets from the unoccupied droplets.
[0352] FIG. 11 shows a schematic example of a microfluidic channel
structure for separating occupied droplets from unoccupied
droplets. As described elsewhere herein, when droplets are
generated, there may be at least a first subset population of
occupied droplets containing one or more biological particles and
at least a second subset population of unoccupied droplets not
containing any biological particles. In some cases, the droplets
may additionally contain one or more barcode carrying beads. For
example, a droplet may have only a biological particle, a droplet
may have only a barcode carrying bead, a droplet may have both a
biological particle and a barcode carrying bead, or a droplet may
have neither biological particles nor barcode carrying beads. In
some cases, the majority of occupied partitions (e.g., droplets)
can include no more than one biological particle per occupied
partition and, in some cases, some of the generated partitions can
be unoccupied (e.g., by any biological particle). In some cases,
though, some of the occupied partitions may include more than one
biological particle. In some cases, the partitioning process may be
controlled such that fewer than 25% of the occupied partitions
contain more than one biological particle, fewer than 20% of the
occupied partitions have more than one biological particle, or
fewer than 10% or even fewer than 5% of the occupied partitions
include more than one biological particle per partition.
[0353] As shown in FIG. 11, the channel structure can include a
channel segment 1100 with an entrance 1102 and exit 1104. In some
instances, the outflow channel 108 of the emulsion carrying the
generated droplets in FIG. 1 can be upstream of the channel segment
1100. A fluid flow unit (not shown) can be configured to facilitate
flow of fluid in the channel structure.
[0354] In operation, a plurality of discrete droplets, each
comprising a first aqueous fluid 1100 can flow as emulsions in a
second fluid 1112, wherein the second fluid 1112 is immiscible to
the first aqueous fluid 1110. The droplets being transported along
channel segment 1100 can comprise a first subset of droplets 1108
that are each occupied with at least a biological particle and/or a
barcode carrying bead and a second subset of droplets 1110 that are
each unoccupied. As described above, a given unoccupied droplet can
have a higher deformability and/or lower surface tension property
than a given occupied droplet, due to the presence of one or more
biological particles in the occupied droplet.
[0355] The channel segment 1100 can comprise a plurality of
entrapment structures 1114. An entrapment structure can define an
aperture. A size of the aperture may be less than a diameter (or
other size dimension) of a droplet. A size of the aperture may be
less than a minimum dimension of a droplet. In some instances, the
size of the aperture can be at most about 90%, 80%, 70%, 60%, 50%,
45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the diameter of a
droplet (in a pre-deformed state). The size of the aperture can be
less than about 5% of the diameter of a droplet. Alternatively, the
size of the aperture can be greater than 50%, 60%, 70%, 80%, 90%,
100%, 150%, or 200% of the diameter of a droplet. The size of the
aperture can be greater than the diameter of the droplet. When a
plurality of droplets comprising both a first subset of occupied
droplets 1108 and a second subset of unoccupied droplets 1110 is
directed to pass through entrapment structures 1114 defining
apertures which is smaller in size than a diameter of a given
droplet in the plurality of droplets, only those droplets deforming
1118 (e.g., unoccupied droplets 1110 having higher deformability
properties) such that at least one dimension of the droplet is less
than a size of the aperture may pass through one or more apertures
defined by the entrapment structures 1114. Because occupied
droplets 1108 may be `harder` or `stiffer,` due to a presence of
one or more biological particles in the droplets, the occupied
droplets may resist deformation, at least from deforming to a size
smaller than a size of the aperture, and remain trapped by the
entrapment structures 1114.
[0356] In some embodiments, there may exist more entrapment
structures 1114 (and thus more apertures) in the channel structure
than there are droplets 1106, 1108 passing through the channel
structure. Beneficially, when the occupied droplets 1108 are
prevented from flowing through the entrapment structures 1114, and
the occupied droplets 1108 clog (or block) some apertures of the
entrapment structures 1114, the unoccupied droplets 1110 may still
deform and flow through other apertures. After sorting, the
entrapment structures 1114 may retain from the plurality of
droplets only the first subset of occupied droplets 1108. The
unoccupied droplets 1104 may flow through all entrapment structures
1114 and exit the channel segment 1100 to a separate compartment,
such as for recycling or discarding. While FIG. 11 shows exemplary
configurations and a layout of entrapment structures in the channel
structure, the configurations and layout of entrapment structures
are not limited as such. For example, an entrapment structure can
be a single plate with a plurality of apertures (e.g., holes)
defined in the plate. The plate can be planar, curved, and/or a
combination thereof. The channel structure and the entrapment
structures 1114 can be configured such that a droplet from the
plurality of droplets passes through at least one entrapment
structure (and aperture defined therein). After entrapment and/or
sorting of the occupied droplets, the fluid flow unit (not shown)
can be configured to reverse a fluid flow direction to collect the
occupied droplets from the entrapment structures.
[0357] In some instances, the fluid flow unit may comprise a
compressor to provide positive pressure at an upstream location to
direct the fluid from the upstream location to flow to a downstream
location. In some instances, the fluid flow unit may comprise a
pump to provide negative pressure at a downstream location to
direct the fluid from an upstream location to flow to the
downstream location. In some instances, the fluid flow unit may
comprise both a compressor and a pump, each at different locations.
In some instances, the fluid flow unit may comprise different
devices at different locations. The fluid flow unit may comprise an
actuator.
[0358] The systems and methods described with respect to FIG. 11
may be used to separate occupied particles (e.g., cell beads) from
unoccupied particles. As described elsewhere herein, a plurality of
particles may comprise a first subset of particles occupied by
biological particles (e.g., cells) and a second subset of particles
unoccupied by biological particles. As described above, a given
unoccupied particle can have a higher deformability and/or lower
surface tension property than a given occupied particle (e.g., cell
bead) due to the presence of one or more biological particles in
the occupied particle. In a channel structure including channel
segment 1100 with an entrance 1102 and exit 1104, and comprising
the plurality of entrapment structures 1114, the plurality of
particles may be directed to flow (e.g., as suspensions in a fluid,
e.g., aqueous fluid) along the channel segment 1100 from entrance
1102 to exit 1104 across the plurality of entrapment structures
1114.
[0359] An entrapment structure can define an aperture. A size of
the aperture may be less than a diameter (or other size dimension)
of a particle. A size of the aperture may be less than a minimum
dimension of a particle. In some instances, the size of the
aperture can be at most about 90%, 80%, 70%, 60%, 50%, 45%, 40%,
35%, 30%, 25%, 20%, 15%, 10%, or 5% of the diameter of a particle
(in a pre-deformed state). The size of the aperture can be less
than about 5% of the diameter of a particle. Alternatively, the
size of the aperture can be greater than 50%, 60%, 70%, 80%, 90%,
100%, 150%, or 200% of the diameter of a particle. The size of the
aperture can be greater than the diameter of the particle. When a
plurality of particles comprising both a first subset of occupied
particles 1108 and a second subset of unoccupied particles 1110 is
directed to pass through entrapment structures 1114 defining
apertures which is smaller in size than a diameter of a given
particle in the plurality of particles, only those particles
deforming 1118 (e.g., unoccupied particles 1110 having higher
deformability properties) such that at least one dimension of the
particle is less than a size of the aperture may pass through one
or more apertures defined by the entrapment structures 1114.
Because occupied particles 1108 (e.g., cell beads) may be harder or
stiffer, due to a presence of one or more biological particles in
the particles, the occupied particles may resist deformation, at
least from deforming to a size smaller than a size of the aperture,
and remain trapped by the entrapment structures 1114.
[0360] In some embodiments, there may exist more entrapment
structures 1114 (and thus more apertures) in the channel structure
than there are particles 1106, 1108 passing through the channel
structure. Beneficially, when the occupied particles 1108 are
prevented from flowing through the entrapment structures 1114, and
the occupied particles 1108 clog (or block) some apertures of the
entrapment structures 1114, the unoccupied particles 1110 may still
deform and flow through other apertures. After sorting, the
entrapment structures 1114 may retain from the plurality of
particles only the first subset of occupied particles 1108. The
unoccupied particles 1104 may flow through all entrapment
structures 1114 and exit the channel segment 1100 to a separate
compartment, such as for recycling or discarding.
[0361] The separation systems and methods disclosed herein (such as
with reference to FIG. 11) may achieve super Poisson loading. For
example, the droplets can be separated into two subsets such that
at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a first subset of
droplets that is isolated are occupied droplets (e.g., containing
at least one biological particle). Such occupancy may be greater
than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, or higher. Alternatively, less than about 97%
of the first subset of droplets can be occupied droplets. In some
instances, at least about 97%, 98%, 99%, or a higher percentage of
a second subset of droplets that is isolated can be unoccupied
droplets (e.g., not containing any biological particle and not
containing any barcode carrying beads). Alternatively, less than
about 97% of the second subset of droplets can be unoccupied
droplets. For example, the plurality of particles can be separated
into two subsets such that at least about 5%, 10%, 15%, 20%, 25%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or
greater of a first subset of particles that is isolated are cell
beads (e.g., containing at least one biological particle). Such
occupancy may be greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher.
Alternatively, less than about 97% of the first subset of particles
can be cell beads. In some instances, at least about 97%, 98%, 99%,
or a higher percentage of a second subset of particles that is
isolated can be unoccupied particles (e.g., not containing any
biological particle and not containing any barcode carrying beads).
Alternatively, less than about 97% of the second subset of
particles can be unoccupied particles.
Microfluidic Architectures
[0362] In an aspect, provided herein are various microfluidic
architectures that can be used in conjunction with the systems and
methods described herein.
[0363] In accordance with certain aspects, beads may be delivered
to droplets. FIG. 12 shows an example of a microfluidic channel
structure 1200 for delivering barcode carrying beads to droplets.
An example of a barcode carrying bead is described with respect to
FIG. 19. The channel structure 1200 can include channel segments
1201, 1202, 1204, 1206 and 1208 communicating at a channel junction
1210. In operation, the channel segment 1201 may transport an
aqueous fluid 1212 that includes a plurality of beads 1214 (e.g.,
with nucleic acid molecules, oligonucleotides, molecular tags)
along the channel segment 1201 into junction 1210. The plurality of
beads 1214 may be sourced from a suspension of beads. For example,
the channel segment 1201 may be connected to a reservoir comprising
an aqueous suspension of beads 1214. The channel segment 1202 may
transport the aqueous fluid 1212 that includes a plurality of
biological particles 1216 along the channel segment 1202 into
junction 1210. The plurality of biological particles 1216 may be
sourced from a suspension of biological particles. For example, the
channel segment 1202 may be connected to a reservoir comprising an
aqueous suspension of biological particles 1216. In some instances,
the aqueous fluid 1212 in either the first channel segment 1201 or
the second channel segment 1202, or in both segments, can include
one or more reagents, as further described below. A second fluid
1218 that is immiscible with the aqueous fluid 1212 (e.g., oil) can
be delivered to the junction 1210 from each of channel segments
1204 and 1206. Upon meeting of the aqueous fluid 1212 from each of
channel segments 1201 and 1202 and the second fluid 1218 from each
of channel segments 1204 and 1206 at the channel junction 1210, the
aqueous fluid 1212 can be partitioned as discrete droplets 1220 in
the second fluid 1218 and flow away from the junction 1210 along
channel segment 1208. The channel segment 1208 may deliver the
discrete droplets to an outlet reservoir fluidly coupled to the
channel segment 1208, where they may be harvested.
[0364] As an alternative, the channel segments 1201 and 1202 may
meet at another junction upstream of the junction 1210. At such
junction, beads and biological particles may form a mixture that is
directed along another channel to the junction 1210 to yield
droplets 1220. The mixture may provide the beads and biological
particles in an alternating fashion, such that, for example, a
droplet comprises a single bead and a single biological
particle.
[0365] Beads, biological particles 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 a single bead and a single biological particle. Such
regular flow profiles may permit the droplets to have an occupancy
(e.g., droplets having beads and biological particles) 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 Ser. No. 12015/0292988, which is entirely incorporated
herein by reference.
[0366] The second fluid 1218 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 1220.
[0367] A discrete droplet that is generated may include an
individual biological particle 1216. A discrete droplet that is
generated may include a barcode or other reagent carrying bead
1214. A discrete droplet generated may include both an individual
biological particle and a barcode carrying bead, such as droplets
1220. In some instances, a discrete droplet may include more than
one individual biological particle or no biological particle. 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).
[0368] Beneficially, a discrete droplet partitioning a biological
particle and a barcode carrying bead may effectively allow the
attribution of the barcode to macromolecular constituents of the
biological particle within the partition. The contents of a
partition may remain discrete from the contents of other
partitions.
[0369] 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 1200 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.
[0370] In accordance with certain aspects, biological particles may
be partitioned along with lysis reagents in order to release the
contents of the biological particles within the partition. In such
cases, the lysis agents can be contacted with the biological
particle suspension concurrently with, or immediately prior to, the
introduction of the biological particles into the partitioning
junction/droplet generation zone, such as through an additional
channel or channels upstream of the channel junction. In accordance
with other aspects, additionally or alternatively, biological
particles may be partitioned along with other reagents, as will be
described further below.
[0371] FIG. 13 shows an example of a microfluidic channel structure
1300 for co-partitioning biological particles and reagents. The
channel structure 1300 can include channel segments 1301, 1302,
1304, 1306 and 1308. Channel segments 1301 and 1302 communicate at
a first channel junction 1309. Channel segments 1302, 1304, 1306,
and 1308 communicate at a second channel junction 1310.
[0372] In an example operation, the channel segment 1301 may
transport an aqueous fluid 1312 that includes a plurality of
biological particles 1314 along the channel segment 1301 into the
second junction 1310. As an alternative or in addition to, channel
segment 1301 may transport beads (e.g., gel beads). The beads may
comprise barcode molecules.
[0373] For example, the channel segment 1301 may be connected to a
reservoir comprising an aqueous suspension of biological particles
1314. Upstream of, and immediately prior to reaching, the second
junction 1310, the channel segment 1301 may meet the channel
segment 1302 at the first junction 1309. The channel segment 1302
may transport a plurality of reagents 1315 (e.g., lysis agents)
suspended in the aqueous fluid 1312 along the channel segment 1302
into the first junction 1309. For example, the channel segment 1302
may be connected to a reservoir comprising the reagents 1315. After
the first junction 1309, the aqueous fluid 1312 in the channel
segment 1301 can carry both the biological particles 1314 and the
reagents 1315 towards the second junction 1310. In some instances,
the aqueous fluid 1312 in the channel segment 1301 can include one
or more reagents, which can be the same or different reagents as
the reagents 1315. A second fluid 1316 that is immiscible with the
aqueous fluid 1312 (e.g., oil) can be delivered to the second
junction 1310 from each of channel segments 1304 and 1306. Upon
meeting of the aqueous fluid 1312 from the channel segment 1301 and
the second fluid 1316 from each of channel segments 1304 and 1306
at the second channel junction 1310, the aqueous fluid 1312 can be
partitioned as discrete droplets 1318 in the second fluid 1316 and
flow away from the second junction 1310 along channel segment 1308.
The channel segment 1308 may deliver the discrete droplets 1318 to
an outlet reservoir fluidly coupled to the channel segment 1308,
where they may be harvested.
[0374] The second fluid 1316 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 1318.
[0375] A discrete droplet generated may include an individual
biological particle 1314 and/or one or more reagents 1315. 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).
[0376] Beneficially, when lysis reagents and biological particles
are co-partitioned, the lysis reagents can facilitate the release
of the contents of the biological particles within the partition.
The contents released in a partition may remain discrete from the
contents of other partitions.
[0377] 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 1300 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 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.
[0378] 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.
[0379] FIG. 14 shows an example of a microfluidic channel structure
for the controlled partitioning of beads into discrete droplets. A
channel structure 1400 can include a channel segment 1402
communicating at a channel junction 1406 (or intersection) with a
reservoir 1404. The reservoir 1404 can be a chamber. Any reference
to "reservoir," as used herein, can also refer to a "chamber." In
operation, an aqueous fluid 1408 that includes suspended beads 1412
may be transported along the channel segment 1402 into the junction
1406 to meet a second fluid 1410 that is immiscible with the
aqueous fluid 1408 in the reservoir 1404 to create droplets 1416,
1418 of the aqueous fluid 1408 flowing into the reservoir 1404. At
the junction 1406 where the aqueous fluid 1408 and the second fluid
1410 meet, droplets can form based on factors such as the
hydrodynamic forces at the junction 1406, flow rates of the two
fluids 1408, 1410, fluid properties, and certain geometric
parameters (e.g., w, h.sub.0, .alpha., etc.) of the channel
structure 1400. A plurality of droplets can be collected in the
reservoir 1404 by continuously injecting the aqueous fluid 1408
from the channel segment 1402 through the junction 1406.
[0380] A discrete droplet generated may include a bead (e.g., as in
occupied droplets 1416). 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 1418). In some instances, a discrete droplet generated may
contain one or more biological particles, as described elsewhere
herein. In some instances, a discrete droplet generated may
comprise one or more reagents, as described elsewhere herein.
[0381] In some instances, the aqueous fluid 1408 can have a
substantially uniform concentration or frequency of beads 1412. The
beads 1412 can be introduced into the channel segment 1402 from a
separate channel (not shown in FIG. 14). The frequency of beads
1412 in the channel segment 1402 may be controlled by controlling
the frequency in which the beads 1412 are introduced into the
channel segment 1402 and/or the relative flow rates of the fluids
in the channel segment 1402 and the separate channel. In some
instances, the beads can be introduced into the channel segment
1402 from a plurality of different channels, and the frequency
controlled accordingly.
[0382] In some instances, the aqueous fluid 1408 in the channel
segment 1402 can comprise biological particles. In some instances,
the aqueous fluid 1408 can have a substantially uniform
concentration or frequency of biological particles. As with the
beads, the biological particles can be introduced into the channel
segment 1402 from a separate channel. The frequency or
concentration of the biological particles in the aqueous fluid 1408
in the channel segment 1402 may be controlled by controlling the
frequency in which the biological particles are introduced into the
channel segment 1402 and/or the relative flow rates of the fluids
in the channel segment 1402 and the separate channel. In some
instances, the biological particles can be introduced into the
channel segment 1402 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 into the channel segment 1402.
The first separate channel introducing the beads may be upstream or
downstream of the second separate channel introducing the
biological particles.
[0383] The second fluid 1410 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.
[0384] In some instances, the second fluid 1410 may not be
subjected to and/or directed to any flow in or out of the reservoir
1404. For example, the second fluid 1410 may be substantially
stationary in the reservoir 1404. In some instances, the second
fluid 1410 may be subjected to flow within the reservoir 1404, but
not in or out of the reservoir 1404, such as via application of
pressure to the reservoir 1404 and/or as affected by the incoming
flow of the aqueous fluid 1408 at the junction 1406. Alternatively,
the second fluid 1410 may be subjected and/or directed to flow in
or out of the reservoir 1404. For example, the reservoir 1404 can
be a channel directing the second fluid 1410 from upstream to
downstream, transporting the generated droplets.
[0385] The channel structure 1400 at or near the junction 1406 may
have certain geometric features that at least partly determine the
sizes of the droplets formed by the channel structure 1400. The
channel segment 1402 can have a height, h.sub.0 and width, w, at or
near the junction 1406. By way of example, the channel segment 1402
can comprise a rectangular cross-section that leads to a reservoir
1404 having a wider cross-section (such as in width or diameter).
Alternatively, the cross-section of the channel segment 1402 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 1404 at or near the junction 1406 can be inclined at
an expansion angle, a. The expansion angle, a, allows the tongue
(portion of the aqueous fluid 1408 leaving channel segment 1402 at
junction 1406 and entering the reservoir 1404 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.
##EQU00010##
[0386] 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.
[0387] In some instances, the expansion angle, a, 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 1408 entering
the junction 1406 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 1408 entering the junction 1406 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
1408 entering the junction 1406 can be less than about 0.01
.mu.L/min. Alternatively, the flow rate of the aqueous fluid 1408
entering the junction 1406 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 1408 entering the junction
1406.
[0388] 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.
[0389] The throughput of droplet generation can be increased by
increasing the points of generation, such as increasing the number
of junctions (e.g., junction 1406) between aqueous fluid 1408
channel segments (e.g., channel segment 1402) and the reservoir
1404. Alternatively or in addition, the throughput of droplet
generation can be increased by increasing the flow rate of the
aqueous fluid 1408 in the channel segment 1402.
[0390] FIG. 15 shows an example of a microfluidic channel structure
for increased droplet generation throughput. A microfluidic channel
structure 1500 can comprise a plurality of channel segments 1502
and a reservoir 1504. Each of the plurality of channel segments
1502 may be in fluid communication with the reservoir 1504. The
channel structure 1500 can comprise a plurality of channel
junctions 1506 between the plurality of channel segments 1502 and
the reservoir 1504. Each channel junction can be a point of droplet
generation. The channel segment 1402 from the channel structure
1400 in FIG. 14 and any description to the components thereof may
correspond to a given channel segment of the plurality of channel
segments 1502 in channel structure 1500 and any description to the
corresponding components thereof. The reservoir 1404 from the
channel structure 1400 and any description to the components
thereof may correspond to the reservoir 1504 from the channel
structure 1500 and any description to the corresponding components
thereof.
[0391] Each channel segment of the plurality of channel segments
1502 may comprise an aqueous fluid 1508 that includes suspended
beads 1512. The reservoir 1504 may comprise a second fluid 1510
that is immiscible with the aqueous fluid 1508. In some instances,
the second fluid 1510 may not be subjected to and/or directed to
any flow in or out of the reservoir 1504. For example, the second
fluid 1510 may be substantially stationary in the reservoir 1504.
In some instances, the second fluid 1510 may be subjected to flow
within the reservoir 1504, but not in or out of the reservoir 1504,
such as via application of pressure to the reservoir 1504 and/or as
affected by the incoming flow of the aqueous fluid 1508 at the
junctions. Alternatively, the second fluid 1510 may be subjected
and/or directed to flow in or out of the reservoir 1504. For
example, the reservoir 1504 can be a channel directing the second
fluid 1510 from upstream to downstream, transporting the generated
droplets.
[0392] In operation, the aqueous fluid 1508 that includes suspended
beads 1512 may be transported along the plurality of channel
segments 1502 into the plurality of junctions 1506 to meet the
second fluid 1510 in the reservoir 1504 to create droplets 1516,
1518. A droplet may form from each channel segment at each
corresponding junction with the reservoir 1504. At the junction
where the aqueous fluid 1508 and the second fluid 1510 meet,
droplets can form based on factors such as the hydrodynamic forces
at the junction, flow rates of the two fluids 1508, 1510, fluid
properties, and certain geometric parameters (e.g., w, h.sub.0, a,
etc.) of the channel structure 1500, as described elsewhere herein.
A plurality of droplets can be collected in the reservoir 1504 by
continuously injecting the aqueous fluid 1508 from the plurality of
channel segments 1502 through the plurality of junctions 1506.
Throughput may significantly increase with the parallel channel
configuration of channel structure 1500. For example, a channel
structure having five inlet channel segments comprising the aqueous
fluid 1508 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, 200, 250, 300, 350, 400, 450, 500, 600,
700, 800, 900, 1000, 1500, 5000 or more parallel or substantially
parallel channel segments.
[0393] The geometric parameters, w, h.sub.0, and a, may or may not
be uniform for each of the channel segments in the plurality of
channel segments 1502. For example, each channel segment may have
the same or different widths at or near its respective channel
junction with the reservoir 1504. For example, each channel segment
may have the same or different height at or near its respective
channel junction with the reservoir 1504. In another example, the
reservoir 1504 may have the same or different expansion angle at
the different channel junctions with the plurality of channel
segments 1502. When the geometric parameters are uniform,
beneficially, droplet size may also be controlled to be uniform
even with the increased throughput. In some instances, when it is
desirable to have a different distribution of droplet sizes, the
geometric parameters for the plurality of channel segments 1502 may
be varied accordingly.
[0394] 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.
[0395] FIG. 16 shows another example of a microfluidic channel
structure for increased droplet generation throughput. A
microfluidic channel structure 1600 can comprise a plurality of
channel segments 1602 arranged generally circularly around the
perimeter of a reservoir 1604. Each of the plurality of channel
segments 1602 may be in fluid communication with the reservoir
1604. The channel structure 1600 can comprise a plurality of
channel junctions 1606 between the plurality of channel segments
1602 and the reservoir 1604. Each channel junction can be a point
of droplet generation. The channel segment 1402 from the channel
structure 1400 in FIG. 14 and any description to the components
thereof may correspond to a given channel segment of the plurality
of channel segments 1602 in channel structure 1600 and any
description to the corresponding components thereof. The reservoir
1404 from the channel structure 1400 and any description to the
components thereof may correspond to the reservoir 1604 from the
channel structure 1600 and any description to the corresponding
components thereof.
[0396] Each channel segment of the plurality of channel segments
1602 may comprise an aqueous fluid 1608 that includes suspended
beads 1612. The reservoir 1604 may comprise a second fluid 1610
that is immiscible with the aqueous fluid 1608. In some instances,
the second fluid 1610 may not be subjected to and/or directed to
any flow in or out of the reservoir 1604. For example, the second
fluid 1610 may be substantially stationary in the reservoir 1604.
In some instances, the second fluid 1610 may be subjected to flow
within the reservoir 1604, but not in or out of the reservoir 1604,
such as via application of pressure to the reservoir 1604 and/or as
affected by the incoming flow of the aqueous fluid 1608 at the
junctions. Alternatively, the second fluid 1610 may be subjected
and/or directed to flow in or out of the reservoir 1604. For
example, the reservoir 1604 can be a channel directing the second
fluid 1610 from upstream to downstream, transporting the generated
droplets.
[0397] In operation, the aqueous fluid 1608 that includes suspended
beads 1612 may be transported along the plurality of channel
segments 1602 into the plurality of junctions 1606 to meet the
second fluid 1610 in the reservoir 1604 to create a plurality of
droplets 1616. A droplet may form from each channel segment at each
corresponding junction with the reservoir 1604. At the junction
where the aqueous fluid 1608 and the second fluid 1610 meet,
droplets can form based on factors such as the hydrodynamic forces
at the junction, flow rates of the two fluids 1608, 1610, fluid
properties, and certain geometric parameters (e.g., widths and
heights of the channel segments 1602, expansion angle of the
reservoir 1604, etc.) of the channel structure 1600, as described
elsewhere herein. A plurality of droplets can be collected in the
reservoir 1604 by continuously injecting the aqueous fluid 1608
from the plurality of channel segments 1602 through the plurality
of junctions 1606. Throughput may significantly increase with the
substantially parallel channel configuration of the channel
structure 1600. 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.
[0398] The reservoir 1604 may have an expansion angle, a (not shown
in FIG. 16) at or near each channel junction. Each channel segment
of the plurality of channel segments 1602 may have a width, w, and
a height, h.sub.0, at or near the channel junction. The geometric
parameters, w, h.sub.0, and .alpha., may or may not be uniform for
each of the channel segments in the plurality of channel segments
1602. For example, each channel segment may have the same or
different widths at or near its respective channel junction with
the reservoir 1604. For example, each channel segment may have the
same or different height at or near its respective channel junction
with the reservoir 1604.
[0399] The reservoir 1604 may have the same or different expansion
angle at the different channel junctions with the plurality of
channel segments 1602. For example, a circular reservoir (as shown
in FIG. 16) 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 1602 at or near the
plurality of channel junctions 1606. When the geometric parameters
are uniform, beneficially, resulting droplet size may be controlled
to be uniform even with the increased throughput. In some
instances, when it is desirable to have a different distribution of
droplet sizes, the geometric parameters for the plurality of
channel segments 1602 may be varied accordingly.
[0400] 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 injected
into the droplets may or may not have uniform size.
[0401] FIG. 17A shows a cross-section view of another example of a
microfluidic channel structure with a geometric feature for
controlled partitioning. A channel structure 1700 can include a
channel segment 1702 communicating at a channel junction 1706 (or
intersection) with a reservoir 1704. In some instances, the channel
structure 1700 and one or more of its components can correspond to
any other channel structure described herein and one or more of its
components. FIG. 17B shows a perspective view of the channel
structure 1700 of FIG. 17A.
[0402] An aqueous fluid 1712 comprising a plurality of particles
1716 may be transported along the channel segment 1702 into the
junction 1706 to meet a second fluid 1714 (e.g., oil, etc.) that is
immiscible with the aqueous fluid 1712 in the reservoir 1704 to
create droplets 720 of the aqueous fluid 1712 flowing into the
reservoir 1704. At the junction 1706 where the aqueous fluid 1712
and the second fluid 1714 meet, droplets can form based on factors
such as the hydrodynamic forces at the junction 1706, relative flow
rates of the two fluids 1712, 1714, fluid properties, and certain
geometric parameters (e.g., .DELTA.h, etc.) of the channel
structure 1700. A plurality of droplets can be collected in the
reservoir 1704 by continuously injecting the aqueous fluid 1712
from the channel segment 1702 at the junction 1706.
[0403] A discrete droplet generated may comprise one or more
particles of the plurality of particles 1716. As described
elsewhere herein, a particle may be any particle, such as a bead,
cell bead, gel bead, biological particle, macromolecular
constituents of biological particle, or other particles.
Alternatively, a discrete droplet generated may not include any
particles.
[0404] In some instances, the aqueous fluid 1712 can have a
substantially uniform concentration or frequency of particles 1716.
As described elsewhere herein (e.g., with reference to FIG. 14),
the particles 1716 (e.g., beads) can be introduced into the channel
segment 1702 from a separate channel (not shown in FIG. 17). The
frequency of particles 1716 in the channel segment 1702 may be
controlled by controlling the frequency in which the particles 1716
are introduced into the channel segment 1702 and/or the relative
flow rates of the fluids in the channel segment 1702 and the
separate channel. In some instances, the particles 1716 can be
introduced into the channel segment 1702 from a plurality of
different channels, and the frequency controlled accordingly. In
some instances, different particles may be introduced via separate
channels. For example, a first separate channel can introduce beads
and a second separate channel can introduce biological particles
into the channel segment 1702. The first separate channel
introducing the beads may be upstream or downstream of the second
separate channel introducing the biological particles.
[0405] In some instances, the second fluid 1714 may not be
subjected to and/or directed to any flow in or out of the reservoir
1704. For example, the second fluid 1714 may be substantially
stationary in the reservoir 1704. In some instances, the second
fluid 1714 may be subjected to flow within the reservoir 1704, but
not in or out of the reservoir 1704, such as via application of
pressure to the reservoir 1704 and/or as affected by the incoming
flow of the aqueous fluid 1712 at the junction 1706. Alternatively,
the second fluid 1714 may be subjected and/or directed to flow in
or out of the reservoir 1704. For example, the reservoir 1704 can
be a channel directing the second fluid 1714 from upstream to
downstream, transporting the generated droplets.
[0406] The channel structure 1700 at or near the junction 1706 may
have certain geometric features that at least partly determine the
sizes and/or shapes of the droplets formed by the channel structure
1700. The channel segment 1702 can have a first cross-section
height, h.sub.1, and the reservoir 1704 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 1706, there is a height
difference of .DELTA.h. 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 1706. In some instances, the cross-section height of the
reservoir may increase in accordance with expansion angle, .beta.,
at or near the junction 1706. The height difference, .DELTA.h,
and/or expansion angle, .beta., can allow the tongue (portion of
the aqueous fluid 1712 leaving channel segment 1702 at junction
1706 and entering the reservoir 1704 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.
[0407] 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.
[0408] In some instances, the flow rate of the aqueous fluid 1712
entering the junction 1706 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 1712 entering the junction 1706 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
1712 entering the junction 1706 can be less than about 0.01
.mu.L/min. Alternatively, the flow rate of the aqueous fluid 1712
entering the junction 1706 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 1712 entering the junction
1706. The second fluid 1714 may be stationary, or substantially
stationary, in the reservoir 1704. Alternatively, the second fluid
1714 may be flowing, such as at the above flow rates described for
the aqueous fluid 1712.
[0409] 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.
[0410] While FIGS. 7A and 7B illustrate the height difference,
.DELTA.h, being abrupt at the junction 1706 (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 1706, 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.
[0411] 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 1208,
reservoir 1604, 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.
[0412] The methods and systems described herein may be used to
greatly increase the efficiency of single 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.
[0413] A variety of applications require the evaluation of the
presence and quantification of different biological particle or
organism types within a population of biological particles,
including, for example, microbiome analysis and characterization,
environmental testing, food safety testing, epidemiological
analysis, e.g., in tracing contamination or the like.
Computer Control Systems
[0414] The present disclosure provides computer control systems
that are programmed to implement methods of the disclosure. FIG. 18
shows a computer system 1801 that is programmed or otherwise
configured to (i) sort occupied droplets from unoccupied droplets
by including field-attractable particles in each droplet and
applying a force field, (ii) sort occupied droplets from unoccupied
droplets by applying a pressure pulse, (iii) sort occupied
particles (e.g., cell beads) from unoccupied particles using
field-attractable particles by applying a force field, (iv) sort
occupied particles (e.g., cell beads) from unoccupied particles by
applying a pressure pulse, (v) selectively polymerize occupied
droplets, and/or (vi) selectively polymerize appropriately sized
droplets. The computer system 1801 can regulate various aspects of
the present disclosure, such as, for example, the timed exposure of
the single biological particle to a variety of chemical or
biological operations, regulating fluid flow rate in one or more
channels in a microfluidic structure, regulating field strength
applied by one or more field application units, regulating pressure
pulses applied by one or more pressure application units, and/or
regulating timing of polymerization application units. The computer
system 1801 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.
[0415] The computer system 1801 includes a central processing unit
(CPU, also "processor" and "computer processor" herein) 1805, which
can be a single core or multi core processor, or a plurality of
processors for parallel processing. The computer system 1801 also
includes memory or memory location 1810 (e.g., random-access
memory, read-only memory, flash memory), electronic storage unit
1815 (e.g., hard disk), communication interface 1820 (e.g., network
adapter) for communicating with one or more other systems, and
peripheral devices 1825, such as cache, other memory, data storage
and/or electronic display adapters. The memory 1810, storage unit
1815, interface 1820 and peripheral devices 1825 are in
communication with the CPU 1805 through a communication bus (solid
lines), such as a motherboard. The storage unit 1815 can be a data
storage unit (or data repository) for storing data. The computer
system 1801 can be operatively coupled to a computer network
("network") 1830 with the aid of the communication interface 1820.
The network 1830 can be the Internet, an internet and/or extranet,
or an intranet and/or extranet that is in communication with the
Internet. The network 1830 in some cases is a telecommunication
and/or data network. The network 1830 can include one or more
computer servers, which can enable distributed computing, such as
cloud computing. The network 1830, in some cases with the aid of
the computer system 1801, can implement a peer-to-peer network,
which may enable devices coupled to the computer system 1801 to
behave as a client or a server.
[0416] The CPU 1805 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
1810. The instructions can be directed to the CPU 1805, which can
subsequently program or otherwise configure the CPU 1805 to
implement methods of the present disclosure. Examples of operations
performed by the CPU 1805 can include fetch, decode, execute, and
writeback.
[0417] The CPU 1805 can be part of a circuit, such as an integrated
circuit. One or more other components of the system 1801 can be
included in the circuit. In some cases, the circuit is an
application specific integrated circuit (ASIC).
[0418] The storage unit 1815 can store files, such as drivers,
libraries and saved programs. The storage unit 1815 can store user
data, e.g., user preferences and user programs. The computer system
1801 in some cases can include one or more additional data storage
units that are external to the computer system 1801, such as
located on a remote server that is in communication with the
computer system 1801 through an intranet or the Internet.
[0419] The computer system 1801 can communicate with one or more
remote computer systems through the network 1830. For instance, the
computer system 1801 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 1801 via the network 1830.
[0420] 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 1801, such as,
for example, on the memory 1810 or electronic storage unit 1815.
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 1805. In some cases, the code can be retrieved from the
storage unit 1815 and stored on the memory 1810 for ready access by
the processor 1805. In some situations, the electronic storage unit
1815 can be precluded, and machine-executable instructions are
stored on memory 1810.
[0421] 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.
[0422] Aspects of the systems and methods provided herein, such as
the computer system 1801, 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.
[0423] 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.
[0424] The computer system 1801 can include or be in communication
with an electronic display 1835 that comprises a user interface
(UI) 1840 for providing, for example, fluid control options (e.g.,
fluid flow rate, timing of applying polymerization source (e.g.,
light), strength of magnetic of electric force field, strength
and/or frequency of pressure pulses, etc.). Examples of UI's
include, without limitation, a graphical user interface (GUI) and
web-based user interface.
[0425] 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 1805. The algorithm can, for example, (i) sort
occupied droplets from unoccupied droplets by including
field-attractable particles in each droplet and applying a force
field, (ii) sort occupied droplets from unoccupied droplets by
applying a pressure pulse, (iii) sort occupied particles (e.g.,
cell beads) from unoccupied particles using field-attractable
particles by applying a force field, (iv) sort occupied particles
(e.g., cell beads) from unoccupied particles by applying a pressure
pulse, (v) selectively polymerize occupied droplets, and/or (vi)
selectively polymerize appropriately sized droplets. The algorithm
can also, for example, generate a plurality of droplets that may or
may not contain biological particles (cells) and/or barcode
carrying beads (particles).
[0426] While preferred embodiments of the present 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. It is not intended that the invention be limited by
the specific examples provided within the specification. While the
invention has been described with reference to the aforementioned
specification, the descriptions and illustrations of the
embodiments herein are not meant to be construed in a limiting
sense. Numerous variations, changes, and substitutions will now
occur to those skilled in the art without departing from the
invention. Furthermore, it shall be understood that all aspects of
the invention are not limited to the specific depictions,
configurations or relative proportions set forth herein which
depend upon a variety of conditions and variables. It should be
understood that various alternatives to the embodiments of the
invention described herein may be employed in practicing the
invention. It is therefore contemplated that the invention shall
also cover any such alternatives, modifications, variations or
equivalents. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
* * * * *