U.S. patent application number 16/094766 was filed with the patent office on 2019-05-02 for immobilization-based systems and methods for genetic analysis and other applications.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to John Heyman, David A. Weitz, Huidan Zhang.
Application Number | 20190127789 16/094766 |
Document ID | / |
Family ID | 60116460 |
Filed Date | 2019-05-02 |
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United States Patent
Application |
20190127789 |
Kind Code |
A1 |
Weitz; David A. ; et
al. |
May 2, 2019 |
IMMOBILIZATION-BASED SYSTEMS AND METHODS FOR GENETIC ANALYSIS AND
OTHER APPLICATIONS
Abstract
The present invention generally relates to microfluidics and
labeled nucleic acids. Certain aspects are generally directed to
containing cells in gels, such as agarose gels, and determining
nucleic acids within the cells, e.g., while contained within the
gels. The nucleic acids may be, for example, genomic DNA, mRNA,
transcriptomes, or the like. In some embodiments, for instance,
both genomic DNA and RNA (e.g., as in a transcriptome) from a cell
may be determined. In some cases, the nucleic acids may be attached
to beads for sequencing or other purposes. Such systems may be
useful, for example, for high-throughput sequencing or other
applications.
Inventors: |
Weitz; David A.; (Bolton,
MA) ; Zhang; Huidan; (Cambridge, MA) ; Heyman;
John; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
60116460 |
Appl. No.: |
16/094766 |
Filed: |
April 19, 2017 |
PCT Filed: |
April 19, 2017 |
PCT NO: |
PCT/US2017/028329 |
371 Date: |
October 18, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62324407 |
Apr 19, 2016 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01F 3/08 20130101; B01J
13/02 20130101; C12Q 1/6827 20130101; C12Q 1/6855 20130101; C12Q
1/68 20130101; C12Q 1/6816 20130101; C12Q 1/6827 20130101; C12Q
2535/122 20130101; C12Q 2563/159 20130101; C12Q 1/6816 20130101;
C12Q 2535/122 20130101; C12Q 2563/159 20130101 |
International
Class: |
C12Q 1/6855 20060101
C12Q001/6855 |
Claims
1. A method, comprising: lysing cells within gel droplets to
release DNA and RNA from the cells; attaching the DNA to a first
set of oligonucleotides; and attaching the RNA to a second set of
oligonucleotides.
2. The method of claim 1, wherein the cells are contained within
the gel droplets at no more than about 1 cell/gel droplet.
3. The method of any one of claim 1 or 2, wherein the cells are
contained within the gel droplets at no more than about 0.1
cell/gel droplet.
4. The method of any one of claims 1-3, wherein the cells are
contained within the gel droplets at no more than about 0.01
cell/gel droplet.
5. The method of any one of claims 1-4, wherein at least about 90%
of the gel droplets contains one cell or no cell.
6. The method of any one of claims 1-5, wherein at least some of
the gel droplets comprise agarose.
7. The method of any one of claims 1-6, wherein the agarose has a
gelation temperature of less than about 60.degree. C.
8. The method of any one of claims 1-7, wherein the agarose has a
gelation temperature of less than about 40.degree. C.
9. The method of any one of claims 1-8, wherein the gel droplets
have an average diameter of no more than about 1 mm.
10. The method of any one of claims 1-9, wherein at least some of
the gel droplets are contained within a microfluidic channel.
11. The method of any one of claims 1-10, comprising lysing at
least some of the cells using a cell lysis reagent.
12. The method of claim 11, wherein the cell lysis reagent
comprises proteinase K.
13. The method of any one of claims 1-12, wherein at least some of
the cells are human cells.
14. The method of any one of claims 1-13, wherein the DNA includes
genomic DNA.
15. The method of claim 14, further comprising fragmenting the
genomic DNA after release from the cells.
16. The method of claim 15, wherein fragmenting the genomic DNA
comprises exposing the genomic DNA to a restriction
endonuclease.
17. The method of any one of claims 1-16, wherein the RNA includes
mRNA.
18. The method of claim 17, further comprising forming cDNA using
the RNA.
19. The method of claim 18, further comprising forming cDNA using
the RNA using a reverse transcriptase.
20. The method of any one of claim 18 or 19, further comprising
attaching the cDNA to oligonucleotides within the gel droplet.
21. The method of any one of claims 1-20, wherein attaching the DNA
to the first set of oligonucleotides comprises ligating at least
some of the DNA to the oligonucleotides.
22. The method of any one of claims 1-21, wherein attaching the RNA
to the first set of oligonucleotides comprises ligating at least
some of the RNA to the oligonucleotides.
23. The method of any one of claims 1-22, wherein first set of
oligonucleotides and/or the second set of oligonucleotides are
attached to particles.
24. The method of claim 23, wherein the particles comprise hydrogel
particles.
25. The method of any one of claim 23 or 24, wherein the particles
have an average diameter of no more than about 1 mm.
26. The method of any one of claims 23-25, wherein the particles
have an average diameter of no more than about 500 micrometers.
27. The method of any one of claims 23-26, wherein the particles
have an average diameter of no more than about 1 micrometer.
28. The method of any one of claims 23-27, wherein the first set of
oligonucleotides further comprises a first set of barcode
sequences.
29. The method of any one of claims 23-28, wherein the second set
of oligonucleotides further comprises a second set of barcode
sequences.
30. The method of claim 29, wherein the first set of barcode
sequences and the second set of barcode sequences have at least one
barcode sequence in common.
31. The method of any one of claim 29 or 30, wherein the first set
of barcode sequences and/or the second set of barcode sequences
comprise a first barcode selected from a pre-defined pool of first
barcodes and a second barcode selected from a pre-defined pool of
second barcodes, such that substantially each of the particles
comprises distinguishable barcode sequences.
32. The method of claim 31, wherein the pre-defined pool of first
barcodes comprises at least about 300 distinguishable barcodes.
33. The method of any one of claims 31-32, wherein the pre-defined
pool of first barcodes comprises at least about 1,000
distinguishable barcodes.
34. The method of any one of claims 31-33, wherein the pre-defined
pool of second barcodes comprises at least about 300
distinguishable barcodes.
35. The method of any one of claims 31-34, wherein the pre-defined
pool of second barcodes comprises at least about 1,000
distinguishable barcodes.
36. The method of any one of claims 31-35, wherein the pre-defined
pool of second barcodes comprises at least about 3,000
distinguishable barcodes.
37. The method of any one of claims 31-36, wherein the
distinguishable barcode sequences comprises at least 10,000
distinguishable barcode sequences.
38. The method of any one of claims 31-37, wherein the
distinguishable barcode sequences comprises at least 100,000
distinguishable barcode sequences.
39. The method of any one of claims 23-38, wherein at least some of
the oligonucleotides are covalently bonded to the particles.
40. The method of claim 39, wherein at least some oligonucleotides
of the first set of oligonucleotides and/or the second set of
oligonucleotides are covalently bonded to the particles via an
acrylic phosphoramidite linkage.
41. The method of any one of claim 39 or 40, wherein at least some
oligonucleotides of the first set of oligonucleotides and/or the
second set of oligonucleotides are covalently bonded to the
particles via an amino linkage.
42. The method of any one of claims 23-41, wherein at least some
oligonucleotides of the first set of oligonucleotides and/or the
second set of oligonucleotides are attached to the particles via a
biotin-steptavidin linkage.
43. The method of any one of claims 23-42, wherein at least some
oligonucleotides of the first set of oligonucleotides and/or the
second set of oligonucleotides comprise a cleavable linker.
44. The method of claim 43, wherein the cleavable linker is a
photocleavable linker.
45. The method of claim 43, wherein the cleavable linker is a
chemically cleavable linker.
46. The method of claim 43, wherein the cleavable linker is an
enzymatically cleavable linker.
47. The method of any one of claims 23-46, further comprising
amplifying at least some oligonucleotides of the first set of
oligonucleotides and/or the second set of oligonucleotides.
48. The method of claim 47, comprising amplifying the
oligonucleotides using PCR.
49. The method of any one of claims 1-48, further comprising
encapsulating the gel droplet within a fluidic droplet prior to
amplifying the oligonucleotides.
50. The method of claim 49, wherein the fluidic droplets contain
the particles.
51. The method of claim 50, wherein the particles are contained
within the fluidic droplets at no more than about 1
particle/droplet.
52. The method of any one of claim 50 or 51, wherein the particles
are contained within the fluidic droplets at no more than about 0.1
particle/droplet.
53. The method of any one of claims 50-52, wherein the particles
are contained within the fluidic droplets at no more than about
0.01 particle/droplet.
54. The method of any one of claims 50-53, wherein at least about
90% of the fluidic droplets contains one particle or no
particle.
55. The method of any one of claims 47-54, further comprising
releasing at least some of the oligonucleotides of the first set of
oligonucleotides and/or the second set of oligonucleotides from the
particles.
56. The method of claim 55, comprising cleaving at least some of
the oligonucleotides from the particles.
57. The method of any one of claim 55 or 56, comprising
photocleaving at least some of the oligonucleotides from the
particles.
58. The method of any one of claims 55-57, comprising releasing the
oligonucleotides from the particles prior to amplifying the
oligonucleotides.
59. The method of any one of claims 47-58, further comprising
bursting the fluidic droplet after amplifying the
oligonucleotides.
60. The method of claim 59, further comprising sequencing at least
some of the first set of oligonucleotides and/or the second set of
oligonucleotides.
61. A method, comprising: lysing a cell within a gel droplet to
release RNA from the cell; forming cDNA using the RNA; and
attaching the cDNA to an oligonucleotide within the gel
droplet.
62. The method of claim 61, comprising attaching the cDNA to the
oligonucleotides using a ligase.
63. The method of any one of claim 61 or 62, comprising exposing
the RNA to a linear polyacrylamide.
64. The method of any one of claims 61-63, comprising forming cDNA
using the RNA by exposing the RNA to a reverse transcriptase.
65. The method of claim 64, wherein the reverse transcriptase adds
three unpaired A bases to the cDNA.
66. The method of claim 65, wherein attaching the cDNA to the
oligonucleotide comprises matching the three unpaired A bases to
the cDNA to an oligonucleotide comprising three unpaired T
bases.
67. The method of any one of claims 61-66, wherein the cells are
contained within the gel droplets at no more than about 1 cell/gel
droplet.
68. The method of any one of claims 61-67, wherein the cells are
contained within the gel droplets at no more than about 0.1
cell/gel droplet.
69. The method of any one of claims 61-68, wherein the cells are
contained within the gel droplets at no more than about 0.01
cell/gel droplet.
70. The method of any one of claims 61-69, wherein at least about
90% of the gel droplets contains one cell or no cell.
71. The method of any one of claims 61-70, wherein at least some of
the gel droplets comprise agarose.
72. The method of any one of claims 61-71, wherein the agarose has
a gelation temperature of less than about 60.degree. C.
73. The method of any one of claims 61-72, wherein the agarose has
a gelation temperature of less than about 40.degree. C.
74. The method of any one of claims 61-73, wherein the gel droplets
have an average diameter of no more than about 1 mm.
75. The method of any one of claims 61-74, comprising lysing at
least some of the cells using a cell lysis reagent.
76. The method of claim 75, wherein the cell lysis reagent
comprises proteinase K.
77. The method of any one of claims 61-76, wherein at least some of
the cells are human cells.
78. The method of any one of claims 61-77, wherein the RNA includes
miRNA.
79. The method of any one of claims 61-78, wherein the RNA includes
mRNA.
80. The method of any one of claims 61-79, wherein at least some of
the oligonucleotides are attached to particles.
81. The method of claim 80, wherein the particles comprise hydrogel
particles.
82. The method of any one of claim 80 or 81, wherein the particles
have an average diameter of no more than about 1 mm.
83. The method of any one of claims 80-82, wherein the particles
have an average diameter of no more than about 500 micrometers.
84. The method of any one of claims 80-83, wherein the particles
have an average diameter of no more than about 1 micrometer.
85. The method of any one of claims 80-84, wherein at least some of
the oligonucleotides are covalently bonded to the particles.
86. The method of claim 85, wherein at least some oligonucleotides
are covalently bonded to the particles via an acrylic
phosphoramidite linkage.
87. The method of any one of claim 85 or 86, wherein at least some
oligonucleotides are covalently bonded to the particles via an
amino linkage.
88. The method of any one of claims 80-87, wherein the
oligonucleotides are attached to the particles via a
biotin-steptavidin linkage.
89. The method of any one of claims 80-88, wherein the
oligonucleotides comprise a cleavable linker.
90. The method of claim 89, wherein the cleavable linker is a
photocleavable linker.
91. The method of claim 89, wherein the cleavable linker is a
chemically cleavable linker.
92. The method of claim 89, wherein the cleavable linker is an
enzymatically cleavable linker.
93. The method of any one of claims 80-92, wherein the
oligonucleotides further comprises a barcode sequence.
94. The method of claim 93, wherein the barcode sequences comprise
a first barcode selected from a pre-defined pool of first barcodes
and a second barcode selected from a pre-defined pool of second
barcodes, such that substantially each of the particles comprises
distinguishable barcode sequences.
95. The method of any one of claim 93 or 94, wherein the
pre-defined pool of first barcodes comprises at least about 300
distinguishable barcodes.
96. The method of claim 95, wherein the pre-defined pool of first
barcodes comprises at least about 1,000 distinguishable
barcodes.
97. The method of any one of claim 95 or 96, wherein the
pre-defined pool of second barcodes comprises at least about 300
distinguishable barcodes.
98. The method of any one of claims 95-97, wherein the pre-defined
pool of second barcodes comprises at least about 1,000
distinguishable barcodes.
99. The method of any one of claims 95-98, wherein the pre-defined
pool of second barcodes comprises at least about 3,000
distinguishable barcodes.
100. The method of any one of claims 95-99, wherein the
distinguishable barcode sequences comprises at least 10,000
distinguishable barcode sequences.
101. The method of any one of claims 95-100, wherein the
distinguishable barcode sequences comprises at least 100,000
distinguishable barcode sequences.
102. The method of any one of claims 80-101, further comprising
amplifying at least some of the oligonucleotides.
103. The method of claim 102, comprising amplifying the
oligonucleotides using PCR.
104. The method of any one of claims 61-103, further comprising
encapsulating the gel droplet within a fluidic droplet prior to
amplifying the oligonucleotides.
105. The method of claim 104, wherein the fluidic droplets contain
the particles.
106. The method of claim 105, wherein the particles are contained
within the fluidic droplets at no more than about 1
particle/droplet.
107. The method of any one of claim 105 or 106, wherein the
particles are contained within the fluidic droplets at no more than
about 0.1 particle/droplet.
108. The method of any one of claims 105-107, wherein the particles
are contained within the fluidic droplets at no more than about
0.01 particle/droplet.
109. The method of any one of claims 105-108, wherein at least
about 90% of the fluidic droplets contains one particle or no
particle.
110. The method of any one of claims 105-109, further comprising
releasing at least some of the oligonucleotides from the
particles.
111. The method of claim 110, comprising cleaving at least some of
the oligonucleotides from the particles.
112. The method of any one of claim 110 or 111, comprising
photocleaving at least some of the oligonucleotides from the
particles.
113. The method of any one of claims 110-112, comprising releasing
the oligonucleotides from the particles prior to amplifying the
oligonucleotides.
114. The method of claim 113, further comprising bursting the
fluidic droplet after amplifying the oligonucleotides.
115. The method of any one of claim 113 or 114, further comprising
sequencing at least some of the oligonucleotides.
116. An article, comprising: a gel droplet comprising a lysed cell,
DNA attached to a first set of oligonucleotides, and RNA attached
to a second set of oligonucleotides.
117. The composition of claim 116, wherein the cells are contained
within the gel droplets at no more than about 1 cell/gel
droplet.
118. The composition of any one of claim 116 or 117, wherein the
cells are contained within the gel droplets at no more than about
0.1 cell/gel droplet.
119. The composition of any one of claims 116-118, wherein the
cells are contained within the gel droplets at no more than about
0.01 cell/gel droplet.
120. The composition of any one of claims 116-119, wherein at least
about 90% of the gel droplets contains one cell or no cell.
121. The composition of any one of claims 116-120, wherein at least
some of the gel droplets comprise agarose.
122. The composition of any one of claims 116-121, wherein the
agarose has a gelation temperature of less than about 60.degree.
C.
123. The composition of any one of claims 116-122, wherein the
agarose has a gelation temperature of less than about 40.degree.
C.
124. The composition of any one of claims 116-123, wherein the gel
droplets have an average diameter of no more than about 1 mm.
125. The composition of any one of claims 116-124, wherein at least
some of the gel droplets are contained within a microfluidic
channel.
126. The composition of any one of claims 116-125, wherein at least
some of the cells are human cells.
127. The composition of any one of claims 116-126, wherein the DNA
includes genomic DNA.
128. The composition of any one of claims 116-127, wherein the RNA
includes mRNA.
129. The composition of any one of claims 116-128, wherein first
set of oligonucleotides and/or the second set of oligonucleotides
are attached to particles.
130. The composition of claim 129, wherein the particles comprise
hydrogel particles.
131. The composition of any one of claim 129 or 130, wherein the
particles have an average diameter of no more than about 1 mm.
132. The composition of any one of claims 129-131, wherein the
particles have an average diameter of no more than about 500
micrometers.
133. The composition of any one of claims 129-132, wherein the
particles have an average diameter of no more than about 1
micrometer.
134. The composition of any one of claims 129-133, wherein the
first set of oligonucleotides further comprises a first set of
barcode sequences.
135. The composition of any one of claims 129-134, wherein the
second set of oligonucleotides further comprises a second set of
barcode sequences.
136. The composition of claim 135, wherein the first set of barcode
sequences and the second set of barcode sequences have at least one
barcode sequence in common.
137. The composition of any one of claims 135 or 136, wherein the
first set of barcode sequences and/or the second set of barcode
sequences comprise a first barcode selected from a pre-defined pool
of first barcodes and a second barcode selected from a pre-defined
pool of second barcodes, such that substantially each of the
particles comprises distinguishable barcode sequences.
138. The composition of claim 137, wherein the pre-defined pool of
first barcodes comprises at least about 300 distinguishable
barcodes.
139. The composition of any one of claim 137 or 138, wherein the
pre-defined pool of first barcodes comprises at least about 1,000
distinguishable barcodes.
140. The composition of any one of claims 137-139, wherein the
pre-defined pool of second barcodes comprises at least about 300
distinguishable barcodes.
141. The composition of any one of claims 137-140, wherein the
pre-defined pool of second barcodes comprises at least about 1,000
distinguishable barcodes.
142. The composition of any one of claims 137-141, wherein the
pre-defined pool of second barcodes comprises at least about 3,000
distinguishable barcodes.
143. The composition of any one of claims 137-142, wherein the
distinguishable barcode sequences comprises at least 10,000
distinguishable barcode sequences.
144. The composition of any one of claims 137-143, wherein the
distinguishable barcode sequences comprises at least 100,000
distinguishable barcode sequences.
145. The composition of any one of claims 129-144, wherein at least
some of the oligonucleotides are covalently bonded to the
particles.
146. The composition of claim 145, wherein at least some
oligonucleotides of the first set of oligonucleotides and/or the
second set of oligonucleotides are covalently bonded to the
particles via an acrylic phosphoramidite linkage.
147. The composition of any one of claim 145 or 146, wherein at
least some oligonucleotides of the first set of oligonucleotides
and/or the second set of oligonucleotides are covalently bonded to
the particles via an amino linkage.
148. The composition of any one of claims 129-147, wherein at least
some oligonucleotides of the first set of oligonucleotides and/or
the second set of oligonucleotides are attached to the particles
via a biotin-steptavidin linkage.
149. The composition of any one of claims 129-148, wherein at least
some oligonucleotides of the first set of oligonucleotides and/or
the second set of oligonucleotides comprise a cleavable linker.
150. The composition of claim 149, wherein the cleavable linker is
a photocleavable linker.
151. The composition of claim 149, wherein the cleavable linker is
a chemically cleavable linker.
152. The composition of claim 149, wherein the cleavable linker is
an enzymatically cleavable linker.
153. The composition of any one of claims 116-152, wherein the gel
droplet is encapsulated within a fluidic droplet.
154. The composition of claim 153, wherein the fluidic droplets
contain the particles.
155. The composition of claim 154, wherein the particles are
contained within the fluidic droplets at no more than about 1
particle/droplet.
156. The composition of any one of claim 154 or 155, wherein the
particles are contained within the fluidic droplets at no more than
about 0.1 particle/droplet.
157. The composition of any one of claims 154-156, wherein the
particles are contained within the fluidic droplets at no more than
about 0.01 particle/droplet.
158. The composition of any one of claims 154-157, wherein at least
about 90% of the fluidic droplets contains one particle or no
particle.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/324,407, filed Apr. 19, 2016,
entitled "Immobilization-Based Systems and Methods for Genetic
Analysis and Other Applications," by Weitz, et al., incorporated
herein by reference in its entirety.
FIELD
[0002] The present invention generally relates to microfluidics and
labeled nucleic acids.
BACKGROUND
[0003] Single-cell transcriptome and genome analysis have been
explored intensively and boosted understanding of various
biological processes. This may revolutionize human health care,
such as significantly improved quality or reduced medical errors.
However, transcriptomes and genomes are located in different
intracellular spaces and have their own unique properties, so
different analysis procedures are usually required when profiling
them. Thus, simultaneous profiling of both in a single cell still
remains challenging. Such a tool would provide a more comprehensive
vision of the cell heterogeneity and may lead to more effective
individual therapy.
SUMMARY
[0004] The present invention generally relates to microfluidics and
labeled nucleic acids. The subject matter of the present invention
involves, in some cases, interrelated products, alternative
solutions to a particular problem, and/or a plurality of different
uses of one or more systems and/or articles.
[0005] The invention, according to a first aspect, is generally
directed to an article. In one set of embodiments, the article
comprises a gel droplet comprising a lysed cell, DNA attached to a
first set of oligonucleotides, and RNA attached to a second set of
oligonucleotides.
[0006] According to another aspect, the present invention is
generally directed to a method. In one set of embodiments, the
method includes acts of lysing cells within gel droplets to release
DNA and RNA from the cells, attaching the DNA to a first set of
oligonucleotides, and attaching the RNA to a second set of
oligonucleotides.
[0007] In another set of embodiments, the method includes acts of
lysing a cell within a gel droplet to release RNA from the cell,
forming cDNA using the RNA, and attaching the cDNA to an
oligonucleotide within the gel droplet.
[0008] In another aspect, the present invention encompasses methods
of making one or more of the embodiments described herein. In still
another aspect, the present invention encompasses methods of using
one or more of the embodiments described herein.
[0009] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control. If two or more documents incorporated
by reference include conflicting and/or inconsistent disclosure
with respect to each other, then the document having the later
effective date shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0011] FIG. 1 illustrates an embodiment of the invention generally
directed to targeting nucleic acids;
[0012] FIG. 2 illustrates another embodiment of the invention
generally directed to targeting nucleic acids;
[0013] FIG. 3 illustrates yet another embodiment of the invention
generally directed to targeting nucleic acids;
[0014] FIG. 4 illustrates still another embodiment of the invention
comprising adapter sequences;
[0015] FIG. 5 illustrate an example of labeled nucleic acids, in
one set of embodiments;
[0016] FIG. 6 illustrates a moiety containing a photocleavable
spacer or linker, in some embodiments of the invention. The
sequence is given by SEQ ID NO: 13;
[0017] FIG. 7 shows production of labeled nucleic acids, in yet
another embodiment; and
[0018] FIG. 8 illustrates an experiment for analyzing encapsulated
cells, in still another embodiment of the invention.
DETAILED DESCRIPTION
[0019] The present invention generally relates to microfluidics and
labeled nucleic acids. Certain aspects are generally directed to
containing cells in gels, such as agarose gels, and determining
nucleic acids within the cells, e.g., while contained within the
gels. The nucleic acids may be, for example, genomic DNA, mRNA,
transcriptomes, or the like. In some embodiments, for instance,
both genomic DNA and RNA (e.g., as in a transcriptome) from a cell
may be determined. In some cases, the nucleic acids may be attached
to beads for sequencing or other purposes. Such systems may be
useful, for example, for high-throughput sequencing or other
applications.
[0020] One aspect of the invention is generally directed to systems
and methods for encapsulating a cell in a gel, and lysing the cell
to access its nucleic acids. In some cases, the nucleic acids
released by the cell may be tagged or attached to oligonucleotides
for subsequent determination or analyses, e.g., using various
sequencing techniques. The nucleic acids to be determined may
include, for example, genomic DNA (gDNA) or mRNA, or in some cases,
both from a single cell may be determined. In some cases, a gel may
be prepared by preparing a microfluidic droplet containing a cell
(or more than one cell), and causing the droplet to form a gel that
encapsulates the cell. The cell may be lysed within the gel to
release nucleic acids; however, the presence of the gel may prevent
or at least reduce the ability of the nucleic acids released by the
lysed cell to exit the gel, and thereby remain contained within the
gel. Other compounds may be used to interact with the nucleic acids
contained within the gel, e.g., for various purposes. For example,
various oligonucleotides may be attached to at least some of the
nucleic acids, e.g., for subsequent analysis.
[0021] One non-limiting example of such a system may be seen in
FIG. 1. In this figure, a plurality of cells is contained within a
plurality of droplets. In some cases, the cells may be encapsulated
at a density of less than 1 cell/droplet (and in some cases, much
less than 1 cell/droplet, e.g., as discussed herein) to ensure that
most or all of the droplets have only zero or one cell present in
them. The droplets also contain components that can be used to form
a gel, e.g., agarose in a liquid state, where the agarose can be
caused to from a gel via cooling the droplet to below the gelling
or solidification temperature of the gel. Other gels are discussed
in further detail below. Those of ordinary skill in the art will be
aware of various techniques for encapsulating cells within
microfluidic droplets; see, for example, U.S. Pat. Nos. 7,708,949,
8,337,778, 8,765,485, or Int. Pat. Apl. Pub. Nos. WO 2004/091763
and WO 2006/096571, each incorporated herein by reference.
[0022] After encapsulation of the cell within a droplet, the
droplet may be solidified to form a gel (or a gel "droplet") The
cells may then be lysed, e.g., upon exposure to a suitable cell
lysis reagent, such as proteinase K (or using other techniques such
as those discussed herein). Due to the presence of the gel, any
nucleic acids released by the cell may stay contained within the
gel. In some embodiments, both genomic DNA and RNA released by a
cell may be determined. The techniques for capturing and labeling
the genomic DNA and the RNA may be different in some cases, e.g.,
to facilitate identification of the DNA and RNA. The RNA may be,
for example, mRNA, siRNA, miRNA, or the like, e.g., as discussed in
more detail below. In some cases, the transcriptome of a cell may
be determined, e.g., by suitably determining sufficient mRNAs to
determine the transcriptome.
[0023] In some embodiments, however, both may be labeled with
oligonucleotides containing unique "barcodes" (which may be
randomly determined, or determined as discussed herein), which may
allow in some cases for hundreds, thousands, tens of thousands, or
even hundreds of thousands or more of different cells to be
barcoded or otherwise labeled in a single experiment. Thus, the
nucleic acids from one gel droplet (or from one cell) can still be
distinguished from those from other droplets (or other cells) using
the oligonucleotide tags. Accordingly, subsequent analysis (e.g.,
sequencing) of the combined pool of nucleic acids may be performed,
and the source of each nucleic acid (e.g., individual cells) may be
determined be determining the different oligonucleotides that are
present. The oligonucleotides may be present within a droplet prior
to formation of a gel from the droplet, or the oligonucleotides may
be added to the gel droplet after it has been formed. The
oligonucleotides may be added before or after cell lysis.
[0024] In some instances, RNA that is released by a cell may be
determined. In one set of embodiments, the RNA may be ligated or
enzymatically attached to oligonucleotides (such as in a barcode,
as mentioned above), e.g., that are present within the gel. Other
examples of attaching or joining nucleic acids together are
discussed in more detail below. In another set of embodiments,
however, the RNA is first converted (i.e., used to produce) to
cDNA, then the cDNA is ligated or enzymatically attached to
oligonucleotides.
[0025] Non-limiting examples of ligases include DNA ligases such as
DNA Ligase I, DNA Ligase II, DNA Ligase III, DNA Ligase IV, T4 DNA
ligase, T7 DNA ligase, T3 DNA Ligase, E. coli DNA Ligase, Taq DNA
Ligase, or the like. Many such ligases may be purchased
commercially. In addition, in some embodiments, two or more nucleic
acids may be ligated together using annealing or a primer extension
method. In addition, in some cases, the nucleic acid may be added
internally of an oligonucleotide, e.g., using transposons or the
like. See, e.g., U.S. Pat. Apl. Ser. No. 62/072,950, incorporated
herein by reference in its entirety.
[0026] In some cases, all of the RNAs (for example, all of the
mRNAs) may be converted into cDNAs (e.g., for the study of the
transcriptome), although in other cases, only certain RNAs may be
converted into cDNA. The cDNA may be tagged with suitable
oligonucleotides, depending on the application.
[0027] A variety of techniques may be used to from cDNA, for
example, exposure to reverse transcriptase. For instance, specific
RNAs (or other nucleic acids) suspected of being within a cell may
be targeted using capture oligonucleotides, e.g., containing
sequences that can recognize specific RNA's. In such a way, only a
portion of the RNA that is released may be converted into cDNA.
However, in other embodiments, reverse transcriptase may be applied
nonspecifically to the RNA, thereby resulting in the production of
cDNA corresponding to all of the RNA. In addition, in some cases,
DNA polymerase may be used to convert single-stranded cDNA (e.g.,
produced by the reverse transcriptase) into double-stranded
cDNA.
[0028] As a non-limiting example, as is shown in FIG. 1, a linear
polyacrylamide containing an oligonucleotide sequence able to
recognize an RNA sequence (or a portion of an RNA sequence) may be
allowed to interact or associate with the RNA, and used to form a
corresponding cDNA sequence, e.g., via a reverse transcriptase
reaction. Linear polyacrylamide can be used to precipitate nucleic
acids, e.g., of the cDNA sequence. In such a fashion, only RNA that
is able to interact with the linear polyacrylamide may be
determined, e.g., through formation of cDNA and subsequent
attachment of a suitable oligonucleotide.
[0029] Thus, in one set of embodiments, RNAs (e.g., released by a
cell) may be converted into DNA (e.g., cDNA) using reverse
transcriptase. The DNA may then be attached to an adapter sequence.
Examples of reverse transcriptases include, but are not limited to
AMV, M-MLV, telomerase reverse transcriptase, or the like. Many
such reverse transcriptases may be readily obtained commercially,
e.g., Superscript, Superscript II, or Superscript III (Life
Technologies).
[0030] FIG. 2 depicts another example, in which a reverse
transcriptase (e.g., Superscript II) that produces an overhang
region comprising three non-templated (or unpaired) A bases on the
end of the cDNA is used. The overhang region (e.g., comprising
adenine in this example) may be matched to sequences that contain a
complementary overhang region (e.g., comprising thymine). The
overhanging region may contain any suitable number of nucleotides,
e.g., 1, 2, 3, 4, 5, etc. The non-templated or unpaired bases on
the end may be used advantageously in some embodiments to allow for
attachment of a suitable oligonucleotide sequence, e.g., via
ligation or other techniques. RNA released by the cell may be used
to produce cDNA using the reverse transcriptase enzyme, and the
presence of the overhang allows oligonucleotides containing a
complementary overhang to be ligated or attached to the cDNA. This
may be used, for example, to differentiate cDNA (which contains the
overhangs) from genomic DNA (which does not contain such overhangs,
and/or contains different overhangs) for the purposes of attaching
oligonucleotides. Accordingly, one set of oligonucleotides (e.g., a
barcode) may be attached to the cDNA while a different set of
oligonucleotides may be attached to the genomic DNA.
[0031] As yet another example, some RNAs are produced containing a
poly-A region (e.g., a poly-A tail). For instance, the reverse
transcriptase may also include non-templated or unpaired bases at
an end of the cDNA, e.g. during the reverse transcriptase process.
For example, some reverse transcriptases add 3 A bases to the 3'
end of the cDNA during the reverse transcriptase process. This can
be used advantageously in some embodiments by exposing the RNA to a
sequence containing a complementary poly-T region that can interact
with the poly-A region of the RNA, as is shown in FIG. 3. Other
sequences may be present in the sequence comprising the poly-T
region, for example, a "ligation" sequence that can be attached or
joined to a suitable oligonucleotide, e.g., by ligation or
enzymatic attachment.
[0032] A non-limiting example of the use of a ligation sequence is
shown with respect to FIG. 4. An oligonucleotide attached to a
particle is cleaved (e.g., photocleaved) to release the
oligonucleotide from the particle. A nucleic acid such as an mRNA
(e.g., having a poly-A tail) is joined to a ligation sequence
(e.g., arising from a gel). A portion of the oligonucleotide may
contain an adapter sequence that is able to interact or associate
with a complementary strand (CS) having a complementary region. For
instance, the oligonucleotide may contain a "universal sequence" or
an adapter sequence that can be used to attach or include any
additional desired sequence to the oligonucleotides (e.g., a
recognition sequence that can be used to recognize another entity,
for example, a complementary sequence). These may be present or
prepared in a bulk phase or within a droplet, such as a
microfluidic droplet.
[0033] The complementary strand may also contain a portion that is
complementary with the ligation sequence, e.g., attached to the
nucleic acid (for example, RNA). Thus, interaction of these,
following by ligation (e.g., with a suitable ligase) may result in
the joining of the nucleic acid to the oligonucleotide.
[0034] In addition, in some instances, DNA (e.g., genomic DNA or
gDNA) that is released by a cell may be determined, in addition to
or instead of RNA. In some cases, e.g., due to the length of
genomic nucleic acid, the genomic nucleic acid may be cut into
smaller fragments, e.g., using restriction endonucleases or other
enzymes able to cut a nucleic acid (e.g., MNase (micrococcal
nuclease) enzymes). Many such enzymes are commercially available;
one such example is NEBNext.RTM. dsDNA Fragmentase.RTM.. The
enzymes may cleave the DNA into smaller fragments specifically or
non-specifically, depending on the enzymes used. In some instances,
the DNA may be fragmented or digested mechanically, via sonication,
nebulization, exposure to shear forces, or by exposure to suitable
chemicals.
[0035] In one set of embodiments, the DNA may be subjected to
bisulfite treatment, e.g., to determine epigenetic information
about the DNA. Treatment of the DNA with bisulfite (or hydrogen
sulfite) converts cytosine residues to uracil, but leaves
5-methylcytosine residues unaffected. Thus, bisulfite treatment
introduces specific changes in the DNA sequence that depend on the
methylation status of individual cytosine residues, yielding
single-nucleotide resolution information about the methylation
status of the DNA, e.g., for epigenetic information.
[0036] In some cases, one or more of the nucleic acids, such as the
genomic DNA fragments, may be attached to an adapter sequence that
can be attached to oligonucleotides such as those discussed herein.
The adapter sequence can be attached to the nucleic acid using
ligases or other techniques such as those described herein. For
example, in some cases, the adapter sequence may contain an
overhang sequence, e.g., one that is complementary to restriction
endonucleases or other enzymes used to cleave or fragment the
genomic DNA. The adapter sequence may also be one that can be
attached to the oligonucleotides, e.g., as discussed herein. For
instance, the adapter sequence may contain poly-A regions or other
overhang sequences such as those described above, that can be used
for attachment to the oligonucleotides.
[0037] In some cases, the adapter sequence may also contain one or
more unique regions, e.g., barcodes such as those described herein.
These may be useful, for example, to allow nucleic acids arising
from genomic DNA to be differentiated from nucleic acids arising
from RNA such as mRNA from the cell.
[0038] One non-limiting example of an adapter sequence can be seen
in FIG. 7. This figure shows a particle (e.g., a hydrogel
particle), one or more barcodes, and a "universal sequence" or an
adapter sequence that can be used to attach or include any
additional desired sequence to the oligonucleotides (e.g., a
recognition sequence that can be used to recognize another entity,
for example, a complementary sequence). These may be present or
prepared in a bulk phase or within a droplet, such as a
microfluidic droplet. In addition, other elements, such as
promoters or enhancers may be present within the oligonucleotide as
well, e.g., as shown in FIG. 7.
[0039] The adaptor sequence may, in some embodiments, be exposed to
a complementary sequence comprising a sequence that is
complementary to the adapter. Other sequences, e.g., a primer, a
promoter, etc. may also be present. Examples of primers, promoters,
etc., are discussed herein. The complementary sequence may be able
to bind to or otherwise associate with at least a portion of the
adapter sequence, such as is shown in FIG. 7. The complementary
sequence may be fully complementary or contain one, two, three, or
more mismatches, relative to the adapter sequence. The adapter
sequence (and its complement) may be of any suitable length, e.g.,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more
nucleotides.
[0040] For instance, is as shown in FIG. 7, the complementary
sequence may include a primer such as a gene-specific inner forward
primer or a gene-specific reverse primer sequence, or other
sequences as discussed herein. These may be useful, for example, to
promote subsequent amplification or incorporation of a nucleic acid
to the oligonucleotide, e.g., attached to the particle. In FIG. 7,
this is shown as the "template" strand. Thus, the template strand
may contain, for instance, nucleic acids arising from RNA (e.g.,
mRNA) or DNA (e.g., genomic DNA) from a lysed cell.
[0041] The primer may be one that is able to interact with the
template, e.g., specifically (such as with a gene-specific inner
forward primer) or nonspecifically. Subsequent amplification or
incorporation, as is shown in FIG. 7, may be used to incorporate
the sequence of the template (or at least a portion thereof) into
the oligonucleotide attached to the particle, thereby producing
particles containing oligonucleotides containing one or more
barcode sequences and a sequence corresponding to at least a
portion of the template.
[0042] Thus, the nucleic acids that are produced (e.g., RNA, cDNA,
gDNA, etc.) may be attached to one or more oligonucleotides. This
may be performed within the gels themselves in some embodiments. In
some cases, the gels may be contained within fluidic droplets and
oligonucleotides present within the fluidic droplets may be allowed
to diffuse into the gel, e.g., for attachment to the various
nucleic acids, e.g., as described above. In some instances, the
gels may be first melted to release the nucleic acids (e.g., into
the surrounding fluidic droplet) for attachment to the
oligonucleotides.
[0043] After being brought together, the nucleic acids may be
bonded to the oligonucleotides, e.g., covalently, through primer
extension, through ligation, or the like. Any of a wide variety of
different techniques may be used, and those of ordinary skill in
the art will be aware of many such techniques. The exact joining or
attachment technique used is not necessarily critical, and can vary
between embodiments. Non-limiting examples include ligases or other
suitable techniques such as those discussed in U.S. Pat. Apl. Ser.
No. 61/981,123, incorporated herein by reference. In some
embodiments, the nucleic acids may be joined or attached with the
oligonucleotide tags and/or amplified using PCR (polymerase chain
reaction) or other amplification techniques. Typically, in PCR
reactions, the nucleic acids are heated to cause dissociation of
the nucleic acids into single strands, and a heat-stable DNA
polymerase (such as Taq polymerase) is used to amplify the nucleic
acid. This process is often repeated multiple times to amplify the
nucleic acids.
[0044] In some embodiments, the oligonucleotides are introduced by
initially joining or attaching the oligonucleotides to a particle
(e.g., a hydrogel or a polymeric particle), then subsequently
releasing the oligonucleotides from the particle after the particle
has been incorporated into a droplet or into the gel. See, e.g.,
U.S. Pat. Apl. Ser. No. 62/072,944, filed Oct. 30, 2014 or a PCT
application filed on Apr. 17, 2015, entitled "Systems and Methods
for Barcoding Nucleic Acids," each incorporated herein by
reference. For example, in certain embodiments, the
oligonucleotides may also contain a cleavable sequence or linker
(e.g., as is shown in FIG. 6), or otherwise be releasable from the
particles.
[0045] The particles may be prepared in some cases such that most
or all of the particles have a uniquely distinguishable
oligonucleotide, relative to other particles having other
distinguishable oligonucleotides). If the particles are present
within the droplets at a density of 1 particle/gel droplet (or
less), then once the oligonucleotides are released from the
particle, then most or all of the gel droplets will contain one
unique oligonucleotide (or no unique oligonucleotide), thus
allowing each gel droplet (and the nucleic acids contained therein)
to be uniquely identified.
[0046] Thus, in some embodiments, the particles may be encapsulated
in droplets, such as microfluidic droplets. Those of ordinary skill
in the art will be aware of techniques for encapsulating particles
within microfluidic droplets; see, for example, U.S. Pat. Nos.
7,708,949, 8,337,778, 8,765,485, or Int. Pat. Apl. Pub. Nos. WO
2004/091763 and WO 2006/096571, each incorporated herein by
reference. In some cases, the particles may be encapsulated at a
density of less than 1 particle/droplet (and in some cases, much
less than 1 particle/droplet) to ensure that most or all of the
droplets have only zero or one particle present in them.
[0047] The particles containing oligonucleotides (which may be
attached to the surface of the particles, or otherwise contained or
incorporated within the particles, etc.) may be used, in some
embodiments, to determine or sequence nucleic acids arising from
cells (or other samples), or for other applications. For instance,
in the non-limiting example of FIG. 5, a population of cells 10 is
desired to be analyzed, e.g., by sequencing their DNA, by
identifying certain proteins or genes that may be suspected of
being present in at least some of the cells, by determining their
mRNA or transcriptome, or the like. Although cells are used in this
example as a source of nucleic acid material, this is by way of
example, and in other embodiments, the nucleic acid may be
introduced into the gels from other sources, or using other
techniques.
[0048] The cells may first be encapsulated within the microfluidic
droplets 40, e.g., using techniques known to those of ordinary
skill in the art. The droplets may be used to from gels
encapsulating the cells, as previously discussed. In some cases,
the cells may be encapsulated at a density of less than 1
cell/droplet (and in some cases, much less than 1 cell/droplet) to
ensure that most or all of the gel droplets have only zero or one
cell present in them. Thus, as is shown in FIG. 1B, each of gel
droplets 41, 42, 43 . . . have either zero or one cell present in
them.
[0049] Also encapsulated in the gel droplets are oligonucleotide
20, present on particles 30. As noted above, particles 30 may be,
for example, microparticles, and may be a hydrogel or a polymeric
particle, or other types of particles such as those described
herein. The particles and the cells may be encapsulated within the
gels simultaneously or sequentially, in any suitable order. In one
set of embodiments, each particle contains a unique
oligonucleotide, although there may be multiple copies of the
oligonucleotide present on a particle. For instance, each of the
oligonucleotides may have one or more barcodes. Thus, for example,
particle 31 contains only copies of oligonucleotide 21, particle 32
contains only copies of oligonucleotide 22, particle 33 contains
only copies of oligonucleotide 33, etc.
[0050] It should be noted that according to certain embodiments of
the invention, the oligonucleotide are initially attached to
particles to facilitate the introduction of only one unique
oligonucleotide to each gel, as is shown in FIG. 5. (In other
embodiments, however, a plurality of oligonucleotides and/or
particles may be present in a gel, e.g., containing the same unique
barcode.) For example, if the particles are present in the gel at a
density of less than 1 particle/gel, then most or all of the gels
will each have only a single particle, and thus only a single type
of oligonucleotide, that is present. Accordingly, as is shown in
FIG. 5, the oligonucleotide may be cleaved or otherwise released
from the particles, e.g., such that each gel 41, 42, 43, . . .
contains a unique oligonucleotide 21, 22, 23, . . . that is
different than the other oligonucleotide that may be present in the
other gels. Thus, each oligonucleotide present within a gel will be
distinguishable from the oligonucleotides that are present in the
other gels. Although light (hv) is used in FIG. 5 to cleave the
oligonucleotides from the particles, it should be understood that
this is by way of example only, and that other methods of cleavage
or release can also be used, e.g., as discussed herein. For
example, in one set of embodiments, agarose particles containing
oligonucleotides (e.g., physically) may be used, and the
oligonucleotides may be released by heating the agarose, e.g.,
until the agarose at least partially liquefies or softens.
[0051] In some cases, the cells are lysed to release nucleic acid
or other materials 51, 52, 53, . . . from the cells, e.g., into the
gels. For example, the cells may be lysed using chemicals or
ultrasound. The cells may release, for instance, DNA, RNA, mRNA,
proteins, enzymes or the like. In some cases, the nucleic acids
that are released may optionally undergo amplification, for
example, by including suitable reagents specific to the
amplification method. Examples of amplification methods known to
those of ordinary skill in the art include, but are not limited to,
polymerase chain reaction (PCR), reverse transcriptase (RT) PCR
amplification, in vitro transcription amplification (IVT), multiple
displacement amplification (MDA), or quantitative real-time PCR
(qPCR).
[0052] Some or all of the nucleic acid or other material 51, 52,
53, . . . may be associated with the oligonucleotides present in
the gels, e.g., by covalently bonding. For example, the nucleic
acid or other material 51, 52, 53 may be ligated or enzymatically
attached to the oligonucleotides present in the gels. Thus, as is
shown in FIG. 1B, gel 41 exhibits nucleic acids 51 attached to
oligonucleotides 21, gel 42 exhibits nucleic acids 52 attached to
oligonucleotides 22, gel 43 exhibits nucleic acids 53 attached to
oligonucleotides 23, etc. Thus, the nucleic acids within each gel
are distinguishable from the nucleic acids within the other gels of
the plurality of gels 50 by way of the oligonucleotides, which are
unique to each gel in this example.
[0053] It should also be understood that although FIG. 5 depicts
cleavage of the oligonucleotides from the particles followed by
lysis of the cells, in other embodiments, these need not
necessarily occur in this order. For example, cell lysis may occur
after cleavage, or both may occur simultaneously.
[0054] In some embodiments, gels 41, 42, 43, . . . may then be
liquefied, or otherwise burst or broken to release their contents,
and in some cases, the nucleic acids present in each gel may be
combined or pooled together, as is shown in FIG. 5. However, since
the nucleic acids are labeled by the different oligonucleotides,
the nucleic acids from one gel (i.e., from one cell) can still be
distinguished from those from other gels (or other cells) using the
oligonucleotides (e.g., by determining barcodes on the
oligonucleotides). Accordingly, subsequent analysis (e.g.,
sequencing) of the combined pool of nucleic acids may be performed,
and the source of each nucleic acid (e.g., individual cells) may be
determined be determining the different oligonucleotides.
[0055] Thus, for example, a population of normal cells and cancer
cells (e.g., arising from a tissue sample or biopsy) may be
analyzed in such a fashion, and the cancer cells may be identified
as having abnormal DNA, even if present in a large pool of normal
cells. For example, due to the ability to track DNA on a cellular
level using the oligonucleotides, the abnormal DNA can still be
identified even if outnumbered by a large volume of normal DNA. As
other non-limiting examples, stem cells may be isolated from normal
cells, or the isolation of rare cell types in a population of
interest may be performed.
[0056] The above discussion is not intended to be limiting; other
embodiments of the invention are also possible for labeling nucleic
acids, e.g., arising from cells. For instance, in certain aspects,
the present invention is generally directed to systems and methods
for encapsulating cells within a gel. Thus, in some cases, cells
may be contained within gels, for example, to protect the cells
during manipulation of the cells. For example, one or more cells
may be contained within gel particles, which can then be exposed to
various conditions, nucleic acids or oligonucleotides, etc., as
discussed herein.
[0057] In some embodiments, additional agents may also be added to
the gels. For example, magnetic particles may be added to some or
all of the gels, e.g., to facilitate later sorting. As another
example, fluorescent agents may be added to some or all of the
gels, e.g., to facilitate identification of certain cells (or other
species to be determined) that are contained within the gels. As a
non-limiting example, in one set of embodiments, one or more cells
may be contained within a gel particle, which may optionally be
contained within a droplet.
[0058] In one set of embodiments, the gel is an agarose gel.
However, other gels are also possible in other embodiments. Other
examples of gels encapsulating cells can be seen in Int. Pat. Apl.
Pub. No. WO 2008/109176, incorporated herein by reference. Further
examples of hydrogels include, but are not limited to
acrylamide-based gels, such as polyacrylamide,
poly-N-isopropylacrylamide, or poly N-isopropylpolyacrylamide. For
example, an aqueous solution of a monomer may be dispersed in a
droplet, and then polymerized, e.g., to form a gel. Another example
is a hydrogel, such as alginic acid that can be gelled by the
addition of calcium ions. In some cases, gelation initiators
(ammonium persulfate and TEMED for acrylamide, or Ca.sup.2+ for
alginate) can be added to a droplet, for example, by co-flow with
the aqueous phase, by co-flow through the oil phase, or by
coalescence of two different drops, e.g., as discussed in U.S.
patent application Ser. No. 11/360,845, filed Feb. 23, 2006,
entitled "Electronic Control of Fluidic Species," by Link, et al.,
published as U.S. Patent Application Publication No. 2007/000342 on
Jan. 4, 2007; or in U.S. patent application Ser. No. 11/698,298,
filed Jan. 24, 2007, entitled "Fluidic Droplet Coalescence," by
Ahn, et al.; each incorporated herein by reference in their
entireties.
[0059] In some embodiments, the gel may be one that is low-melt,
e.g., the gel may melt at temperatures below about 100.degree. C.,
below about 80.degree. C., below about 70.degree. C., or below
about 60.degree. C. in some cases. In some cases, the gel may be
chosen to be able to solidify upon exposure to relatively low
temperatures, e.g., below about 60.degree. C., below about
50.degree. C., below about 40.degree. C., below about 35.degree.
C., below 30.degree. C., below about 25.degree. C., or below about
20.degree. C. For example, low-melt agarose gels may be readily
obtained commercially.
[0060] In some embodiments, as non-limiting examples, the average
diameter of the gel may be less than about 1 mm, less than about
500 micrometers, less than about 300 micrometers, less than about
200 micrometers, less than about 100 micrometers, less than about
75 micrometers, less than about 50 micrometers, less than about 30
micrometers, less than about 25 micrometers, less than about 20
micrometers, less than about 15 micrometers, less than about 10
micrometers, less than about 5 micrometers, less than about 3
micrometers, less than about 2 micrometers, less than about 1
micrometer, less than about 500 nm, less than about 300 nm, less
than about 100 nm, or less than about 50 nm. The average diameter
of the gel may also be at least about 30 nm, at least about 50 nm,
at least about 100 nm, at least about 300 nm, at least about 500
nm, at least about 1 micrometer, at least about 2 micrometers, at
least about 3 micrometers, at least about 5 micrometers, at least
about 10 micrometers, at least about 15 micrometers, or at least
about 20 micrometers in certain cases. Combinations of these are
also possible, e.g., the gel may have an average diameter of
between about 25 micrometers and about 100 micrometers. In some
embodiments, a gel may be prepared by containing a cell (or other
suitable species) within a microfluidic droplet with a gel
precursor. For example, the gel may contain agarose which can form
a gel upon exposure to a suitable temperature, or some gels may be
prepared by containing a precursor such as disclosed herein and
exposing the precursor to a suitable gelation initiator.
[0061] If cells are used, the cells may arise from any suitable
source. For instance, the cells may be any cells for which nucleic
acid from the cells is desired to be studied or sequenced, etc.,
and may include one, or more than one, cell type. The cells may be
for example, from a specific population of cells, such as from a
certain organ or tissue (e.g., cardiac cells, immune cells, muscle
cells, cancer cells, etc.), cells from a specific individual or
species (e.g., human cells, mouse cells, bacteria, etc.), cells
from different organisms, cells from a naturally-occurring sample
(e.g., pond water, soil, etc.), or the like. In some cases, the
cells may be dissociated from tissue.
[0062] In some cases, a gel encapsulating a cell (or more than one
cell) may be prepared by preparing a droplet, such as a
microfluidic droplet, containing the cell, then causing the droplet
to solidify or harden into a gel. For example, agarose gel may be
hardened by cooling to a suitable temperature.
[0063] The gel (or droplet) may have a volume of, for example less
than about 1 ml, less than about 300 microliters, less than about
100 microliters, less than about 30 microliters, less than about 10
microliters, less than about 3 microliters, less than about 1
microliter, less than about 500 nl, less than about 300 nl, less
than about 100 nl, less than about 50 nl, less than about 30 nl, or
less than about 10 nl.
[0064] In one set of embodiments, the gel or droplet may, on the
average, contain less than one cell in it. For example, the average
loading rate may be less than about 1 particle/gel (or droplet),
less than about 0.9 cell/gel, less than about 0.8 cell/gel, less
than about 0.7 cell/gel, less than about 0.6 cell/gel, less than
about 0.5 cell/gel, less than about 0.4 cell/gel, less than about
0.3 cell/gel, less than about 0.2 cell/gel, less than about 0.1
cell/gel, less than about 0.05 cell/gel, less than about 0.03
cell/gel, less than about 0.02 cell/gel, or less than about 0.01
cell/gel. As discussed, droplets may be produced containing cells,
then converted into gel droplets containing the cells; thus, the
average loading rate for gels and droplets may be substantially the
same. In some cases, lower loading rates may be chosen to minimize
the probability that a gel (or droplet) will be produced having two
or more cells in it. Thus, for example, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95%, at least about 97%, at least about
98%, or at least about 99% of the gels (or droplets) may contain
either no cell or only one cell.
[0065] In some cases, a relatively large number of gels or droplets
may be created, e.g., at least about 10, at least about 30, at
least about 50, at least about 100, at least about 300, at least
about 500, at least about 1,000, at least about 3,000, at least
about 5,000, at least about 10,000, at least about 30,000, at least
about 50,000, at least about 100,000 gels or droplets, etc.
[0066] In some embodiments, cells may be lysed within the gels. The
cell may release nucleic acids upon lysis, for example, DNA or RNA.
The DNA may be genomic DNA in some cases. The RNA may be, for
instance, mRNA, siRNA, miRNA, tRNA, rRNA, or the like. In one set
of embodiments, the cells may be lysed within the droplets, e.g.,
to release DNA and/or RNA from the cell, and/or to produce a cell
lysate. For instance, the cells may be lysed via exposure to a
lysing chemical or a cell lysis reagent (e.g., a surfactant such as
Triton-X or SDS, an enzyme such as lysozyme, lysostaphin, zymolase,
cellulase, mutanolysin, glycanases, proteases, mannase, proteinase
K, etc.), or a physical condition (e.g., ultrasound, ultraviolet
light, mechanical agitation, etc.). If a lysing chemical is used,
the lysing chemical may be introduced into the gel before or after
formation of the gel, e.g., through picoinjection or other methods
such as those discussed in U.S. patent application Ser. No.
13/379,782, filed Dec. 21, 2011, entitled "Fluid Injection,"
published as U.S. Pat. Apl. Pub. No. 2012/0132288 on May 31, 2012,
incorporated herein by reference in its entirety, through fusion of
a droplet with a droplet containing the chemical or enzyme, or
through other techniques known to those of ordinary skill in the
art. In some cases, lysing a cell will cause the cell to release
its contents, e.g., cellular nucleic acids, proteins, enzymes,
sugars, etc.
[0067] As discussed above, certain aspects of the invention are
generally directed to systems and methods for attaching nucleic
acids (e.g., released from cells, or otherwise present within a gel
or a droplet) to oligonucleotides, such as those described herein.
The oligonucleotides may be attached to a particle in some
embodiments, although they need not be in other embodiments. In
some cases, the gel and the oligonucleotides may be contained
within a fluidic droplet. As previously discussed, in some cases,
there may be a plurality of droplets containing various
oligonucleotides such that the oligonucleotides of one droplet are
distinguishable from oligonucleotides in other droplets, e.g., due
to the presence of one or more barcodes. In some embodiments, after
attaching the nucleic acids to the oligonucleotides, they may be
sequenced or otherwise analyzed. In some cases, they may also be
combined or merged together, e.g., by bursting or otherwise
disrupting the droplets, then sequenced or otherwise analyzed
together.
[0068] For instance, as discussed herein, nucleic acids arising
from a cell (e.g., within a gel droplet) may be attached to an
oligonucleotide, e.g., for subsequent sequencing or analysis. As
discussed below, the oligonucleotide may be attached to or
otherwise incorporated or contained within a particle. In one set
of embodiments, the nucleic acid may be added to the
oligonucleotide using a ligase or other suitable enzyme that can
directly attach the nucleic acid to the oligonucleotides, e.g., to
a free end of the oligonucleotide. See, e.g., U.S. Pat. Apl. Ser.
No. 62/072,944, filed Oct. 30, 2014 or a PCT application filed on
Apr. 17, 2015, entitled "Systems and Methods for Barcoding Nucleic
Acids," each incorporated herein by reference.
[0069] In some embodiments, the nucleic acid and the
oligonucleotide may have straight or "sticky" ends, e.g.,
containing overhangs of unpaired nucleotides that may be
complementary. In some cases, restriction enzymes may be used to
prepare the ends of the nucleic acids prior to joining.
[0070] Thus, for example, the nucleic acid may contain a portion of
unpaired nucleotides, and the oligonucleotide may contain a
complementary portion of unpaired nucleotides. As a non-limiting
example, the overhang may be an A and the complement may be a T.
The overhanging region may contain any suitable number of
nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. The overhang
region may contain only a single nucleotide (e.g., A, AA, AAA,
etc.) or a random or any other sequence of suitable nucleotides. In
some cases, the overhang may be created using a suitable enzyme,
e.g., a restriction endonuclease or a reverse transcriptase.
[0071] For instance, in one non-limiting embodiment, the 3' end of
a barcoded primer is terminated with a poly-T sequences that may be
used to capture cellular mRNA for whole-transcriptome profiling.
The resulting library combining all cells can optionally be
enriched using PCR-based methods or using hybridization
capture-based methods (such as Agilent SureSelect), e.g., to allow
sequencing of only a sub-set of genes of interest. In another
embodiment, the 3' end of the barcoded primers may terminate with a
random DNA sequence that can be used to capture the RNA in the
cell. In another embodiment, the 3' end of the barcoded primers may
terminate with a specific DNA sequence, e.g., that can be used to
capture DNA or RNA species ("genes") of interest, or to hybridize
to a DNA probe that is delivered into the droplets in addition to
the particles or microspheres, for example, together with the
enzyme reagents. In another embodiment, a particle or microsphere
may carry a number of different primers to target several genes of
interest. Yet another embodiment is directed to optimization of the
size of droplets and the concentration of reaction components
required for droplet barcoding.
[0072] In another set of embodiments, a nucleic acid may be
attached to an oligonucleotide using a sequence that is
complementary to an adapter sequence on the oligonucleotide, where
the complementary sequence contains a primer that can be used to
amplify or incorporate a desired or arbitrary sequence (e.g., a
template) to the oligonucleotide.
[0073] For instance, in some embodiments, a template may be
introduced into a droplet and amplified within the droplet. If the
droplet contains oligonucleotides, then the amplification process
may also be used to attach the template to the oligonucleotide,
e.g., via a complementary sequence containing primers able to
recognize at least a portion of the template. For example, the
oligonucleotide may contain a "universal" or adapter sequence,
which is complementary to a complementary sequence containing a
portion complementary to the adapter sequence and a primer able to
recognize at least a portion of the template. Upon amplification
within the droplet, the oligonucleotide may thus be extended, e.g.,
to contain the template. Thus, upon amplification, the template
sequence may become incorporated into the oligonucleotide, e.g., as
is shown in FIG. 1, and the primer may be used to facilitate
amplification or joining of a template strand or other sequences to
the oligonucleotide. For example, this process may be facilitated
using primers such as gene-specific primers (forward or reverse)
within the complementary sequence.
[0074] The oligonucleotide to which the nucleic acid is attached to
may contain, for example, barcode sequences, recognition sequences,
cleavable linkages, random sequences, or other sequences such as
any of those discussed herein. For example, in one set of
embodiments, the nucleic acids may be attached to specific
oligonucleotides (e.g., barcodes") that can be used to distinguish
nucleic acids from one source (e.g., from a cell contained within a
droplet) from those from other sources (e.g., from other cells).
One or more than one barcode may be present on an
oligonucleotide.
[0075] In some embodiments, the oligonucleotides may comprise a
"barcode" or a unique sequence. The sequence may be selected such
that some or most of the oligonucleotides (e.g., present on a
particle and/or in a droplet) have the unique sequence (or
combination of sequences that is unique), but other
oligonucleotides (e.g., on other particles or droplets) do not have
the unique sequence or combination of sequences. Thus, for example,
the sequences may be used to uniquely identify or distinguish a
droplet, or nucleic acid contained arising from the droplet (e.g.,
from a lysed cell) from other droplets, or other nucleic acids
(e.g., released from other cells) arising from other droplets.
[0076] The sequences may be of any suitable length. The length of
the barcode sequence is not critical, and may be of any length
sufficient to distinguish the barcode sequence from other barcode
sequences. One, two, or more "barcode" sequence may be present in
an oligonucleotide, as discussed above. A barcode sequence may have
a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 nt. More than 25 nucleotides may also be
present in some cases.
[0077] In some cases, the unique or barcode sequences may be taken
from a "pool" of potential barcode sequences. If more than one
barcode sequence is present in an oligonucleotide, the barcode
sequences may be taken from the same, or different pools of
potential barcode sequences. The pool of sequences may be selected
using any suitable technique, e.g., randomly, or such that the
sequences allow for error detection and/or correction, for example,
by being separated by a certain distance (e.g., Hamming distance)
such that errors in reading of the barcode sequence can be
detected, and in some cases, corrected. The pool may have any
number of potential barcode sequences, e.g., at least 100, at least
300, at least 500, at least 1,000, at least 3,000, at least 5,000,
at least 10,000, at least 30,000, at least 50,000, at least
100,000, at least 300,000, at least 500,000, or at least 1,000,000
barcode sequences.
[0078] Thus, some embodiments of the present invention are
generally directed to barcoded nucleic acids attached to particles
or microspheres. For example, one set of embodiments is generally
directed to particles or microspheres carrying nucleic acid
fragments (each encoding a barcode, a primer, and/or other
sequences possibly used for capture, amplification and/or
sequencing of nucleic acids). Microspheres may refer to a hydrogel
particle (polyacrylamide, agarose, etc.), or a colloidal particle
(polystyrene, magnetic or polymer particle, etc.) of 1 to 500
micrometer in size, or other dimensions such as those described
herein. The microspheres may be porous in some embodiments. Other
suitable particles or microspheres that can be used are discussed
in more detail herein.
[0079] The preparation of particles or microspheres, in some cases,
may rely on the covalent attachment or other techniques of
incorporation of an initial DNA oligonucleotide to the particles or
microspheres, followed by enzymatic extension of each
oligonucleotide by one or more barcodes selected, e.g., at random,
from a pre-defined pool. The final number of possible unique
barcodes may depend in some cases on the size of the pre-defined
barcode pool and/or on the number of extension steps. For example,
using a pool of 384 pre-defined barcodes and 2 extension steps,
each particle or microsphere carries one of 384.sup.2=147,456
possible barcodes; using 3 extension steps, each particle or
microsphere carries one of 384.sup.3=56,623,104 possible barcodes;
and so on. Other numbers of steps may also be used in some cases;
in addition, each pool may have various numbers of pre-defined
barcodes (not just 384), and the pools may have the same or
different numbers of pre-defined barcodes. The pools may include
the same and/or different sequences.
[0080] Accordingly, in some embodiments, the possible barcodes that
are used are formed from one or more separate "pools" of barcode
elements that are then joined together to produce the final
barcode, e.g., using a split-and-pool approach. A pool may contain,
for example, at least about 300, at least about 500, at least about
1,000, at least about 3,000, at least about 5,000, or at least
about 10,000 distinguishable barcodes. For example, a first pool
may contain x.sub.1 elements and a second pool may contain x.sub.2
elements; forming a barcode containing an element from the first
pool and an element from the second pool may yield, e.g.,
x.sub.1x.sub.2 possible barcodes that could be used. It should be
noted that x.sub.1 and x.sub.2 may or may not be equal. This
process can be repeated any number of times; for example, the
barcode may include elements from a first pool, a second pool, and
a third pool (e.g., producing x.sub.1x.sub.2x.sub.3 possible
barcodes), or from a first pool, a second pool, a third pool, and a
fourth pool (e.g., producing x.sub.1x.sub.2x.sub.3x.sub.4 possible
barcodes), etc. There may also be 5, 6, 7, 8, or any other suitable
number of pools. Accordingly, due to the potential number of
combinations, even a relatively small number of barcode elements
can be used to produce a much larger number of distinguishable
barcodes.
[0081] In some cases, such use of multiple pools, in combination,
may be used to create substantially large numbers of useable
barcodes, without having to separately prepare and synthesize large
numbers of barcodes individually. For example, in many prior art
systems, requiring 100 or 1,000 barcodes would require the
individual synthesis of 100 or 1,000 barcodes. However, if larger
numbers of barcodes are needed, e.g., for larger numbers of cells
to be studied, then correspondingly larger numbers of barcodes
would need to be synthesized. Such systems become impractical and
unworkable at larger numbers, such as 10,000, 100,000, or 1,000,000
barcodes. However, by using separate "pools" of barcodes, larger
numbers of barcodes can be achieved without necessarily requiring
each barcode to be individually synthesized. As a non-limiting
example, a first pool of 1,000 distinguishable barcodes (or any
other suitable number) and a second pool of 1,000 distinguishable
barcodes can be synthesized, requiring the synthesis of 2,000
barcodes (or only 1,000 if the barcodes are re-used in each pool),
yet they may be combined to produce 1,000.times.1,000=1,000,000
distinguishable barcodes, e.g., where each distinguishable barcode
comprises a first barcode taken from the first pool and a second
barcode taken from the second pool. Using 3, 4, or more pools to
assemble the barcode may result in even larger numbers of barcodes
that may be prepared, without substantially increasing the total
number of distinguishable barcodes that would need to be
synthesized.
[0082] Thus, in some cases, some or all of the gels may be
distinguishable, e.g., on the basis of the oligonucleotides (e.g.,
which may comprise one or more unique sequences or barcodes). In
some cases, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, at
least about 97%, at least about 98%, or at least about 99% of the
gels may be distinguishable.
[0083] The oligonucleotide may be of any suitable length or
comprise any suitable number of nucleotides. The oligonucleotide
may comprise DNA, RNA, and/or other nucleic acids such as PNA,
and/or combinations of these and/or other nucleic acids. In some
cases, the oligonucleotide is single stranded, although it may be
double stranded in other cases. For example, the oligonucleotide
may have a length of at least about 10 nt, at least about 30 nt, at
least about 50 nt, at least about 100 nt, at least about 300 nt, at
least about 500 nt, at least about 1000 nt, at least about 3000 nt,
at least about 5000 nt, at least about 10,000 nt, etc. In some
cases, the oligonucleotide may have a length of no more than about
10,000 nt, no more than about 5000 nt, no more than about 3000 nt,
no more than about 1000 nt, no more than about 500 nt, no more than
about 300 nt, no more than about 100 nt, no more than about 50 nt,
etc. Combinations of any of these are also possible, e.g., the
oligonucleotide may be between about 10 nt and about 100 nt. The
length of the oligonucleotide is not critical, and a variety of
lengths may be used in various embodiments.
[0084] The oligonucleotide may also contain a variety of sequences.
For example, the oligonucleotide may contain one or more primer
sequences, one or more unique or "barcode" sequences as discussed
herein, one or more promoter sequences, one or more spacer
sequences, or the like. The oligonucleotide may also contain, in
some embodiments one or more cleavable spacers, e.g.,
photocleavable linker. The oligonucleotide may in some embodiments
be attached to a particle chemically (e.g., via a linker) or
physically (e.g., without necessarily requiring a linker), e.g.,
such that the oligonucleotides can be removed from the particle via
cleavage. Other examples include portions that may be used to
increase the bulk (or length) of the oligonucleotides (e.g., using
specific sequences or nonsense sequences), to facilitate handling
(for example, an oligonucleotide may include a poly-A tail), to
increase selectivity of binding (e.g., as discussed below), to
facilitate recognition by an enzyme (e.g., a suitable ligase), to
facilitate identification, or the like. Examples of these and/or
other sequences are described in further detail herein.
[0085] In one set of embodiments, the oligonucleotide tag may
comprise an antibody, e.g., that can specifically bind to a protein
suspected of being present in the cell (or droplet). For example,
the droplet may contain one or more antibodies tagged with an
oligonucleotide tag as described herein.
[0086] In some cases, the oligonucleotide may contain one or more
promoter sequences, e.g., to allow for production of the
oligonucleotide, to allow for enzymatic amplification, or the like.
Those of ordinary skill in the art will be aware of primer
sequences, e.g., P5 or P7. Many such primer sequences are available
commercially. Examples of promoters include, but are not limited
to, T7 promoters, T3 promoters, or SP6 promoters.
[0087] In some cases, the oligonucleotide may contain one or more
primer sequences. Typically, a primer is a single-stranded or
partially double-stranded nucleic acid (e.g., DNA) that serves as a
starting point for nucleic acid synthesis, allowing polymerase
enzymes such as nucleic acid polymerase to extend the primer and
replicate the complementary strand. A primer may be complementary
to and to hybridize to a target nucleic acid. In some embodiments,
a primer is a synthetic primer. In some embodiments, a primer is a
non-naturally-occurring primer. A primer typically has a length of
10 to 50 nucleotides. For example, a primer may have a length of 10
to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50,
20 to 40, or 20 to 30 nucleotides. In some embodiments, a primer
has a length of 18 to 24 nucleotides. Examples of primers include,
but are not limited to, P5 primer, P7 primer, PE1 primer, PE2
primer, A19 primer, or other primers discussed herein.
[0088] In some cases, the oligonucleotide may contain nonsense or
random sequences, e.g., to increase the mass or size of the
oligonucleotide. The random sequence can be of any suitable length,
and there may be one or more than one present. As non-limiting
examples, the random sequence may have a length of 10 to 40, 10 to
30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or
20 to 30 nucleotides.
[0089] Other sequences may also be present as well, e.g., primers
such as PE-1, or a gene-specific inner forward primer or a
gene-specific reverse primer sequence, or other sequences as
discussed herein. Additional examples of primers include, but are
not limited to, P5 primer, P7 primer, PE1 primer, PE2 primer, A19
primer, or other primers discussed herein. These may be useful, for
example, to promote amplification of the nucleic acid, or for other
applications. In addition, as mentioned, the oligonucleotides may
also contain a cleavable sequence or linker, or otherwise be
releasable from the particles.
[0090] In some cases, the oligonucleotide may comprise one or more
sequences able to specifically bind a gene or other entity. For
example, in one set of embodiments, the oligonucleotide may
comprise a sequence able to recognize mRNA, e.g., one containing a
poly-T sequence (e.g., having several T's in a row, e.g., 4, 5, 6,
7, 8, or more T's).
[0091] In one set of embodiments, the oligonucleotide may contain
one or more cleavable linkers, e.g., that can be cleaved upon
application of a suitable stimulus. For example, the cleavable
sequence may be a photocleavable linker that can be cleaved by
applying light or a suitable chemical or enzyme. A non-limiting
example of a photocleavable linker can be seen in FIG. 4. In some
cases, for example, a plurality of particles (containing
oligonucleotides on their surfaces) may be prepared and added to
droplets, e.g., such that, on average, each droplet contains one
particle, or less (or more) in some cases. After being added to the
droplet, the oligonucleotides may be cleaved from the particles,
e.g., using light or other suitable cleavage techniques, to allow
the oligonucleotides to become present in solution, i.e., within
the interior of the droplet. In such fashion, oligonucleotides can
be easily loaded into droplets by loading of the particles into the
droplets, then cleaved off to allow the oligonucleotides to be in
solution, e.g., to interact with nucleotides or other species, such
as is discussed herein.
[0092] A variety of techniques may be used for preparing
oligonucleotides such as those discussed herein. These may be
prepared in bulk and/or in one or more droplets, such as
microfluidic droplets. In some cases, the oligonucleotides may be
prepared in droplets, e.g., to ensure that the barcodes and/or
oligonucleotides within each droplet are unique. In addition, in
some embodiments, particles may be prepared containing
oligonucleotides with various barcodes in separate droplets, and
the particles may then be given or sold to a user who then adds the
nucleic acids to the oligonucleotides, e.g., as described
above.
[0093] In some cases, an oligonucleotide comprising DNA and/or
other nucleic acids may be attached to particles and delivered to
the droplets. In some cases, the oligonucleotides are attached to
particles to control their delivery into droplets, e.g., such that
a droplet will typically have at most one particle in it. In some
cases, upon delivery into a droplet, the oligonucleotide may be
removed from the particle, e.g., by cleavage, by degrading the
particle, etc. However, it should be understood that in other
embodiments, a droplet may contain 2, 3, or any other number of
particles, which may have oligonucleotides that are the same or
different.
[0094] The oligonucleotide may be attached to a particle, e.g., as
discussed herein. In some embodiments, a particle may comprise only
one oligonucleotide, although multiple copies of the
oligonucleotide may be present on the particle; other particles may
comprise different oligonucleotides that are distinguishable, e.g.,
using the barcode sequences described herein.
[0095] Any suitable method may be used to attach the
oligonucleotide to the particle. The exact method of attachment is
not critical, and may be, for instance, chemical or physical. For
example, the oligonucleotide may be covalently bonded to the
particle via a biotin-steptavidin linkage, an amino linkage, or an
acrylic phosphoramidite linkage. See, e.g., FIG. 6 for an example
of an acrylic phosphoramidite linkage. In another set of
embodiments, the oligonucleotide may be incorporated into the
particle, e.g., physically, where the oligonucleotide may be
released by altering the particle. Thus, in some cases, the
oligonucleotide need not have a cleavable linkage. For instance, in
one set of embodiments, an oligonucleotide may be incorporated into
particle, such as an agarose particle, upon formation of the
particle. Upon degradation of the particle (for example, by heating
the particle until it begins to soften, degrade, or liquefy), the
oligonucleotide may be released from the particle.
[0096] The particle is a microparticle in certain aspects of the
invention. The particle may be of any of a wide variety of types;
as discussed, the particle may be used to introduce a particular
oligonucleotide into a droplet, and any suitable particle to which
oligonucleotides can associate with (e.g., physically or
chemically) may be used. The exact form of the particle is not
critical. The particle may be spherical or non-spherical, and may
be formed of any suitable material. In some cases, a plurality of
particles is used, which have substantially the same composition
and/or substantially the same average diameter. The "average
diameter" of a plurality or series of particles is the arithmetic
average of the average diameters of each of the particles. Those of
ordinary skill in the art will be able to determine the average
diameter (or other characteristic dimension) of a plurality or
series of particles, for example, using laser light scattering,
microscopic examination, or other known techniques. The average
diameter of a single particle, in a non-spherical particle, is the
diameter of a perfect sphere having the same volume as the
non-spherical particle. The average diameter of a particle (and/or
of a plurality or series of particles) may be, for example, less
than about 1 mm, less than about 500 micrometers, less than about
200 micrometers, less than about 100 micrometers, less than about
75 micrometers, less than about 50 micrometers, less than about 25
micrometers, less than about 10 micrometers, or less than about 5
micrometers in some cases. The average diameter may also be at
least about 1 micrometer, at least about 2 micrometers, at least
about 3 micrometers, at least about 5 micrometers, at least about
10 micrometers, at least about 15 micrometers, or at least about 20
micrometers in certain cases.
[0097] The particle may be, in one set of embodiments, a hydrogel
particle. See, e.g., Int. Pat. Apl. Pub. No. WO 2008/109176,
entitled "Assay and other reactions involving droplets"
(incorporated herein by reference) for examples of hydrogel
particles, including hydrogel particles containing DNA. Examples of
hydrogels include, but are not limited to agarose or
acrylamide-based gels, such as polyacrylamide,
poly-N-isopropylacrylamide, or poly N-isopropylpolyacrylamide. For
example, an aqueous solution of a monomer may be dispersed in a
droplet, and then polymerized, e.g., to form a gel. Another example
is a hydrogel, such as alginic acid that can be gelled by the
addition of calcium ions. In some cases, gelation initiators
(ammonium persulfate and TEMED for acrylamide, or Ca.sup.2+ for
alginate) can be added to a droplet, for example, by co-flow with
the aqueous phase, by co-flow through the oil phase, or by
coalescence of two different drops, e.g., as discussed in U.S.
patent application Ser. No. 11/360,845, filed Feb. 23, 2006,
entitled "Electronic Control of Fluidic Species," by Link, et al.,
published as U.S. Patent Application Publication No. 2007/000342 on
Jan. 4, 2007; or in U.S. patent application Ser. No. 11/698,298,
filed Jan. 24, 2007, entitled "Fluidic Droplet Coalescence," by
Ahn, et al.; each incorporated herein by reference in their
entireties.
[0098] In another set of embodiments, the particles may comprise
one or more polymers. Exemplary polymers include, but are not
limited to, polystyrene (PS), polycaprolactone (PCL), polyisoprene
(PIP), poly(lactic acid), polyethylene, polypropylene,
polyacrylonitrile, polyimide, polyamide, and/or mixtures and/or
co-polymers of these and/or other polymers. In addition, in some
cases, the particles may be magnetic, which could allow for the
magnetic manipulation of the particles. For example, the particles
may comprise iron or other magnetic materials. The particles could
also be functionalized so that they could have other molecules
attached, such as proteins, nucleic acids or small molecules. Thus,
some embodiments of the present invention are directed to a set of
particles defining a library of, for example, nucleic acids,
proteins, small molecules, or other species such as those described
herein. In some embodiments, the particle may be fluorescent.
[0099] In some aspects, particles such as those discussed herein
containing oligonucleotides may be contained within a droplet and
the oligonucleotides released from the particle into the interior
of the droplet. The droplet may also contain nucleic acid (e.g.,
produced by lysing a cell), which can be bound to or recognized by
the oligonucleotides. The particles and the cells may be introduced
within the droplets during and/or after formation of the droplets,
and may be added simultaneously or sequentially (in any suitable
order). As mentioned, in some embodiments, the particles and the
cells may be placed within droplets such that the droplets
typically would contain, on average, no more than one particle and
no more than one cell. In addition, in some cases, the droplet may
be caused to form a gel, e.g., encapsulating the cell and/or
particle (which may also be a gel, as noted above).
[0100] In one set of embodiments, droplets are formed containing a
cell or other source of nucleic acid, and a particle, e.g.,
comprising an oligonucleotide as described above. Any suitable
method may be chosen to create droplets, and a wide variety of
different techniques for forming droplets will be known to those of
ordinary skill in the art. For example, a junction of channels may
be used to create the droplets. The junction may be, for instance,
a T-junction, a Y-junction, a channel-within-a-channel junction
(e.g., in a coaxial arrangement, or comprising an inner channel and
an outer channel surrounding at least a portion of the inner
channel), a cross (or "X") junction, a flow-focusing junction, or
any other suitable junction for creating droplets. See, for
example, International Patent Application No. PCT/US2004/010903,
filed Apr. 9, 2004, entitled "Formation and Control of Fluidic
Species," by Link, et al., published as WO 2004/091763 on Oct. 28,
2004, or International Patent Application No. PCT/US2003/020542,
filed Jun. 30, 2003, entitled "Method and Apparatus for Fluid
Dispersion," by Stone, et al., published as WO 2004/002627 on Jan.
8, 2004, each of which is incorporated herein by reference in its
entirety. In some embodiments, the junction may be configured and
arranged to produce substantially monodisperse droplets. The
droplets may also be created on the fluidic device, and/or the
droplets may be created separately then brought to the device.
[0101] In certain cases, the gel or other particles may be
contained within a fluidic droplet. The fluidic droplet may also
contain other species, e.g., oligonucleotides or enzymes (such as
those used to produce cDNA, cleave nucleic acids, ligation, or the
like). In some cases, the fluidic droplets are microfluidic.
Fluidic droplets are discussed in additional detail below. In some
cases, the gel or other particles and the oligonucleotides may be
encapsulated into a fluidic droplet, e.g., sequentially and/or
simultaneously. The gel or other particles and the oligonucleotides
may be encapsulated into a fluidic droplet at formation of the
fluidic droplet, and/or after the fluidic droplet has been formed,
for example, through picoinjection or other methods such as those
discussed in U.S. patent application Ser. No. 13/379,782, filed
Dec. 21, 2011, entitled "Fluid Injection," published as U.S. Pat.
Apl. Pub. No. 2012/0132288 on May 31, 2012, incorporated herein by
reference in its entirety, through fusion of the fluidic droplets,
or through other techniques known to those of ordinary skill in the
art.
[0102] In some embodiments, oligonucleotides or other nucleic acids
contained within the fluidic droplets may be amplified within the
droplets. In some cases, oligonucleotides or other nucleic acids
may be joined together and or amplified using PCR or other
amplification techniques, e.g., within a fluidic droplet.
[0103] In one set of embodiments, the PCR may be performed within
the fluidic droplets. For example, the droplets may contain a
polymerase (such as Taq polymerase), and DNA nucleotides, and the
droplets may be processed (e.g., via repeated heated and cooling)
to amplify the nucleic acid within the droplets. The polymerase and
nucleotides may be added to a droplet at any suitable point. Those
of ordinary skill in the art will be aware of suitable PCR
techniques and variations, such as assembly PCR or polymerase
cycling assembly, which may be used in some embodiments to produce
an amplified nucleic acid
[0104] In one set of embodiments, PCR or nucleic acid amplification
may be performed within the fluidic droplets. For example, the
fluidic droplets may contain a polymerase (such as Taq polymerase),
and DNA nucleotides, and the fluidic droplets may be processed
(e.g., via repeated heated and cooling) to amplify nucleic acids or
oligonucleotides within the droplets. The polymerase and
nucleotides may be added at any suitable point, e.g., before,
during, or after various nucleic acids encoding various conditions
are added to the fluidic droplets. For instance, a droplet may
contain polymerase and DNA nucleotides, which is fused to the
fluidic droplet to allow amplification to occur. Those of ordinary
skill in the art will be aware of suitable PCR techniques and
variations, such as assembly PCR or polymerase cycling assembly,
which may be used in some embodiments to produce an amplified
nucleic acid. Non-limiting examples of such procedures are also
discussed below. In addition, in some cases, suitable primers may
be used to initiate polymerization, e.g., P5 and P7, or other
primers known to those of ordinary skill in the art. In some
embodiments, primers may be added to the droplets, or the primers
may be present on one or more of the nucleic acids within the
droplets. Those of ordinary skill in the art will be aware of
suitable primers, many of which can be readily obtained
commercially.
[0105] In some cases, the fluidic droplets may be burst, broken, or
otherwise disrupted. A wide variety of methods for "breaking"
droplets are available to those of ordinary skill in the art, and
the exact method chosen is not critical. For example, droplets
contained in a carrying fluid may be disrupted using techniques
such as mechanical disruption or ultrasound.
[0106] Nucleic acids (labeled with oligonucleotide tags) from
different droplets may then be pooled or combined together or
analyzed, e.g., sequenced, amplified, etc. The nucleic acids from
different droplets, may however, remain distinguishable due to the
presence of different oligonucleotide tags (e.g., containing
different barcodes) that were present in each droplet prior to
disruption.
[0107] In certain embodiments, after attaching oligonucleotides
(e.g., containing barcodes), the nucleic acids from different
droplets may be pooled together for subsequent analysis, e.g., by
sequencing, such as high-throughput sequencing. In some cases, the
droplets may be burst to release the nucleic acids.
[0108] For example, the nucleic acids may be sequenced using a
variety of techniques and instruments, many of which are readily
available commercially. Examples of such techniques include, but
are not limited to, chain-termination sequencing,
sequencing-by-hybridization, Maxam-Gilbert sequencing,
dye-terminator sequencing, chain-termination methods, Massively
Parallel Signature Sequencing (Lynx Therapeutics), polony
sequencing, pyrosequencing, sequencing by ligation, ion
semiconductor sequencing, DNA nanoball sequencing, single-molecule
real-time sequencing, nanopore sequencing, microfluidic Sanger
sequencing, digital RNA sequencing ("digital RNA-seq"), etc. The
exact sequencing method chosen is not critical.
[0109] In addition, in some cases, the droplets may also contain
one or more DNA-tagged antibodies, e.g., to determine proteins in
the cell, e.g., by suitable tagging with DNA. Thus, for example, a
protein may be detected in a plurality of cells as discussed
herein, using DNA-tagged antibodies specific for the protein.
[0110] Additional details regarding systems and methods for
manipulating droplets in a microfluidic system in accordance with
various aspects of the invention follow, e.g., for determining
droplets (or species within droplets), sorting droplets, etc. For
example, various systems and methods for screening and/or sorting
droplets are described in U.S. patent application Ser. No.
11/360,845, filed Feb. 23, 2006, entitled "Electronic Control of
Fluidic Species," by Link, et al., published as U.S. Patent
Application Publication No. 2007/000342 on Jan. 4, 2007,
incorporated herein by reference. As a non-limiting example, by
applying (or removing) a first electric field (or a portion
thereof), a droplet may be directed to a first region or channel;
by applying (or removing) a second electric field to the device (or
a portion thereof), the droplet may be directed to a second region
or channel; by applying a third electric field to the device (or a
portion thereof), the droplet may be directed to a third region or
channel; etc., where the electric fields may differ in some way,
for example, in intensity, direction, frequency, duration, etc.
[0111] In certain embodiments of the invention, sensors are
provided that can sense and/or determine one or more
characteristics of the fluidic droplets, and/or a characteristic of
a portion of the fluidic system containing the fluidic droplet
(e.g., the liquid surrounding the fluidic droplet) in such a manner
as to allow the determination of one or more characteristics of the
fluidic droplets. Characteristics determinable with respect to the
droplet and usable in the invention can be identified by those of
ordinary skill in the art. Non-limiting examples of such
characteristics include fluorescence, spectroscopy (e.g., optical,
infrared, ultraviolet, etc.), radioactivity, mass, volume, density,
temperature, viscosity, pH, concentration of a substance, such as a
biological substance (e.g., a protein, a nucleic acid, etc.), or
the like.
[0112] In some cases, the sensor may be connected to a processor,
which in turn, cause an operation to be performed on the fluidic
droplet, for example, by sorting the droplet, adding or removing
electric charge from the droplet, fusing the droplet with another
droplet, splitting the droplet, causing mixing to occur within the
droplet, etc., for example, as previously described. For instance,
in response to a sensor measurement of a fluidic droplet, a
processor may cause the fluidic droplet to be split, merged with a
second fluidic droplet, etc.
[0113] One or more sensors and/or processors may be positioned to
be in sensing communication with the fluidic droplet. "Sensing
communication," as used herein, means that the sensor may be
positioned anywhere such that the fluidic droplet within the
fluidic system (e.g., within a channel), and/or a portion of the
fluidic system containing the fluidic droplet may be sensed and/or
determined in some fashion. For example, the sensor may be in
sensing communication with the fluidic droplet and/or the portion
of the fluidic system containing the fluidic droplet fluidly,
optically or visually, thermally, pneumatically, electronically, or
the like. The sensor can be positioned proximate the fluidic
system, for example, embedded within or integrally connected to a
wall of a channel, or positioned separately from the fluidic system
but with physical, electrical, and/or optical communication with
the fluidic system so as to be able to sense and/or determine the
fluidic droplet and/or a portion of the fluidic system containing
the fluidic droplet (e.g., a channel or a microchannel, a liquid
containing the fluidic droplet, etc.). For example, a sensor may be
free of any physical connection with a channel containing a
droplet, but may be positioned so as to detect electromagnetic
radiation arising from the droplet or the fluidic system, such as
infrared, ultraviolet, or visible light. The electromagnetic
radiation may be produced by the droplet, and/or may arise from
other portions of the fluidic system (or externally of the fluidic
system) and interact with the fluidic droplet and/or the portion of
the fluidic system containing the fluidic droplet in such as a
manner as to indicate one or more characteristics of the fluidic
droplet, for example, through absorption, reflection, diffraction,
refraction, fluorescence, phosphorescence, changes in polarity,
phase changes, changes with respect to time, etc. As an example, a
laser may be directed towards the fluidic droplet and/or the liquid
surrounding the fluidic droplet, and the fluorescence of the
fluidic droplet and/or the surrounding liquid may be determined.
"Sensing communication," as used herein may also be direct or
indirect. As an example, light from the fluidic droplet may be
directed to a sensor, or directed first through a fiber optic
system, a waveguide, etc., before being directed to a sensor.
[0114] Non-limiting examples of sensors useful in the invention
include optical or electromagnetically-based systems. For example,
the sensor may be a fluorescence sensor (e.g., stimulated by a
laser), a microscopy system (which may include a camera or other
recording device), or the like. As another example, the sensor may
be an electronic sensor, e.g., a sensor able to determine an
electric field or other electrical characteristic. For example, the
sensor may detect capacitance, inductance, etc., of a fluidic
droplet and/or the portion of the fluidic system containing the
fluidic droplet.
[0115] As used herein, a "processor" or a "microprocessor" is any
component or device able to receive a signal from one or more
sensors, store the signal, and/or direct one or more responses
(e.g., as described above), for example, by using a mathematical
formula or an electronic or computational circuit. The signal may
be any suitable signal indicative of the environmental factor
determined by the sensor, for example a pneumatic signal, an
electronic signal, an optical signal, a mechanical signal, etc.
[0116] In one set of embodiments, a fluidic droplet may be directed
by creating an electric charge and/or an electric dipole on the
droplet, and steering the droplet using an applied electric field,
which may be an AC field, a DC field, etc. As an example, an
electric field may be selectively applied and removed (or a
different electric field may be applied, e.g., a reversed electric
field) as needed to direct the fluidic droplet to a particular
region. The electric field may be selectively applied and removed
as needed, in some embodiments, without substantially altering the
flow of the liquid containing the fluidic droplet. For example, a
liquid may flow on a substantially steady-state basis (i.e., the
average flowrate of the liquid containing the fluidic droplet
deviates by less than 20% or less than 15% of the steady-state flow
or the expected value of the flow of liquid with respect to time,
and in some cases, the average flowrate may deviate less than 10%
or less than 5%) or other predetermined basis through a fluidic
system of the invention (e.g., through a channel or a
microchannel), and fluidic droplets contained within the liquid may
be directed to various regions, e.g., using an electric field,
without substantially altering the flow of the liquid through the
fluidic system.
[0117] In some embodiments, the fluidic droplets may be screened or
sorted within a fluidic system of the invention by altering the
flow of the liquid containing the droplets. For instance, in one
set of embodiments, a fluidic droplet may be steered or sorted by
directing the liquid surrounding the fluidic droplet into a first
channel, a second channel, etc.
[0118] In another set of embodiments, pressure within a fluidic
system, for example, within different channels or within different
portions of a channel, can be controlled to direct the flow of
fluidic droplets. For example, a droplet can be directed toward a
channel junction including multiple options for further direction
of flow (e.g., directed toward a branch, or fork, in a channel
defining optional downstream flow channels). Pressure within one or
more of the optional downstream flow channels can be controlled to
direct the droplet selectively into one of the channels, and
changes in pressure can be effected on the order of the time
required for successive droplets to reach the junction, such that
the downstream flow path of each successive droplet can be
independently controlled. In one arrangement, the expansion and/or
contraction of liquid reservoirs may be used to steer or sort a
fluidic droplet into a channel, e.g., by causing directed movement
of the liquid containing the fluidic droplet. The liquid reservoirs
may be positioned such that, when activated, the movement of liquid
caused by the activated reservoirs causes the liquid to flow in a
preferred direction, carrying the fluidic droplet in that preferred
direction. For instance, the expansion of a liquid reservoir may
cause a flow of liquid towards the reservoir, while the contraction
of a liquid reservoir may cause a flow of liquid away from the
reservoir. In some cases, the expansion and/or contraction of the
liquid reservoir may be combined with other flow-controlling
devices and methods, e.g., as described herein. Non-limiting
examples of devices able to cause the expansion and/or contraction
of a liquid reservoir include pistons and piezoelectric components.
In some cases, piezoelectric components may be particularly useful
due to their relatively rapid response times, e.g., in response to
an electrical signal. In some embodiments, the fluidic droplets may
be sorted into more than two channels.
[0119] As mentioned, certain embodiments are generally directed to
systems and methods for sorting fluidic droplets in a liquid, and
in some cases, at relatively high rates. For example, a property of
a droplet may be sensed and/or determined in some fashion (e.g., as
further described herein), then the droplet may be directed towards
a particular region of the device, such as a microfluidic channel,
for example, for sorting purposes. In some cases, high sorting
speeds may be achievable using certain systems and methods of the
invention. For instance, at least about 10 droplets per second may
be determined and/or sorted in some cases, and in other cases, at
least about 20 droplets per second, at least about 30 droplets per
second, at least about 100 droplets per second, at least about 200
droplets per second, at least about 300 droplets per second, at
least about 500 droplets per second, at least about 750 droplets
per second, at least about 1,000 droplets per second, at least
about 1,500 droplets per second, at least about 2,000 droplets per
second, at least about 3,000 droplets per second, at least about
5,000 droplets per second, at least about 7,500 droplets per
second, at least about 10,000 droplets per second, at least about
15,000 droplets per second, at least about 20,000 droplets per
second, at least about 30,000 droplets per second, at least about
50,000 droplets per second, at least about 75,000 droplets per
second, at least about 100,000 droplets per second, at least about
150,000 droplets per second, at least about 200,000 droplets per
second, at least about 300,000 droplets per second, at least about
500,000 droplets per second, at least about 750,000 droplets per
second, at least about 1,000,000 droplets per second, at least
about 1,500,000 droplets per second, at least about 2,000,000 or
more droplets per second, or at least about 3,000,000 or more
droplets per second may be determined and/or sorted.
[0120] In some aspects, a population of relatively small droplets
may be used. In certain embodiments, as non-limiting examples, the
average diameter of the droplets may be less than about 1 mm, less
than about 500 micrometers, less than about 300 micrometers, less
than about 200 micrometers, less than about 100 micrometers, less
than about 75 micrometers, less than about 50 micrometers, less
than about 30 micrometers, less than about 25 micrometers, less
than about 20 micrometers, less than about 15 micrometers, less
than about 10 micrometers, less than about 5 micrometers, less than
about 3 micrometers, less than about 2 micrometers, less than about
1 micrometer, less than about 500 nm, less than about 300 nm, less
than about 100 nm, or less than about 50 nm. The average diameter
of the droplets may also be at least about 30 nm, at least about 50
nm, at least about 100 nm, at least about 300 nm, at least about
500 nm, at least about 1 micrometer, at least about 2 micrometers,
at least about 3 micrometers, at least about 5 micrometers, at
least about 10 micrometers, at least about 15 micrometers, or at
least about 20 micrometers in certain cases. The "average diameter"
of a population of droplets is the arithmetic average of the
diameters of the droplets.
[0121] In some embodiments, the droplets may be of substantially
the same shape and/or size (i.e., "monodisperse"), or of different
shapes and/or sizes, depending on the particular application. In
some cases, the droplets may have a homogenous distribution of
cross-sectional diameters, i.e., the droplets may have a
distribution of diameters such that no more than about 5%, no more
than about 2%, or no more than about 1% of the droplets have a
diameter less than about 90% (or less than about 95%, or less than
about 99%) and/or greater than about 110% (or greater than about
105%, or greater than about 101%) of the overall average diameter
of the plurality of droplets. Some techniques for producing
homogenous distributions of cross-sectional diameters of droplets
are disclosed in International Patent Application No.
PCT/US2004/010903, filed Apr. 9, 2004, entitled "Formation and
Control of Fluidic Species," by Link et al., published as WO
2004/091763 on Oct. 28, 2004, incorporated herein by reference.
[0122] Those of ordinary skill in the art will be able to determine
the average diameter of a population of droplets, for example,
using laser light scattering or other known techniques. The
droplets so formed can be spherical, or non-spherical in certain
cases. The diameter of a droplet, in a non-spherical droplet, may
be taken as the diameter of a perfect mathematical sphere having
the same volume as the non-spherical droplet.
[0123] In some embodiments, one or more droplets may be created
within a channel by creating an electric charge on a fluid
surrounded by a liquid, which may cause the fluid to separate into
individual droplets within the liquid. In some embodiments, an
electric field may be applied to the fluid to cause droplet
formation to occur. The fluid can be present as a series of
individual charged and/or electrically inducible droplets within
the liquid. Electric charge may be created in the fluid within the
liquid using any suitable technique, for example, by placing the
fluid within an electric field (which may be AC, DC, etc.), and/or
causing a reaction to occur that causes the fluid to have an
electric charge.
[0124] The electric field, in some embodiments, is generated from
an electric field generator, i.e., a device or system able to
create an electric field that can be applied to the fluid. The
electric field generator may produce an AC field (i.e., one that
varies periodically with respect to time, for example,
sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that
is constant with respect to time), a pulsed field, etc. Techniques
for producing a suitable electric field (which may be AC, DC, etc.)
are known to those of ordinary skill in the art. For example, in
one embodiment, an electric field is produced by applying voltage
across a pair of electrodes, which may be positioned proximate a
channel such that at least a portion of the electric field
interacts with the channel. The electrodes can be fashioned from
any suitable electrode material or materials known to those of
ordinary skill in the art, including, but not limited to, silver,
gold, copper, carbon, platinum, copper, tungsten, tin, cadmium,
nickel, indium tin oxide ("ITO"), etc., as well as combinations
thereof.
[0125] In another set of embodiments, droplets of fluid can be
created from a fluid surrounded by a liquid within a channel by
altering the channel dimensions in a manner that is able to induce
the fluid to form individual droplets. The channel may, for
example, be a channel that expands relative to the direction of
flow, e.g., such that the fluid does not adhere to the channel
walls and forms individual droplets instead, or a channel that
narrows relative to the direction of flow, e.g., such that the
fluid is forced to coalesce into individual droplets. In some
cases, the channel dimensions may be altered with respect to time
(for example, mechanically or electromechanically, pneumatically,
etc.) in such a manner as to cause the formation of individual
droplets to occur. For example, the channel may be mechanically
contracted ("squeezed") to cause droplet formation, or a fluid
stream may be mechanically disrupted to cause droplet formation,
for example, through the use of moving baffles, rotating blades, or
the like. Other techniques of creating droplets include, for
example mixing or vortexing of a fluid.
[0126] Certain embodiments are generally directed to systems and
methods for splitting a droplet into two or more droplets. For
example, a droplet can be split using an applied electric field.
The droplet may have a greater electrical conductivity than the
surrounding liquid, and, in some cases, the droplet may be
neutrally charged. In certain embodiments, in an applied electric
field, electric charge may be urged to migrate from the interior of
the droplet to the surface to be distributed thereon, which may
thereby cancel the electric field experienced in the interior of
the droplet. In some embodiments, the electric charge on the
surface of the droplet may also experience a force due to the
applied electric field, which causes charges having opposite
polarities to migrate in opposite directions. The charge migration
may, in some cases, cause the drop to be pulled apart into two
separate droplets.
[0127] Some embodiments of the invention generally relate to
systems and methods for fusing or coalescing two or more droplets
into one droplet, e.g., where the two or more droplets ordinarily
are unable to fuse or coalesce, for example, due to composition,
surface tension, droplet size, the presence or absence of
surfactants, etc. In certain cases, the surface tension of the
droplets, relative to the size of the droplets, may also prevent
fusion or coalescence of the droplets from occurring.
[0128] As a non-limiting example, two droplets can be given
opposite electric charges (i.e., positive and negative charges, not
necessarily of the same magnitude), which can increase the
electrical interaction of the two droplets such that fusion or
coalescence of the droplets can occur due to their opposite
electric charges. For instance, an electric field may be applied to
the droplets, the droplets may be passed through a capacitor, a
chemical reaction may cause the droplets to become charged, etc.
The droplets, in some cases, may not be able to fuse even if a
surfactant is applied to lower the surface tension of the droplets.
However, if the droplets are electrically charged with opposite
charges (which can be, but are not necessarily of, the same
magnitude), the droplets may be able to fuse or coalesce. As
another example, the droplets may not necessarily be given opposite
electric charges (and, in some cases, may not be given any electric
charge), and are fused through the use of dipoles induced in the
droplets that causes the droplets to coalesce. Also, the two or
more droplets allowed to coalesce are not necessarily required to
meet "head-on." Any angle of contact, so long as at least some
fusion of the droplets initially occurs, is sufficient. See also,
e.g., U.S. patent application Ser. No. 11/698,298, filed Jan. 24,
2007, entitled "Fluidic Droplet Coalescence," by Ahn, et al.,
published as U.S. Patent Application Publication No. 2007/0195127
on Aug. 23, 2007, incorporated herein by reference in its
entirety.
[0129] In one set of embodiments, a fluid may be injected into a
droplet. The fluid may be microinjected into the droplet in some
cases, e.g., using a microneedle or other such device. In other
cases, the fluid may be injected directly into a droplet using a
fluidic channel as the droplet comes into contact with the fluidic
channel. Other techniques of fluid injection are disclosed in,
e.g., International Patent Application No. PCT/US2010/040006, filed
Jun. 25, 2010, entitled "Fluid Injection," by Weitz, et al.,
published as WO 2010/151776 on Dec. 29, 2010; or International
Patent Application No. PCT/US2009/006649, filed Dec. 18, 2009,
entitled "Particle-Assisted Nucleic Acid Sequencing," by Weitz, et
al., published as WO 2010/080134 on Jul. 15, 2010, each
incorporated herein by reference in its entirety.
[0130] A variety of materials and methods, according to certain
aspects of the invention, can be used to form articles or
components such as those described herein, e.g., channels such as
microfluidic channels, chambers, etc. For example, various articles
or components can be formed from solid materials, in which the
channels can be formed via micromachining, film deposition
processes such as spin coating and chemical vapor deposition, laser
fabrication, photolithographic techniques, etching methods
including wet chemical or plasma processes, and the like. See, for
example, Scientific American, 248:44-55, 1983 (Angell, et al).
[0131] In one set of embodiments, various structures or components
of the articles described herein can be formed of a polymer, for
example, an elastomeric polymer such as polydimethylsiloxane
("PDMS"), polytetrafluoroethylene ("PTFE" or Teflon.RTM.), or the
like. For instance, according to one embodiment, a microfluidic
channel may be implemented by fabricating the fluidic system
separately using PDMS or other soft lithography techniques (details
of soft lithography techniques suitable for this embodiment are
discussed in the references entitled "Soft Lithography," by Younan
Xia and George M. Whitesides, published in the Annual Review of
Material Science, 1998, Vol. 28, pages 153-184, and "Soft
Lithography in Biology and Biochemistry," by George M. Whitesides,
Emanuele Ostuni, Shuichi Takayama, Xingyu Jiang and Donald E.
Ingber, published in the Annual Review of Biomedical Engineering,
2001, Vol. 3, pages 335-373; each of these references is
incorporated herein by reference).
[0132] Other examples of potentially suitable polymers include, but
are not limited to, polyethylene terephthalate (PET), polyacrylate,
polymethacrylate, polycarbonate, polystyrene, polyethylene,
polypropylene, polyvinylchloride, cyclic olefin copolymer (COC),
polytetrafluoroethylene, a fluorinated polymer, a silicone such as
polydimethylsiloxane, polyvinylidene chloride, bis-benzocyclobutene
("BCB"), a polyimide, a fluorinated derivative of a polyimide, or
the like. Combinations, copolymers, or blends involving polymers
including those described above are also envisioned. The device may
also be formed from composite materials, for example, a composite
of a polymer and a semiconductor material.
[0133] In some embodiments, various structures or components of the
article are fabricated from polymeric and/or flexible and/or
elastomeric materials, and can be conveniently formed of a
hardenable fluid, facilitating fabrication via molding (e.g.
replica molding, injection molding, cast molding, etc.). The
hardenable fluid can be essentially any fluid that can be induced
to solidify, or that spontaneously solidifies, into a solid capable
of containing and/or transporting fluids contemplated for use in
and with the fluidic network. In one embodiment, the hardenable
fluid comprises a polymeric liquid or a liquid polymeric precursor
(i.e. a "prepolymer"). Suitable polymeric liquids can include, for
example, thermoplastic polymers, thermoset polymers, waxes, metals,
or mixtures or composites thereof heated above their melting point.
As another example, a suitable polymeric liquid may include a
solution of one or more polymers in a suitable solvent, which
solution forms a solid polymeric material upon removal of the
solvent, for example, by evaporation. Such polymeric materials,
which can be solidified from, for example, a melt state or by
solvent evaporation, are well known to those of ordinary skill in
the art. A variety of polymeric materials, many of which are
elastomeric, are suitable, and are also suitable for forming molds
or mold masters, for embodiments where one or both of the mold
masters is composed of an elastomeric material. A non-limiting list
of examples of such polymers includes polymers of the general
classes of silicone polymers, epoxy polymers, and acrylate
polymers. Epoxy polymers are characterized by the presence of a
three-membered cyclic ether group commonly referred to as an epoxy
group, 1,2-epoxide, or oxirane. For example, diglycidyl ethers of
bisphenol A can be used, in addition to compounds based on aromatic
amine, triazine, and cycloaliphatic backbones. Another example
includes the well-known Novolac polymers. Non-limiting examples of
silicone elastomers suitable for use according to the invention
include those formed from precursors including the chlorosilanes
such as methylchlorosilanes, ethylchlorosilanes,
phenylchlorosilanes, dodecyltrichlorosilanes, etc.
[0134] Silicone polymers are used in certain embodiments, for
example, the silicone elastomer polydimethylsiloxane. Non-limiting
examples of PDMS polymers include those sold under the trademark
Sylgard by Dow Chemical Co., Midland, Mich., and particularly
Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers
including PDMS have several beneficial properties simplifying
fabrication of various structures of the invention. For instance,
such materials are inexpensive, readily available, and can be
solidified from a prepolymeric liquid via curing with heat. For
example, PDMSs are typically curable by exposure of the
prepolymeric liquid to temperatures of about, for example, about
65.degree. C. to about 75.degree. C. for exposure times of, for
example, about an hour. Also, silicone polymers, such as PDMS, can
be elastomeric and thus may be useful for forming very small
features with relatively high aspect ratios, necessary in certain
embodiments of the invention. Flexible (e.g., elastomeric) molds or
masters can be advantageous in this regard.
[0135] One advantage of forming structures such as microfluidic
structures or channels from silicone polymers, such as PDMS, is the
ability of such polymers to be oxidized, for example by exposure to
an oxygen-containing plasma such as an air plasma, so that the
oxidized structures contain, at their surface, chemical groups
capable of cross-linking to other oxidized silicone polymer
surfaces or to the oxidized surfaces of a variety of other
polymeric and non-polymeric materials. Thus, structures can be
fabricated and then oxidized and essentially irreversibly sealed to
other silicone polymer surfaces, or to the surfaces of other
substrates reactive with the oxidized silicone polymer surfaces,
without the need for separate adhesives or other sealing means. In
most cases, sealing can be completed simply by contacting an
oxidized silicone surface to another surface without the need to
apply auxiliary pressure to form the seal. That is, the
pre-oxidized silicone surface acts as a contact adhesive against
suitable mating surfaces. Specifically, in addition to being
irreversibly sealable to itself, oxidized silicone such as oxidized
PDMS can also be sealed irreversibly to a range of oxidized
materials other than itself including, for example, glass, silicon,
silicon oxide, quartz, silicon nitride, polyethylene, polystyrene,
glassy carbon, and epoxy polymers, which have been oxidized in a
similar fashion to the PDMS surface (for example, via exposure to
an oxygen-containing plasma). Oxidation and sealing methods useful
in the context of the present invention, as well as overall molding
techniques, are described in the art, for example, in an article
entitled "Rapid Prototyping of Microfluidic Systems and
Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy et
al.), incorporated herein by reference.
[0136] Thus, in certain embodiments, the design and/or fabrication
of the article may be relatively simple, e.g., by using relatively
well-known soft lithography and other techniques such as those
described herein. In addition, in some embodiments, rapid and/or
customized design of the article is possible, for example, in terms
of geometry. In one set of embodiments, the article may be produced
to be disposable, for example, in embodiments where the article is
used with substances that are radioactive, toxic, poisonous,
reactive, biohazardous, etc., and/or where the profile of the
substance (e.g., the toxicology profile, the radioactivity profile,
etc.) is unknown. Another advantage to forming channels or other
structures (or interior, fluid-contacting surfaces) from oxidized
silicone polymers is that these surfaces can be much more
hydrophilic than the surfaces of typical elastomeric polymers
(where a hydrophilic interior surface is desired). Such hydrophilic
channel surfaces can thus be more easily filled and wetted with
aqueous solutions than can structures comprised of typical,
unoxidized elastomeric polymers or other hydrophobic materials.
[0137] The following documents are each incorporated herein by
reference in its entirety for all purposes: U.S. Pat. Apl. Ser. No.
61/980,541, entitled "Methods and Systems for Droplet Tagging and
Amplification," by Weitz, et al.; U.S. Pat. Apl. Ser. No.
61/981,123, entitled "Systems and Methods for Droplet Tagging," by
Bernstein, et al.; Int. Pat. Apl. Pub. No. WO 2004/091763, entitled
"Formation and Control of Fluidic Species," by Link et al.; Int.
Pat. Apl. Pub. No. WO 2004/002627, entitled "Method and Apparatus
for Fluid Dispersion," by Stone et al.; Int. Pat. Apl. Pub. No. WO
2006/096571, entitled "Method and Apparatus for Forming Multiple
Emulsions," by Weitz et al.; Int. Pat. Apl. Pub. No. WO
2005/021151, entitled "Electronic Control of Fluidic Species," by
Link et al.; Int. Pat. Apl. Pub. No. WO 2011/056546, entitled
"Droplet Creation Techniques," by Weitz, et al.; Int. Pat. Apl.
Pub. No. WO 2010/033200, entitled "Creation of Libraries of
Droplets and Related Species," by Weitz, et al.; U.S. Pat. Apl.
Pub. No. 2012-0132288, entitled "Fluid Injection," by Weitz, et
al.; Int. Pat. Apl. Pub. No. WO 2008/109176, entitled "Assay And
Other Reactions Involving Droplets," by Agresti, et al.; and Int.
Pat. Apl. Pub. No. WO 2010/151776, entitled "Fluid Injection," by
Weitz, et al.; and U.S. Pat. Apl. Ser. No. 62/072,944, entitled
"Systems and Methods for Barcoding Nucleic Acids," by Weitz, et
al.
[0138] In addition, the following are incorporated herein by
reference in their entireties: U.S. Pat. Apl. Ser. No. 61/981,123
filed Apr. 17, 2014; Int. Pat. Apl. Ser. No. PCT/US15/026338, filed
Apr. 17, 2015, entitled "Systems and Methods for Droplet Tagging";
U.S. Pat. Apl. Ser. No. 61/981,108 filed Apr. 17, 2014; Int. Pat.
Apl. Ser. No. PCT/US15/26422, filed on Apr. 17, 2015, entitled
"Methods and Systems for Droplet Tagging and Amplification"; Int.
Pat. Apl. Ser. No. PCT/US16/027734, filed on Apr. 15, 2016,
entitled "Barcoding Systems and Methods for Gene Sequencing and
Other Applications"; U.S. Pat. Apl. Ser. No. 62/072,944, filed Oct.
30, 2014; and Int. Pat. Apl. Ser. No. PCT/US15/26443, filed on Apr.
17, 2015, entitled "Systems and Methods for Barcoding Nucleic
Acids." Also incorporated herein by reference in its entirety is
U.S. Provisional Patent Application Ser. No. 62/324,407, filed Apr.
19, 2016, entitled "Immobilization-Based Systems and Methods for
Genetic Analysis and Other Applications," by Weitz, et al.
[0139] The following examples are intended to illustrate certain
embodiments of the present invention, but do not exemplify the full
scope of the invention.
Example 1
[0140] This example shows a high-throughput and cost-effective
method for the simultaneous analysis of RNA and DNA from single
cells. Single cells are first co-encapsulated with lysis buffer
into a liquid, low-melting temperature agarose using a
microfluidics drop maker, and the cells are immobilized within a
gel by cooling the agarose to form a solid (or gel) phase, e.g.,
gel particles. In the agarose gel, a linear polymer conjugated to
either oligo d(T) or gene specific primers is added to capture
cellular RNAs, followed by reverse transcription (RT) to copy the
mRNA information onto agarose gel particles. To achieve the genomic
DNA information, genomic DNA (gDNA) is treated and further
encapsulation and amplification is performed within the gel drops.
Since the pore size of agarose gel particles is relatively small,
gene fragments from a single cell may be kept in the same agarose
gel particle during various treatments. Meanwhile, a large size of
monoclonal DNA-based barcoding beads library is prepared; there are
about 10.sup.8 same barcoding sequences on each bead. The barcoding
beads are merged with the drops containing products from cellular
RNA and DNA, and ligated with the barcodes on the beads, thereby
generating a library of barcoding beads containing all the gene
information from a single cell. Next-gene sequencing may then be
utilized to obtain the information from, e.g., thousands of single
cells.
[0141] 1. Fabrication of microfluidics devices.
Polydimethylsiloxane (PDMS) microfluidic devices were fabricated
using standard soft lithographic methods. The microfluidic channel
walls were rendered hydrophobic by treating them with Aquapel. In
the PDMS device for the pico-injection experiment, electrodes were
designed as channels. These channels were filled with Indalloy 19
(51In, 32.5 Bi, 16.5 Sn; 0.020 inch diameter), a low melting point
metal alloy, by pushing the wire into the punched holes on an
80.degree. C. hot plate. Electrical connections were made using
eight-pin terminal blocks. See, e.g., Int. Pat. Apl. Pub. Nos. WO
2004/091763, WO 2004/002627, WO 2005/021151, or WO 2010/151776,
each incorporated herein by reference in its entirety.
[0142] 2. Synthesis of monoclonal barcoded beads. The full length
of barcode sequence is 5'-/5Acryd/iSpPC/-PE1 illumina
adapter-barcode 1-linker sequence-barcode 2-universal sequence
ending with T-3'. To synthesize the monoclonal barcoded beads, the
protocol described in U.S. Pat. Apl. Ser. No. 62/072,944
(incorporated herein by reference in its entirety) was used with an
additional hybridization step. Briefly, a microfluidics drop maker
was used to encapsulate acrylamide:bis-acrylamide solution,
acrydite-modified DNA primer and ammonium persulfate into
micron-sized water-in-oil drops. To catalyze acrylamide
polymerization, TEMED was added in the continuous oil phase. The
generated drops were then incubated at 65.degree. C. for overnight,
and then the oil washed away. In the resulting acrylamide gel
beads, there were millions of incorporated 5'-/5Acryd/iSpPC/-PE1
illumina adapters.
[0143] In the next step, the combinatorial barcode-1 and barcode-2
were sequentially synthesized onto the gel beads by extension and
split-pool method. Finally, DNA fragments were hybridized with the
complementary sequence to the universal sequence to form a partial
double-stranded DNA with an overhanging T at the 3' end.
[0144] 3. Simultaneous target RNA (small number) and genomic DNA
sequencing (see FIG. 1).
[0145] 3.1. Co-encapsulate cells with lysis buffer and target RNA
capture oligonucleotides: Acrydited target RNA capture
oligonucleotides were grafted onto linear polyacrylamide (LPA) by
copolymerization with acrylamide. A total of 10 microliters of 1 mM
acrydited target RNA capture oligonucleotides was mixed with 10
microliters of 40% acrylamide in 80 microliters of DI water. Then,
1.4 microliters of 10% APS and 1.4 microliters of 10% TEMED were
added in the solution. The solution is immediately mixed well and
kept in a vacuum to remove air and the polymerization reaction took
about 12 min to complete.
[0146] Cell lysis cocktail solution was prepared with the following
final concentrations: 20 mM Tris-HCl, 4% Tween 20, 1 mg/mL
Proteinase K and 1% low melting temperature agarose gel. The cells
were mixed with 2% low melting temperature agarose gel at a 1:1
ratio. In the agarose gel, 2 micromolar target RNA capture
oligonucleotides on LPA were used. To avoid amplification of
genomic DNA, reverse and forward primers were designed to span two
adjacent exons of RNA. The cell-agarose solution and lysis buffer
was injected into two inlets of a co-encapsulation drop maker. The
generated drops were collected on ice and subsequently incubated at
37.degree. C. for 10 min and 50.degree. C. for 30 min to lyse
cells. To capture the target RNAs, the temperature was decreased to
25.degree. C. at 0.1.degree. C./min, and then the drops were put on
ice to solidify the agarose gel.
[0147] 3.2 Break emulsion and RT to generate first-strand cDNA: To
break the emulsion, 1H,1H,2H,2H-perfluoro-1-octanol (PFO) was added
to the emulsion and centrifuged to collect the agarose gel
particles. The gel particles were washed twice with 10 mM Tris-HCl
and mixed with 2.times. SuperScript II reverse transcriptase
cocktail at a 1:1 ratio. The 2.times. SuperScript II reverse
transcriptase cocktail was prepared with the following final
concentrations: 2.times.RT buffer, 20 mM DTT, 4 units/microliters
RNasin and 20 units/microliters SuperScript II reverse
transcriptase. The mixture was incubated at 42.degree. C. for 40
min, followed by gel washing twice with 10 mM Tris-HCl.
[0148] 3.3 Digest RNA: RNase H was used to digest the RNA molecules
on the hybrid of RNA and first-strand cDNA.
[0149] 3.4 Digest double-stranded genomic DNA (dsgDNA): Using
NEBNext.RTM. dsDNA Fragmentase.RTM., the genomic DNA was digested
to yield around 300-bp DNA fragments. The 20 microliters reaction
solution contained 1.times. reaction buffer, 2 microliters
fragmentase and 10 microliters agarose gel particles containing
cells. These were incubated at 37.degree. C. for 30 min and then 5
microliters of 0.5M EDTA was added to stop the digestion
reaction.
[0150] 3.5. Add a universal adapter on one side: The resulting
beads performed end repair and dA tailing, and then ligated with
universal adapters which are made by annealing the Miseq common
oligonucleotide.
[0151] 3.6 Re-emulsify the gel particles and amplify: Using a
re-emulsification microfluidics device, the gel particles were
re-encapsulated with a PCR mixture. Using this scheme, hundreds of
gDNA genes could be amplified with forward primers and universal
adapter sequences, and small amount of cDNA (<5) with
gene-specific forward and reverse primers.
[0152] 3.7 Label amplicons with barcoded gel beads: To label
amplicons in each drop with barcoded gel beads, the thermocycled
drops were flowed to a microfluidic pico-injection device and the
barcoded gel beads injected to the drop by electro-coalescence.
Drops were spaced on chip by oil with 1% w/w surfactant. The device
electrodes were connected to a high voltage TREK 2210 amplifier
which supplied a 100 V sine wave at a frequency of 25 kHz. The flow
rates used to inject the drops were chosen to ensure that one
barcode drop fuses with a barcoded gel beads at a 1:1 ratio.
Typical flow rates fulfilling these requirements were 150
microliters/hr for the oil, 40 microliters/hr for thermocycled
drops and 40 microliters/hr for the barcoded gel beads. In the
solution of barcoded gel beads, T4 DNA ligase and 2.times. ligation
buffer was added. The drops were collected and incubated at
16.degree. C. for overnight to perform ligation.
[0153] 3.8. Breaking the thermocycled drops: During ligation, the
barcode sequences on the gel beads in each merged drop were ligated
with the amplicon from a single template. Thus the emulsion could
be broken without losing the information regarding the gDNA
associated with every barcoded fragment. To break the emulsion
drops in each tube for sequencing, PFO was added to the emulsion
and gently centrifuged to separate the phases. PCR products were in
the liquid phase.
[0154] 3.9. Library preparation and 2ndGS: The PCR products were
cleaned using AMPure XP Beads (Life Technologies, Grand Island,
N.Y.) and eluted into 10 microliters of 0.1M Tris-HCl. To add the
full-length adapter for Miseq sequencing, the purified PCR products
were amplified with the full-length primer pairs in 25 microliters
of PCR cocktail. The cocktail contained 0.2 microliters of KAPA2G
Fast DNA Polymerase, 1.times.KAPA2G buffer, 0.4 mM dNTPs, 1
microliters of forward and reverse primers and 2 microliters of
ligation products. The thermocycling condition was 95.degree. C.
for 2 min, 10 cycles of 95.degree. C. for 15 s, 56.degree. C. for
15 s, and 72.degree. C. for 2 s, followed by 72.degree. C. for 5
min. The PCR products were cleaned again using beads and quantified
using Qubit. The concentration was then adjusted and the sequence
sampled using MiSeq system.
Example 2
[0155] This example shows simultaneous target RNA (large number)
and genomic DNA sequencing. See FIG. 2.
[0156] 4.1. Co-encapsulate cells and lysis buffer and capture the
whole transcriptome: Acrydited oligo dT was grafted onto LPA by
copolymerization with acrylamide. The embedding method was the same
as section 3.1.
[0157] 4.2. Break emulsion and RT to generate first-strand cDNA:
The emulsions were broken and centrifuged to collect the agarose
gel particles. The gel particles are washed and mixed with 2.times.
SuperScript II reverse transcriptase cocktail at a 1:1 ratio,
followed by incubating at 42.degree. C. for 40 min. During
Superscript II reverse transcription, non-templated three A bases
were added to the 3' end of the cDNA.
[0158] 4.3. Digest RNA: Same as section 3.3.
[0159] 4.4. Digest dsgDNA: Same as section 3.4.
[0160] 4.5. Add a universal adapter on one side of the dsgDNA and
sscDNA: The same method
as described in section 3.5 was used to add a universal adapter on
one side of the dsgDNA. To add a universal adapter on one side of
sscDNA, another adapter with three T's overhanging was created,
which can pair with the three A on the 3' end of the first-strand
cDNA. This adapter also included a UMI sequence for calculation of
expression level.
[0161] 4.6. Re-emulsify the gel particle and amplify: Using this
scheme, hundreds of gDNA genes with forward primers and universal
adapter sequences, and hundreds of cDNA with forward primers and
their universal adapter sequences could be amplified.
[0162] 4.7-4.9. Same as section 3.7-3.9.
Example 3
[0163] This example shows co-encapsulate cells and lysis buffer and
capture the whole transcriptome. See FIG. 3.
[0164] 5.1. Acrydited oligo dT was grafted onto LPA by
copolymerization with acrylamide. The embedding method was the same
as section 3.1. Upstream of Oligo dT, a ligation sequence (LS) was
added.
[0165] 5.2. Break emulsion and RT to generate first-strand cDNA:
Same as section 4.2.
[0166] 5.3. Digest RNA: Same as section 3.3.
[0167] 5.5. Digest genomic DNA, ligate with a sequencing adapter on
one end: Same as section 3.4.
[0168] 5.6. Re-emulsify the gel particles and amplify: Using a
re-emulsification microfluidics device, the gel particles were
re-encapsulated with a PCR mixture. In this PCR mixture,
gene-specific forward and different universal primers for the
amplification of target gDNA were added.
[0169] 5.7. Label amplicons with barcoded gel beads: (1) Ligate
amplicons from gDNA with barcoded gel beads. Same at section 3.7.
(2) Ligate first-strand cDNA with barcoded gel beads. These were
incubated to ligate the ligation sequence (LS) on the cDNA to the
corresponding complementary sequence (CS) annealed to the barcoding
DNA sequence.
[0170] 5.8. Next steps included breaking the thermocycled drops,
and adding reverse transcription enzyme mix and an oligo with
sequence: 3'-TTT-PE2-T7 RNApol binding site. The oligonucleotide
will pair to the non-templated A bases added to the 3' end of the
cDNA during Superscript II reverse transcription and the reverse
transcriptase will extend from the final non-templated A base to
append the oligo complement. T7 amplification of mRNA and RT, PCR
amplification of both gDNA and mRNA were performed.
[0171] 5.9. Library preparation and 2ndGS: Same as section 3.9.
Example 4
[0172] In this example, cells were first encapsulated into a
droplet containing agarose, and the droplet solidified to form a
gel particle by exposure to ice. Then, whole genome amplification
(WGA) was performed using multiple displacement amplification. To
demonstrate the success of amplification, the gel particles were
stained with propidium iodide (PI). Compared with the samples not
exposed to WGA, highly dense staining was observed at the places
where cells were present. See FIG. 8.
[0173] After being digested for 10 min, the WGA products were in
the range of 200-400 bp, which was useful for the following
procedure for amplification. The gel particles went through end
repair and adaptor ligation, and then were encapsulated into
droplets again, followed by in-drop PCR. Strong amplification was
observed, as can be seen in FIG. 8.
[0174] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0175] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0176] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0177] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0178] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0179] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0180] When the word "about" is used herein in reference to a
number, it should be understood that still another embodiment of
the invention includes that number not modified by the presence of
the word "about."
[0181] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0182] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
13131DNAArtificial SequenceSynthetic Polynucleotide 1ccatctcatc
cctgcgtgtc tccgactcag t 31248DNAArtificial SequenceSynthetic
Polynucleotide 2ctgagtcgga cacgcaacag gggatagaca aggcacacag
gggatagg 48354DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(1)..(1)Modified by
/5Acryd/misc_feature(29)..(34)n is a, c, g, t or
umisc_feature(54)..(54)n is a, c, or g 3cgatgacgta atacgactca
ctatagggnn nnnntttttt tttttttttt tttn 54454DNAArtificial
SequenceSynthetic Polynucleotidemisc_feature(1)..(1)Modified by
/5Acryd/misc_feature(29)..(34)n is a, c, g, t or u 4cgatgacgta
atacgactca ctatagggnn nnnntgagca gggtctagag caga 54533DNAArtificial
SequenceSynthetic Polynucleotide 5acactctttc cctacacgac gctcttccga
tct 3368DNAArtificial SequenceSynthetic Polynucleotide 6aaacaaac
8711DNAArtificial SequenceSynthetic Polynucleotide 7tcggtcattc a
11815DNAArtificial SequenceSynthetic Polynucleotide 8ctgtctctta
tacac 15914DNAArtificial SequenceSynthetic Polynucleotide
9atctgacgct gccg 141030DNAArtificial SequenceSynthetic
Polynucleotide 10tcggcagcgt cagatgtgta taagagacag
301121DNAArtificial SequenceSynthetic Polynucleotide 11gtctcggcat
tcctgctgaa c 211220DNAArtificial SequenceSynthetic Polynucleotide
12taatacgact cactataggg 201364DNAArtificial SequenceSynthetic
Polynucleotide 13cgatgacgta atacgactca ctatagggat accaccatgg
ctctttccct acacgacgct 60cttc 64
* * * * *