PARTITIONING AND PROCESSING OF ANALYTES AND OTHER SPECIES

• US 20140227684A1
• Filed: 02/07/2014
• Published: 08/14/2014
• Est. Priority Date: 02/08/2013
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10x Technology Inc.
• Inventors: Hindson, Benjamin, Saxonov, Serge, Ness, Kevin, Hardenbol, Paul, Masquelier, Donald, Hindson, Christopher, Jarosz, Mima, Schnall-Levin, Michael
• Granted Patent: US 9,644,204 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Claims

1. A composition comprising a plurality of capsules, said capsules situated within droplets in an emulsion, wherein said capsules are configured to release their contents into said droplets upon the application of a stimulus.

2. The composition of claim 1, wherein at least one of said capsules and said droplets comprise a species selected from the group consisting of a reagent and an analyte.

4. The composition of claim 2, wherein said reagent is selected from the group consisting of a protein, a polynucleotide, an enzyme, an antibody, a barcode, an adapter, a buffer, a small molecule, a detergent, a dye, a polymer and combinations thereof.

5. The composition of claim 4, wherein said enzyme is selected from the group consisting of a proteinase, a restriction enzyme, a ligase, a polymerase, a fragmentase, a reverse transcriptase, a transposase, and combinations thereof.

6. The composition of claim 5, wherein said restriction enzyme is a restriction enzyme that is a rare cutter.

7. The composition of claim 4, wherein said barcode is an oligonucleotide barcode.

8. The composition of claim 2, wherein said analyte is selected from the group consisting of a cell, a polynucleotide, a chromosome, a protein, a peptide, a polysaccharide, a sugar, a lipid, a small molecule, and combinations thereof.

9. The composition of claim 8, wherein said analyte is a polynucleotide.

10. The composition of claim 9, wherein said polynucleotide is selected from the group consisting of DNA, RNA, cDNA, and combinations thereof.

12. The composition of claim 9, wherein the amount of said polynucleotide in said composition is an amount sufficient to provide about 100-200X sequence coverage.

13. The composition of claim 8, wherein each capsule comprises, on average, about one chromosome.

14. The composition of claim 1, wherein each droplet comprises, on average, about 1 capsule per droplet.

15. The composition of claim 1, wherein each droplet can hold, at most, a single capsule.

16. The composition of claim 1, wherein at least one of said capsules comprises a further partition.

17. The composition of claim 16, wherein said further partition is selected from the group consisting of a capsule and a droplet in an emulsion.

18. The composition of claim 1, wherein at least one of said capsules has a shell selected from the group consisting of a polymeric shell, a hydrogel, a hydrophilic shell, a hydrophobic shell, a shell with a net positive charge, a shell with a net negative charge, a shell with a neutral charge, and combinations thereof.

19. The composition of claim 1, wherein said stimulus is selected from the group consisting of a chemical stimulus, a bulk stimulus, a biological stimulus, a light stimulus, a thermal stimulus, a magnetic stimulus, and combinations thereof.

20. The composition of claim 19, wherein said thermal stimulus comprises causing said composition to reach a temperature of at least 32 degrees Celsius.

21. The composition of claim 1, wherein said stimulus is selected from the group consisting of a change in pH, a change in ion concentration, reduction of disulfide bonds, and combinations thereof.

26. The composition of claim 1, wherein said droplets comprise a fluid that is of a lesser density than the density of said capsules.

27. The composition of claim 1, wherein said droplets comprise a fluid that is of a greater density than the density of said capsules.

28. The composition of claim 1, wherein said capsules are produced by a method selected from the group consisting of emulsification polymerization, layer-by-layer assembly with polyelectrolytes, coacervation, internal phase separation, flow focusing, and combinations thereof.

29. The composition of claim 1, wherein said stimulus is applied to said capsules.

30. The composition of claim 1, wherein said stimulus is applied to said droplets.

3. (canceled)

11. (canceled)

22-25. -25. (canceled)

31. A composition comprising a plurality of outer capsules, said outer capsules comprising at least one inner capsule, wherein said at least one inner capsule is configured to release its contents into at least one outer capsule among said plurality of outer capsules upon the application of a stimulus.

32-77. -77. (canceled)

78. A device comprising a plurality of partitions, wherein at least one partition of said plurality of partitions comprises a capsule, wherein said capsule is situated within a droplet in an emulsion, wherein said capsule is configured to release its contents into said droplet upon the application of a stimulus.

79-88. -88. (canceled)

89. A device comprising a plurality of partitions, wherein at least one partition of said plurality of partitions comprises an outer capsule, said outer capsule comprising at least one inner capsule, wherein said at least one inner capsule is configured to release its contents into said outer capsule upon the application of a stimulus.

90-109. -109. (canceled)

110. A method comprising:

a. providing a plurality of capsules, said capsules situated within droplets in an emulsion, wherein said capsules are configured to release their contents into said droplets upon the application of a stimulus; and

b. providing a stimulus to cause said capsules to release their contents into said droplets.

111-117. -117. (canceled)

118. A method comprising:

a. providing a plurality of inner capsules, said inner capsules situated within outer capsules, wherein said inner capsules are configured to release their contents into said outer capsules upon the application of a stimulus; and

b. providing a stimulus to cause said inner capsules to release their contents into said outer capsules.

119-133. -133. (canceled)

134. A method comprising:

a. providing a sample comprising a cell;

b. lysing said cell, thereby generating a lysate; and

c. partitioning said lysate into a capsule.

135-155. -155. (canceled)

156. A kit for generating capsules within droplets in an emulsion, said kit comprising reagents for generating capsules, reagents for generating an emulsion, and instructions for generating said capsules within droplets in an emulsion.

157-158. -158. (canceled)

159. A kit for generating capsules within capsules, said kit comprising reagents for generating inner capsules, reagents for generating outer capsules, and instructions for generating capsules within capsules.

160-163. -163. (canceled)

164. A method of partitioning polynucleotides comprising:

a. isolating polynucleotides from a source of polynucleotides;

b. partitioning said polynucleotides at a predetermined coverage amount, to produce a plurality of partitions, wherein at least one partition comprises a mixture of non-overlapping polynucleotides, thereby generating partitioned polynucleotides; and

c. fragmenting said partitioned polynucleotides, thereby generating fragmented polynucleotides.

165-168. -168. (canceled)

169. A method of fragmenting a polynucleotide comprising:

a. providing a polynucleotide;

b. encapsulating said polynucleotide, thereby generating an encapsulated polynucleotide; and

c. fragmenting said polynucleotide, thereby generating an encapsulated fragmented polynucleotide.

170-174. -174. (canceled)