Partitioning and processing of analytes and other species

• US 9,644,204 B2
• Filed: 02/07/2014
• Issued: 05/09/2017
• Est. Priority Date: 02/08/2013
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Saxonov, Serge, Ness, Kevin, Hardenbol, Paul, Hindson, Christopher, Masquelier, Donald, Jarosz, Mirna, Schnall-Levin, Michael
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/175,935
• Publication Number:
  US 20140227684A1
• Time in Patent Office: 1,187 Days
• 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 to provide said contents in said droplets in said emulsion, 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.

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.

3. 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.

4. The composition of claim 3, 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.

5. The composition of claim 4, wherein said restriction enzyme is a restriction enzyme that is a rare cutter.

6. The composition of claim 3, wherein said barcode is an oligonucleotide barcode.

7. 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.

8. The composition of claim 7, wherein said analyte is a polynucleotide.

9. The composition of claim 8, wherein said polynucleotide is selected from the group consisting of DNA, RNA, cDNA, and combinations thereof.

10. The composition of claim 8, wherein the amount of said polynucleotide in said composition is an amount sufficient to provide about 100-200X sequence coverage.

11. The composition of claim 7, wherein each capsule comprises, on average, about one chromosome.

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

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

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

15. The composition of claim 14, wherein said further partition is selected from the group consisting of a capsule and a droplet in an emulsion.

16. 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.

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

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

19. 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.

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

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

27. The composition of claim 1, wherein said contents comprise at least 10,000 barcoded oligonucleotides releasably attached to each of said capsules.

33. The composition of claim 1, wherein said capsules are gels.

22. A composition comprising a plurality of outer capsules in an emulsion, 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 to provide said contents in said outer capsule in said emulsion, 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.

28. The composition of claim 22, wherein said contents comprise at least 10,000 barcoded oligonucleotides releasably attached to said inner capsule.

34. The composition of claim 22, wherein said inner capsule is a gel.

35. The composition of claim 22, wherein at least one of said inner capsules and said outer capsules comprise a species selected from the group consisting of a reagent and an analyte.

36. The composition of claim 35, wherein said reagent is selected from the group consisting of a protein, a polynucleotide, an enzyme, an antibody, a barcode, an adaptor, a buffer, a small molecule, a detergent, a dye, a polymer and combinations thereof.

37. The composition of claim 36, 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.

38. The composition of claim 36, wherein said barcode is an oligonucleotide barcode.

39. The composition of claim 35, 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.

40. The composition of claim 39, wherein said analyte is a polynucleotide.

23. 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 to provide said contents in said droplet in said emulsion, 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.

29. The device of claim 23, wherein said contents comprise at least 10,000 barcoded oligonucleotides releasably attached to said capsule.

24. A device comprising a plurality of partitions, wherein at least one partition of said plurality of partitions comprises an outer capsule in an emulsion, 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 to provide said contents in said outer capsule in said emulsion, 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.

30. The device of claim 24, wherein said contents comprise at least 10,000 barcoded oligonucleotides releasably attached to said inner capsule.

25. 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, 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; and

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

31. The method of claim 25, wherein said contents comprise at least 10,000 barcoded oligonucleotides releasably attached to each of said capsule.

26. A method comprising:

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

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

32. The method of claim 26, wherein said contents comprise at least 10,000 barcoded oligonucleotides releasably attached to each of said inner capsule.