METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180179590A1
• Filed: 09/29/2017
• Published: 06/28/2018
• Est. Priority Date: 12/22/2016
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Belgrader, Phillip, Bent, Zachary, Gopalan, Vijay Kumar Sreenivasa, Harada, Josephine, Hindson, Christopher, Lenji, Mohammad Rahimi, McDermott, Geoffrey, Meer, Elliott, Mikkelsen, Tarjei Sigurd, O'Keeffe, Christopher Joachim, Pfeiffer, Katherine, Price, Andrew D., Ryvkin, Paul, Saxonov, Serge, Stuelpnagel, John R., Terry, Jessica Michele
• Granted Patent: US 10,011,872 B1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• 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 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

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 method for analyte characterization, comprising:

(a) providing a plurality of partitions, wherein a partition of said plurality of partitions comprises a plurality of barcode molecules and a plurality of analytes, wherein said plurality of barcode molecules is coupled to a bead, wherein (i) a first individual barcode molecule of said plurality of barcode molecules comprises a first nucleic acid barcode sequence and is capable of coupling to a first analyte of said plurality of analytes, and (ii) a second individual barcode molecule of said plurality of barcode molecules comprises a second nucleic acid barcode sequence and is capable of coupling to a second analyte of said plurality of analytes, wherein said first analyte and said second analyte are different types of analytes; and

(b) in said partition,

(i) using at least a portion of said first individual barcode molecule and said first analyte to synthesize a first nucleic acid molecule comprising (1) at least a portion of said first nucleic acid barcode sequence or a complement of said portion of said first nucleic acid barcode sequence and (2) a first sequence of said first analyte or a complement of said first sequence; and

(ii) using at least a portion of said second individual barcode molecule and said second analyte to synthesize a second nucleic acid molecule comprising (1) at least a portion of said second nucleic acid barcode sequence or a complement of said portion of said second nucleic acid barcode sequence and (2) a second sequence of said second analyte or a complement of said second sequence; and

(c) removing or releasing said first nucleic acid molecule and said second nucleic acid molecule from said partition,

wherein, after (a), said first individual barcode molecule or said second individual barcode molecule is released from said bead.

2. The method of claim 1, further comprising subjecting said first nucleic acid molecule or derivative thereof and said second nucleic acid molecule or derivative thereof to sequencing to characterize said first analyte or said second analyte.

3. The method of claim 2, further comprising repeating (a)-(c) based on a characterization of said first analyte or said second analyte from said sequencing.

4. The method of claim 2, further comprising selecting an analyte for characterization based on a characterization of said first analyte or said second analyte obtained from said sequencing upon repeating (a)-(c).

5. The method of claim 1, wherein said first individual barcode molecule or said second individual barcode molecule comprises an identifier sequence that is different from identifier sequences associated with other nucleic acid molecules in said partition.

6. The method of claim 1, wherein said first analyte is a nucleic acid molecule.

7. The method of claim 1, wherein said first analyte is a labelling agent capable of coupling to a surface feature of a cell.

8. The method of claim 7, wherein said first individual barcode molecule or said second individual barcode molecule is capable of coupling to said labelling agent via a nucleic acid molecule of said labelling agent.

9. The method of claim 7, wherein said surface feature is a receptor, an antigen or a protein.

11. The method of claim 7, wherein said partition comprises said cell or one or more components of said cell.

12. The method of claim 11, wherein said partition comprises a single cell.

13. The method of claim 1, wherein said first nucleic acid molecule or said second nucleic molecule comprises a third nucleic acid barcode sequence.

14. The method of claim 13, wherein said third nucleic acid barcode sequence is derived from a third nucleic acid molecule.

15. The method of claim 14, wherein said third nucleic acid molecule is coupled to a labelling agent capable of binding to a surface feature of a cell.

16. The method of claim 1, wherein said first analyte and said second analyte are different types of nucleic acid molecules.

17. The method of claim 16, wherein said first analyte is a messenger ribonucleic acid molecule and said second analyte is a deoxyribonucleic acid molecule.

18. The method of claim 16, wherein (i) said first individual barcode molecule comprises a first priming sequence capable of hybridizing to said first analyte;

or (ii) said second individual barcode molecule comprises a second priming sequence capable of hybridizing to said second analyte.

19. The method of claim 1, wherein said first analyte is a nucleic acid molecule and said second analyte is a labelling agent capable of coupling to a surface feature of a cell.

20. The method of claim 19, wherein said first analyte is a ribonucleic acid molecule.

21. The method of claim 19, wherein (i) said first individual barcode molecule comprises a first priming sequence capable of hybridizing to said first analyte;

or (ii) said second individual barcode molecule comprises a second priming sequence capable of hybridizing to a nucleic acid molecule of said labelling agent.

22. The method of claim 1, wherein said first nucleic acid barcode sequence and said second nucleic acid barcode sequence are identical.

23. The method of claim 1, wherein said first analyte comprises a nucleic acid sequence that encodes at least a portion of a V(D)J sequence of an immune cell receptor, or a complement of said nucleic sequence.

24. The method of claim 1, wherein said first analyte is a nucleic acid molecule comprising a nucleic acid sequence that is capable of functioning as a component of a gene editing reaction.

26. The method of claim 1, wherein said bead is a gel bead.

28. The method of claim 1, wherein said partition further comprises an agent capable of releasing said first individual barcode molecule or said second individual barcode from said bead.

29. The method of claim 1, wherein said plurality of partitions is a plurality of droplets.

30. The method of claim 1, further comprising performing one or more reactions involving said first nucleic acid molecule or said second nucleic acid molecule subsequent to removing or releasing said first nucleic acid molecule and said second nucleic acid molecule from said partition.

32. The method of claim 1, wherein, after (a), said first individual barcode molecule and said second individual barcode molecule are released from said bead.

33. The method of claim 1, wherein, prior to or during (b), said first individual barcode molecule or said second individual barcode molecule is released from said bead.

34. The method of claim 1, wherein said first nucleic acid barcode sequence and said second nucleic acid barcode sequence are different.

35. The method of claim 1, wherein (i) said first analyte comprises a nucleic acid sequence that encodes at least a portion of a V(D)J sequence of an immune cell receptor or complement of said nucleic acid sequence, and (ii) said second analyte comprises a nucleic acid molecule coupled to a major histocompatibility complex.

36. The method of claim 1, wherein said partition comprises a switch oligonucleotide that aids in synthesizing said first nucleic acid molecule or said second nucleic acid molecule.

37. The method of claim 7, wherein said labelling agent comprises an antibody.

38. The method of claim 7, wherein said labelling agent comprises an antibody fragment.

39. The method of claim 7, wherein said labelling agent comprises a protein.

40. The method of claim 7, wherein said labelling agent comprises a major histocompatibility complex.

41. The method of claim 7, wherein said labelling agent comprises a plurality of major histocompatibility complex molecules.

42. The method of claim 7, wherein said labeling is among a plurality of labelling agents in said partition.

43. The method of claim 42, wherein said plurality of labelling agents comprises a plurality of major histocompatibility complex molecules.

44. The method of claim 24, wherein said second analyte is a messenger ribonucleic acid molecule.

45. The method of claim 1, wherein said first analyte is a ribonucleic acid molecule or a deoxyribonucleic acid molecule.

46. The method of claim 1, wherein said first analyte is a ribonucleic acid molecule.

47. The method of claim 1, wherein said partition comprises an agent that releases said first individual barcode molecule and said second individual barcode molecule from said bead.

48. The method of claim 26 wherein said partition comprises an agent that degrades said gel bead to release said first individual barcode molecule and said second individual barcode molecule from said bead.

49. The method of claim 1, wherein said bead comprises a disulfide bond.

50. The method of claim 26, wherein said gel bead comprises a disulfide bond.

51. The method of claim 1, wherein said first individual barcode molecule or said second individual barcode molecule is coupled to said bead via a disulfide bond.

52. The method of claim 51, wherein said first individual barcode molecule and said second individual barcode molecule are each coupled to said bead via a disulfide bond.

53. The method of claim 1, wherein said first analyte is capable of coupling to a protein.

54. The method of claim 53, wherein said second analyte is a ribonucleic acid molecule.

55. The method of claim 54, wherein said ribonucleic acid molecule is a messenger ribonucleic acid molecule.

56. The method of claim 18, wherein said first priming sequence or said second priming sequence comprises a poly-thymine (poly-T) sequence.

57. The method of claim 56, wherein each of said first priming sequence and said second priming sequence comprises said poly-T sequence.

58. The method of claim 1, wherein said plurality of partitions is a plurality of wells.

10. (canceled)

25. (canceled)

27. (canceled)

31. (canceled)