Methods and systems for processing polynucleotides

• US 10,480,029 B2
• Filed: 03/22/2018
• Issued: 11/19/2019
• Est. Priority Date: 12/22/2016
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Bent, Zachary, Harada, Josephine, Hindson, Christopher, Meer, Elliott, Mikkelsen, Tarjei Sigurd, O'Keeffe, Christopher Joachim, Pfeiffer, Katherine, Price, Andrew D., Ryvkin, Paul, Saxonov, Serge, Stuelpnagel, John R., Terry, Jessica Michele, Ziraldo, Solongo Batjargal
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/933,299
• Publication Number:
  US 20180282803A1
• Time in Patent Office: 607 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 multi-analyte processing or analysis, comprising:

(a) providing a mixture comprising a first analyte, a second analyte, a labelling agent comprising a reporter oligonucleotide molecule that comprises a reporter barcode sequence associated with said labelling agent, and a support having coupled thereto

(i) a first barcode molecule comprising a first oligonucleotide sequence configured to couple to said reporter oligonucleotide molecule, and

(ii) a second barcode molecule comprising a second oligonucleotide sequence configured to couple to said second analyte,

 

wherein said second analyte is a different type of analyte than said first analyte,

 

wherein said first oligonucleotide sequence is different from said second oligonucleotide sequence, and

 

wherein said labelling agent is configured to couple to said first analyte;

(b) in said mixture, (i) using said reporter oligonucleotide molecule and said first oligonucleotide sequence of said first barcode molecule to synthesize a first barcoded product, and (ii) using said second analyte and said second oligonucleotide sequence of said second barcode molecule to synthesize a second barcoded product; and

(c) determining a sequence of (i) said first barcoded product or derivative thereof and (ii) said second barcoded product or derivative thereof, to identify said first analyte and/or said second analyte.

2. The method of claim 1, wherein said support is a bead.

3. The method of claim 2, wherein said bead is a gel bead.

4. The method of claim 1, wherein, after (a), said first barcode molecule and/or said second barcode molecule is released from said support.

5. The method of claim 1, wherein said second analyte is selected from the group consisting of a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, and an additional labelling agent configured to couple to a protein.

6. The method of claim 1, wherein said first analyte is a surface feature of a cell.

7. The method of claim 5, wherein said additional labelling agent further comprises a second reporter oligonucleotide molecule comprising a second reporter barcode sequence wherein said second barcode molecule is configured to couple to said second reporter oligonucleotide molecule.

8. The method of claim 1, wherein said first barcoded product comprises said reporter barcode sequence.

9. The method of claim 1, wherein said labelling agent is an antibody.

10. The method of claim 1, further comprising, prior to (c), removing (i) said first barcoded product or derivative thereof and (ii) said second barcoded product or derivative thereof from said mixture.

11. The method of claim 1, wherein said first barcode molecule is a first nucleic acid molecule comprising a first nucleic acid barcode sequence, and wherein said second barcode molecule is a second nucleic acid molecule comprising a second nucleic acid barcode sequence.

12. The method of claim 11, wherein said first nucleic acid barcode sequence comprises a first barcode sequence segment, and wherein said second nucleic acid barcode sequence comprises a second barcode sequence segment that is identical to said first barcode sequence segment.

13. The method of claim 11, wherein said first barcoded product and said second barcoded product are nucleic acid molecules, wherein said first barcoded product comprises said first barcode sequence, and wherein said second barcoded product comprises said second barcode sequence.

14. The method of claim 13, wherein said characterizing comprises sequencing (i) said first barcoded product or a derivative thereof and (ii) said second barcoded product or a derivative thereof, to identify said first barcode sequence and said second barcode sequence.

15. The method of claim 1, wherein said first analyte is a protein.

16. The method of claim 11, wherein said first oligonucleotide sequence of said first barcode molecule comprises a first nucleic acid priming sequence capable of hybridizing to said reporter oligonucleotide molecule, and wherein said second oligonucleotide sequence of said second barcode molecule comprises a second nucleic acid priming sequence capable of hybridizing to said second analyte.

17. The method of claim 16, wherein said second nucleic acid priming sequence is a poly-T sequence, a random N-mer sequence, or a sequence complementary to a sequence of an additional reporter oligonucleotide coupled to a second labelling agent, which second labelling agent is configured to couple to a protein in said mixture.

18. The method of claim 1, wherein said mixture is in a partition among a plurality of partitions.

19. The method of claim 18, wherein said partition is a droplet and said plurality of partitions is a plurality of droplets.

20. The method of claim 18, wherein said partition is a well and said plurality of partitions is a plurality of wells.

21. The method of claim 18, wherein said partition comprises said first analyte, said second analyte, said labelling agent, and said support.

22. The method of claim 21, wherein said partition further comprises a cell, wherein said first analyte and/or said second analyte are associated with said cell.

23. The method of claim 21, wherein said first barcode molecule is a nucleic acid molecule comprising a first nucleic acid barcode sequence, wherein said second barcode molecule is a nucleic acid molecule comprising a second nucleic acid barcode sequence, and wherein said first barcode molecule and said second barcode molecule are releasably attached to said support.

24. The method of claim 23, wherein said support is a bead.

25. The method of claim 24, wherein said bead is a gel bead.

26. The method of claim 23, wherein after (a), said first barcode molecule and said second barcode molecule are released from said support.

27. The method of claim 26, wherein said first barcode molecule and said second barcode molecule are released upon application of a chemical stimulus.

28. The method of claim 27, wherein said partition further comprises said chemical stimulus.

29. The method of claim 1, wherein said first barcode molecule or said second barcode molecule comprises one or more functional sequences selected from the group consisting of an adapter sequence, a primer sequence, a primer binding sequence, a unique molecular identification (UMI) sequence, and a sequence configured to couple to a flow cell of a sequencer.

30. The method of claim 6, wherein said surface feature is a receptor, an antigen, or a protein.

31. The method of claim 18, further comprising removing or releasing said first barcoded product or derivative thereof and said second barcoded product or derivative thereof from said partition.

32. The method of claim 18, wherein said partition comprises an agent that releases said first barcode molecule and said second barcode molecule from said support.

33. The method of claim 18, wherein said partition comprises an agent that degrades said support to release said first barcode molecule and said second barcode molecule from said support.

34. The method of claim 1, wherein said second 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.

35. The method of claim 1, wherein said second analyte comprises a nucleic acid molecule coupled to a major histocompatibility complex.

36. The method of claim 1, wherein said mixture comprises a switch oligonucleotide that aids in synthesizing said first barcoded product or said second barcoded product.

37. The method of claim 36, wherein said switch oligonucleotide comprises a 3′

poly-guanine (poly-G) sequence.

38. The method of claim 1, wherein said labelling agent comprises an antibody fragment.

39. The method of claim 1, wherein said labelling agent comprises a protein.

40. The method of claim 1, wherein said labelling agent comprises a major histocompatibility complex.

41. The method of claim 1, wherein said labelling agent comprises a plurality of major histocompatibility complex molecules.

42. The method of claim 5, wherein said second analyte is a RNA molecule, and said RNA molecule is a messenger RNA (mRNA) molecule.

43. The method of claim 1, wherein said support comprises a disulfide bond.

44. The method of claim 2, wherein said bead comprises a disulfide bond.

45. The method of claim 3, wherein said gel bead comprises a disulfide bond.

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

47. The method of claim 1, wherein said reporter oligonucleotide molecule comprises one or more functional sequences.

48. The method of claim 47, wherein said one or more functional sequences are selected from the group consisting of an adapter sequence, a primer sequence, a primer binding sequence, a unique molecular identifier (UMI), and a sequence configured to attach to a flow cell of a sequencer.

49. The method of claim 1, wherein said labelling agent comprises an aptamer.

50. The method of claim 49, wherein said reporter barcode sequence identifies said aptamer.

51. The method of claim 3, wherein said gel bead is a degradable gel bead.

52. The method of claim 51, wherein said degradable gel bead comprises a labile bond degradable upon application of a stimulus.

53. The method of claim 52, wherein said stimulus is a thermal stimulus, chemical stimulus, enzymatic stimulus, or photo-stimulus.

54. The method of claim 53, wherein said stimulus is a chemical stimulus comprising a reducing agent.

55. The method of claim 53, wherein said gel bead is a degradable gel bead.

56. The method of claim 55, wherein said degradable gel bead comprises a labile bond degradable upon application of a stimulus.

57. The method of claim 56, wherein said stimulus is a thermal stimulus, chemical stimulus, enzymatic stimulus, or photo-stimulus.

58. The method of claim 57, wherein said stimulus is a chemical stimulus comprising a reducing agent.