METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180282803A1
• Filed: 03/22/2018
• Published: 10/04/2018
• Est. Priority Date: 12/22/2016
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Belgrader, Phillip, Bent, Zachary, Bharadwaj, Rajiv, Gopalan, Vijay Kumar Sreenivasa, Harada, Josephine, Hindson, Christopher, Lenji, Mohammad Rahimi, Lucero, Michael Ybarra, 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, Wheeler, Tobias Daniel, Wu, Indira, Ziraldo, Solongo Batjargal
• Granted Patent: US 10,480,029 B2
• 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

1-30. -30. (canceled)

31. A method for multi-analyte processing or analysis, comprising:

(a) providing a mixture comprising a first analyte, a second analyte that is different than said first analyte, and a support having coupled thereto (i) a first barcode molecule capable of coupling to said first analyte and (ii) a second barcode molecule capable of coupling to said second analyte;

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

(c) characterizing (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.

32. The method of claim 31, wherein said support is a bead.

33. The method of claim 32, wherein said bead is a gel bead.

34. The method of claim 31, wherein, after (a), said first barcode molecule and/or said second barcode molecule is released from said support.

35. The method of claim 31, wherein said first analyte and said second analyte are differently selected from the group consisting of a deoxyribonucleic acid molecule, a ribonucleic acid molecule, and a labelling agent capable of coupling to a protein.

36. The method of claim 35, wherein said protein is a surface feature of a cell.

37. The method of claim 35, wherein said labelling agent further comprises a labelling agent barcode molecule, wherein said first barcode molecule or said second barcode molecule is capable of coupling to said labelling agent barcode molecule.

38. The method of claim 37, wherein said labelling agent barcode molecule is used to synthesize said first barcoded product or said second barcoded product.

39. The method of claim 37, wherein said labelling agent is an antibody.

40. The method of claim 31, 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.

41. The method of claim 31, 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.

42. The method of claim 41, 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.

43. The method of claim 41, 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.

44. The method of claim 43, 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.

45. The method of claim 41, wherein said first or said second analyte is a labelling agent capable of coupling to a protein, and wherein said labelling agent comprises a labelling agent nucleic acid molecule coupled thereto.

46. The method of claim 45, wherein said labelling agent nucleic acid molecule coupled to said labeling agent comprises a peptide barcode sequence, and wherein said peptide barcode sequence identifies said protein.

47. The method of claim 41, wherein said first barcode molecule comprises a first priming sequence capable of hybridizing to said first analyte, and wherein said second barcode molecule comprises a second priming sequence capable of hybridizing to said second analyte.

48. The method of claim 47, wherein said first priming sequence or said second priming sequence is an oligo(dT) sequence, a random N-mer sequence, or a sequence complementary to a sequence in a labelling agent nucleic acid molecule coupled to a labelling agent, which labelling agent is capable of coupling to a protein in said mixture.

49. The method of claim 31, wherein said mixture is in a partition among a plurality of partitions.

50. The method of claim 49, wherein said partition is a droplet and said plurality of partitions is a plurality of droplets.

51. The method of claim 49, wherein said partition is a well and said plurality of partitions is a plurality of wells.

52. The method of claim 49, wherein said partition comprises said first analyte, said second analyte, and said support.

53. The method of claim 52, wherein said partition further comprises a cell, wherein said first analyte and/or said second analyte are associated with said cell.

54. The method of claim 52, 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.

55. The method of claim 54, wherein said support is a bead.

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

57. The method of claim 54, wherein after (a), said first barcode molecule and said second barcode molecule are released from said support.

58. The method of claim 57, wherein said first barcode molecule and said second barcode molecule are released upon application of a chemical stimulus.

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

60. The method of claim 58, wherein said partition further comprises said chemical stimulus.