Methods and systems for processing polynucleotides

• US 10,344,329 B2
• Filed: 07/25/2018
• Issued: 07/09/2019
• Est. Priority Date: 06/26/2014
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mirna, Saxonov, Serge
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/045,474
• Publication Number:
  US 20180327839A1
• Time in Patent Office: 349 Days
• 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 2525/191 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 2537/149 Sequential reactions
• 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 2565/629 being a microfluidic device

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 cellular analysis, comprising:

(a) causing an analyte of a cell to contact a cell feature binding group to provide said cell feature binding group coupled to said analyte, wherein said cell feature binding group is associated with a first nucleic acid molecule comprising a first barcode sequence; and

(b) using said first nucleic acid molecule and a second nucleic acid molecule to generate a third nucleic acid molecule, wherein said second nucleic acid molecule comprises a second barcode sequence, and wherein said third nucleic acid molecule comprises (i) said first barcode sequence or complement thereof, and (ii) said second barcode sequence or complement thereof.

2. The method of claim 1, further comprising processing said third nucleic acid molecule or derivative thereof to generate data indicative of said first barcode sequence and said second barcode sequence, and using said data to identify said analyte as being of said cell or as having originated from said cell.

3. The method of claim 2, wherein said processing comprises sequencing said third nucleic acid molecule or derivative thereof.

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

5. The method of claim 4, wherein said protein is a cell surface protein.

6. The method of claim 1, wherein said cell comprises said cell feature binding group coupled to said analyte, and wherein said cell is co-partitioned with a bead in a partition among a plurality of partitions, wherein said bead comprises said second barcode sequence coupled thereto.

7. The method of claim 6, wherein said second barcode sequence is releasably attached to said bead.

8. The method of claim 7, further comprising releasing said second barcode sequence from said bead.

9. The method of claim 7, wherein said second barcode sequence is releasably coupled to said bead through a chemical cross-linker.

10. The method of claim 7, wherein said second barcode sequence is releasably coupled to said bead through a disulfide bond.

11. The method of claim 7, wherein said partition further comprises an agent capable of subjecting said second barcode sequence to release from said bead.

12. The method of claim 11, wherein said agent is a reducing agent.

13. The method of claim 6, wherein said bead is a gel bead.

14. The method of claim 6, wherein said partition is a droplet and said plurality of partitions is a plurality of droplets.

15. The method of claim 6, wherein said partition is a well and said plurality of partitions is a plurality of wells.

16. The method of claim 6, wherein said third nucleic acid molecule is generated in said partition, and wherein, subsequent to (b), said third nucleic acid molecule or derivative thereof is released from said partition.

17. The method of claim 1, wherein said second nucleic acid molecule comprises a unique molecular identification sequence.

18. The method of claim 1, wherein said second nucleic acid molecule comprises a sequence complementary to a sequence of said first nucleic acid molecule.

19. The method of claim 1, wherein said second nucleic acid molecule comprises a primer sequence.

20. The method of claim 1, wherein said first nucleic acid molecule or said second nucleic acid molecule comprises (i) a sequence configured to attach to a flow cell of a sequencer, (ii) or a complement of said sequence.

21. The method of claim 1, further comprising subjecting said third nucleic acid molecule or derivative thereof to a polymerase chain reaction.

22. The method of claim 1, further comprising adding one or more functional sequences to said third nucleic acid molecule or derivative thereof, wherein said one or more functional sequences are configured to permit attachment of said third nucleic acid molecule to a flow cell of a sequencer.

23. The method of claim 1, wherein (b) comprises performing a nucleic acid extension reaction using said first nucleic acid molecule and said second nucleic acid molecule to yield said third nucleic acid molecule.

31. The method of claim 1, wherein said cell feature binding group is an antibody or antibody fragment.

32. The method of claim 1, wherein said cell feature binding group is a cell surface receptor binding molecule.

33. The method of claim 1, wherein said analyte is coupled to said cell feature binding group by a covalent attachment.

34. The method of claim 1, wherein said analyte is coupled to said cell feature binding group by a non-covalent association.

35. The method of claim 1, wherein said cell is a tumor cell.

36. The method of claim 1, wherein said cell is a neural cell.

37. The method of claim 4, wherein said protein is a cluster of differentiation (CD) protein.

38. The method of claim 4, wherein said protein is a cell surface receptor.

39. The method of claim 4, wherein said protein is an antibody.

40. The method of claim 1, further comprising subjecting said third nucleic acid molecule or derivative thereof to a nucleic acid amplification reaction.

41. The method of claim 1, further comprising adding one or more functional sequences to said third nucleic acid molecule or derivative thereof, wherein said one or more functional sequences is a sequencing primer binding site.

42. The method of claim 1, further comprising processing said third nucleic acid molecule or derivative thereof to generate data indicative of said first barcode sequence and said second barcode sequence, and using said data to measure a quantity of said analyte in said cell.

43. The method of claim 42, wherein said processing comprises sequencing said third nucleic acid molecule or derivative thereof.

44. The method of claim 1, wherein (b) comprises (i) hybridizing said second nucleic acid molecule to said first nucleic acid molecule;

and (ii) extending said second nucleic acid molecule in a nucleic acid extension reaction to generate said third nucleic acid molecule.

45. The method of claim 44, wherein said first nucleic acid molecule comprises a first poly-adenine (poly-A) sequence, said second nucleic acid molecule comprises a first poly-thymine (poly-T) sequence, and said hybridizing occurs via hybridization between said first poly-A sequence and said first poly-T sequence.

46. The method of claim 1, further comprising using a messenger ribonucleic acid RNA (mRNA) molecule of said cell and a fourth nucleic acid molecule comprising said second barcode sequence to generate a fifth nucleic acid molecule comprising (i) a nucleic acid sequence corresponding to said mRNA molecule and (ii) said second barcode sequence.

47. The method of claim 46, wherein said fourth nucleic acid molecule comprises a poly-T sequence, wherein said mRNA molecule comprises a poly-A sequence, and said fifth nucleic acid molecule is generated by hybridizing said poly-A sequence of said mRNA molecule to said poly-T sequence of said fourth nucleic acid molecule and extending said fourth nucleic acid molecule in a nucleic acid extension reaction.

48. The method of claim 47, wherein said nucleic acid extension reaction is performed with the aid of a reverse transcriptase.

49. The method of claim 47, wherein said fifth nucleic acid molecule comprises deoxyribonucleic acid (DNA) complementary to at least a portion of said mRNA molecule.

50. The method of claim 47, further comprising sequencing said fifth nucleic acid molecule or derivative thereof to identify said nucleic acid sequence corresponding to said mRNA molecule and said second barcode sequence.

51. The method of claim 1, further comprising using a complementary deoxyribonucleic acid (cDNA) molecule, complementary to at least a portion of a messenger ribonucleic acid RNA (mRNA) molecule of said cell, and a fourth nucleic acid molecule comprising said second barcode sequence to generate a fifth nucleic acid molecule comprising (i) a nucleic acid sequence corresponding to said mRNA molecule and (ii) said second barcode sequence.

52. The method of claim 1, wherein said first nucleic acid molecule comprises a primer sequence.

53. The method of claim 1, wherein said first barcode sequence is associated with said cell feature binding group.

24. A method for cellular analysis, comprising:

(a) causing an analyte of a cell to contact a cell-binding ligand to provide said cell-binding ligand coupled to said analyte, wherein said cell-binding ligand is associated with a first nucleic acid molecule comprising a first barcode sequence; and

(b) using said first nucleic acid molecule and a second nucleic acid molecule to generate a third nucleic acid molecule, wherein said second nucleic acid molecule comprises a second barcode sequence, and wherein said third nucleic acid molecule comprises (i) said first barcode sequence or complement thereof, and (ii) said second barcode sequence or complement thereof.

25. The method of claim 24, further comprising processing said third nucleic acid molecule or derivative thereof to generate data indicative of said first barcode sequence and said second barcode sequence, and using said data to identify said analyte as being of said cell or as having originated from said cell.

26. The method of claim 25, wherein said processing comprises sequencing said third nucleic acid molecule or derivative thereof.

27. The method of claim 24, wherein said analyte is a protein.

28. The method of claim 27, wherein said protein is a cell surface protein.

29. The method of claim 24, wherein said cell comprises said cell-binding ligand coupled to said analyte, and wherein said cell is partitioned with a bead in a partition among a plurality of partitions, which bead comprises said second barcode sequence coupled thereto.

30. The method of claim 29, wherein said bead is a gel bead.

54. The method of claim 24, wherein said cell-binding ligand is an antibody or an antibody fragment.

55. The method of claim 24, wherein said cell-binding ligand is a cell surface receptor binding molecule.

56. The method of claim 24, wherein said analyte is coupled to said cell-binding ligand by a covalent attachment.

57. The method of claim 24, wherein said analyte is coupled to said cell-binding ligand by a non-covalent association.

58. The method of claim 24, wherein said cell is a tumor cell.

59. The method of claim 24, wherein said cell is a neural cell.

60. The method of claim 27, wherein said protein is a cluster of differentiation (CD) protein.

61. The method of claim 27, wherein said protein is a cell surface receptor.

62. The method of claim 27, wherein said protein is an antibody.

63. The method of claim 29, wherein said second barcode sequence is releasably coupled to said bead through a chemical cross-linker.

64. The method of claim 29, wherein said second barcode sequence is releasably coupled to said bead through a disulfide bond.

65. The method of claim 29, wherein said partition further comprises an agent capable of subjecting said second barcode sequence to release from said bead to yield said second barcode sequence disassociated from said bead.

66. The method of claim 65, wherein said agent is a reducing agent.

67. The method of claim 29, wherein said partition is a droplet and said plurality of partitions is a plurality of droplets.

68. The method of claim 29, wherein said partition is a well and said plurality of partitions is a plurality of wells.

69. The method of claim 29, wherein said third nucleic acid molecule is generated in said partition, and wherein subsequent to (b), said third nucleic acid molecule or derivative thereof is released from said partition.

70. The method of claim 24, wherein said second nucleic acid molecule comprises a unique molecular identification sequence.

71. The method of claim 24, wherein said second nucleic acid molecule is at least partially complementary to said first nucleic acid molecule.

72. The method of claim 24, wherein said first nucleic acid molecule comprises a primer sequence.

73. The method of claim 24, wherein said first nucleic acid molecule comprises an adapter sequence configured to attach to a flow cell of a sequencer.

74. The method of claim 24, further comprising subjecting said third nucleic acid molecule or derivative thereof to a polymerase chain reaction.

75. The method of claim 24, further comprising adding one or more functional sequences to said third nucleic acid molecule or derivative thereof, wherein said one or more functional sequences are configured to permit attachment to a flow cell of a sequencer.

76. The method of claim 24, wherein (b) comprises performing a nucleic acid extension reaction using said first nucleic acid molecule and said second nucleic acid molecule to yield said third nucleic acid molecule.

77. The method of claim 24, further comprising subjecting said third nucleic acid molecule or derivative thereof to a nucleic acid amplification reaction.

78. The method of claim 24, further comprising adding one or more functional sequences to said third nucleic acid molecule or derivative thereof, wherein said one or more functional sequences is a sequencing primer binding site.

79. The method of claim 24, further comprising processing said third nucleic acid molecule or derivative thereof to generate data indicative of said first barcode sequence and said second barcode sequence, and using said data to measure a quantity of said analyte in said cell.

80. The method of claim 79, wherein said processing comprises sequencing said third nucleic acid molecule or derivative thereof.

81. The method of claim 24, wherein (b) comprises (i) hybridizing said second nucleic acid molecule to said first nucleic acid molecule;

and (ii) extending said second nucleic acid molecule in a nucleic acid extension reaction to generate said third nucleic acid molecule.

82. The method of claim 81, wherein said first nucleic acid molecule comprises a first poly-adenine (poly-A) sequence, said second nucleic acid molecule comprises a first poly-thymine (poly-T) sequence, and said hybridizing occurs via hybridization between said first poly-A sequence and said first poly-T sequence.

83. The method of claim 24, further comprising using a messenger ribonucleic acid RNA (mRNA) molecule of said cell and a fourth nucleic acid molecule comprising said second barcode sequence to generate a fifth nucleic acid molecule comprising (i) a nucleic acid sequence corresponding to said mRNA molecule and (ii) said second barcode sequence.

84. The method of claim 83, wherein said fourth nucleic acid molecule comprises a poly-T sequence, wherein said mRNA molecule comprises a poly-A sequence, and said fifth nucleic acid molecule is generated by hybridizing said poly-A sequence of said mRNA molecule to said poly-T sequence of said fourth nucleic acid molecule and extending said fourth nucleic acid molecule in a nucleic acid extension reaction.

85. The method of claim 84, wherein said nucleic acid extension reaction is performed with the aid of a reverse transcriptase.

86. The method of claim 84, wherein said fifth nucleic acid molecule comprises deoxyribonucleic acid (DNA) complementary to at least a portion of said mRNA molecule.

87. The method of claim 84, further comprising sequencing said fifth nucleic acid molecule or derivative thereof to identify said nucleic acid sequence corresponding to said mRNA molecule and said second barcode sequence.

88. The method of claim 24, further comprising using a complementary deoxyribonucleic acid (cDNA) molecule, complementary to at least a portion of a messenger ribonucleic acid RNA (mRNA) molecule of said cell, and a fourth nucleic acid molecule comprising said second barcode sequence to generate a fifth nucleic acid molecule comprising (i) a nucleic acid sequence corresponding to said mRNA molecule and (ii) said second barcode sequence.

89. The method of claim 24, wherein said first barcode sequence is associated with said cell-binding ligand.

90. A method for cellular analysis, comprising:

(a) causing a first protein of a cell to contact a first cell-binding ligand to provide said first cell-binding ligand coupled to said first protein, wherein said first cell-binding ligand is associated with a first nucleic acid molecule comprising a first barcode sequence;

(b) causing a second protein of said cell to contact a second cell-binding ligand to provide said second cell-binding ligand coupled to said second protein, wherein said second cell-binding ligand is associated with a second nucleic acid molecule comprising a second barcode sequence;

(c) using said first nucleic acid molecule and a third nucleic acid molecule to generate a fourth nucleic acid molecule, wherein said third nucleic acid molecule comprises a third barcode sequence, and wherein said fourth nucleic acid molecule comprises (i) said first barcode sequence or complement thereof, and (ii) said third barcode sequence or complement thereof; and

(d) using said second nucleic acid molecule and a fifth nucleic acid molecule to generate a sixth nucleic acid molecule, wherein said fifth nucleic acid molecule comprises a fourth barcode sequence, and wherein said sixth nucleic acid molecule comprises (i) said second barcode sequence or complement thereof, and (ii) said fourth barcode sequence or complement thereof.

91. The method of claim 90, wherein said first cell-binding ligand is a first antibody and said second cell-binding ligand is a second antibody.

92. The method of claim 90, wherein said cell is co-partitioned with a bead in a partition among a plurality of partitions, wherein said third barcode sequence or said fourth barcode sequence are coupled to said bead.

93. The method of claim 92, wherein said third barcode sequence and said fourth barcode sequence are identical.

94. The method of claim 92, wherein said partition is a droplet and said plurality of partitions is a plurality of droplets.

95. The method of claim 92, wherein said partition is a well and said plurality of partitions is a plurality of wells.

96. The method of claim 90, further comprising causing a nucleic acid molecule from said cell or derivative thereof to contact a seventh nucleic acid molecule to generate an eighth nucleic acid molecule, wherein said seventh nucleic acid molecule comprises said third barcode sequence, and wherein said eighth nucleic acid molecule comprises said third barcode sequence or complement thereof.

97. The method of claim 96, wherein said nucleic acid molecule from said cell or derivative thereof is a ribonucleic acid (RNA) molecule.

98. The method of claim 97, wherein said RNA molecule is a messenger RNA (mRNA) molecule.

99. The method of claim 96, wherein said nucleic acid molecule from said cell or derivative thereof is a deoxyribonucleic acid (DNA) molecule.

100. The method of claim 90, wherein said cell is co-partitioned with a bead in a partition among a plurality of partitions, wherein said third barcode sequence and said fourth barcode sequence are coupled to said bead.

101. The method of claim 100, wherein said third barcode sequence and said fourth barcode sequence comprise different barcode sequences.

102. The method of claim 90, wherein said first barcode sequence is associated with said first cell-binding ligand.

103. The method of claim 90, wherein said second barcode sequence is associated with said second cell-binding ligand.

104. The method of claim 90, wherein said first nucleic acid molecule comprises a primer sequence.

105. The method of claim 90, wherein said second nucleic acid molecule comprises a primer sequence.