Methods and systems for processing polynucleotides

• US 10,676,789 B2
• Filed: 12/21/2017
• Issued: 06/09/2020
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Saxonov, Serge, Ness, Kevin, Hardenbol, Paul, Schnall-Levin, Michael, Jarosz, Mirna
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/850,241
• Publication Number:
  US 20180274027A1
• Time in Patent Office: 901 Days
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

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 nucleic acid sequence analysis, comprising:

(a) providing a plurality of partitions, a plurality of sample polynucleotide molecules and a plurality of barcode molecules,

wherein said plurality of barcode molecules comprise a plurality of barcode sequences, wherein at least 1,000 partitions of said plurality of partitions each comprise (i) a sample polynucleotide molecule from said plurality of sample polynucleotide molecules, and (ii) a barcode molecule from said plurality of barcode molecules,

wherein said plurality of barcode sequences are different across said at least 1,000 partitions;

(b) using said plurality of sample polynucleotide molecules and said plurality of barcode molecules in said at least 1,000 partitions to generate a plurality of barcoded polynucleotide molecules,

wherein said plurality of barcoded polynucleotide molecules comprises said plurality of barcode sequences that are different across said at least 1,000 partitions; and

(c) determining sequences of said plurality of barcoded polynucleotide molecules.

2. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 10,000 partitions.

3. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 100,000 partitions.

4. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 1,000,000 partitions.

5. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 10,000,000 partitions.

6. The method of claim 1, wherein said at least 1,000 partitions is 10,000 to 100,000 partitions.

7. The method of claim 1, wherein a partition of said at least 1,000 partitions comprises at least 1,000 barcode molecules of said plurality of barcode molecules.

8. The method of claim 7, wherein each of said at least 1,000 barcode molecules comprise a common barcode sequence.

9. The method of claim 7, wherein said partition comprises at least 20,000 barcode molecules of said plurality of barcode molecules.

10. The method of claim 9, wherein each of said at least 20,000 barcode molecules comprise a common barcode sequence.

11. The method of claim 1, wherein said plurality of barcode molecules is coupled to a plurality of beads.

12. The method of claim 11, wherein said plurality of beads is a plurality of solid particles.

13. The method of claim 11, wherein said plurality of beads is a plurality of gel beads.

14. The method of claim 1, further comprising, subsequent to (b), subjecting said plurality of barcoded polynucleotide molecules to release from said plurality of partitions.

15. The method of claim 1, further comprising, prior to (c), subjecting said plurality of barcoded polynucleotide molecules to nucleic acid amplification.

16. The method of claim 1, wherein at least one partition of said plurality of partitions is free of barcode molecules.

17. The method of claim 1, wherein said at least 1,000 different barcodes molecules includes 1,000 to 10,000 different barcode molecules.

18. The method of claim 1, wherein said at least 1,000 different barcode molecules includes 1,000 to 100,000 different barcode molecules.

19. The method of claim 1, wherein said at least 1,000 different barcode molecules includes 10,000 to 100,000 different barcode molecules.

20. The method of claim 1, wherein said plurality of barcode molecules comprises at least 10,000 different barcode molecules.

21. The method of claim 1, wherein said plurality of barcode molecules comprises at least 100,000 different barcode molecules.

22. The method of claim 1, wherein a partition of said at least 1,000 partitions comprises a cell.

23. The method of claim 1, further comprising fragmenting a nucleic acid molecule into said plurality of sample polynucleotide molecules.

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

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

26. The method of claim 1, wherein said at least 1,000 partitions are a subset of said plurality of partitions.

27. The method of claim 1, wherein said at least 1,000 partitions comprises at least 10,000 partitions, and wherein each of said at least 10,000 partitions has a different barcode sequence of said plurality of barcode sequences.

28. The method of claim 1, wherein said at least 1,000 partitions comprises at least 100,000 partitions, and wherein each of said at least 100,000 partitions has a different barcode sequence of said plurality of barcode sequences.

29. The method of claim 1, further comprising subjecting said plurality of sample polynucleotide molecules and said plurality of barcode molecules in said at least 1,000 partitions to nucleic acid amplification under conditions sufficient to generate said plurality of barcoded polynucleotide molecules.

30. The method of claim 1, wherein (c) comprises subjecting said plurality of barcoded polynucleotide molecules or derivatives thereof to nucleic acid sequencing.

31. The method of claim 1, wherein said plurality of sample polynucleotide molecules are a plurality of messenger ribonucleic acid (mRNA) molecules.

32. The method of claim 31, wherein (b) comprises reverse transcribing said plurality of mRNA molecules in the presence of said plurality of barcode molecules to generate said plurality of barcoded polynucleotide molecules.

33. The method of claim 1, wherein said plurality of sample polynucleotide molecules comprise deoxyribonucleic acid (DNA) molecules.

34. The method of claim 1, wherein said plurality of sample polynucleotide molecules comprises ribonucleic acid (RNA) molecules.

35. The method of claim 11, wherein a bead of said plurality of beads comprises a bond that is cleavable upon application of a stimulus.

36. The method of claim 35, wherein said bond is a disulfide bond or a peptide bond.

37. The method of claim 11, further comprising exposing a bead of said beads to a stimulus to release barcode molecules of said plurality of barcode molecules.

38. The method of claim 37, wherein a partition of said at least 1,000 partitions comprises said stimulus.

39. The method of claim 37, wherein said stimulus initiates depolymerization of a component of said bead.

40. The method of claim 37, wherein said stimulus is selected from the group consisting of a reducing agent, a change in pH, an osmotic trigger, and a change in ion concentration.

41. The method of claim 11, wherein said beads comprise cross-linked polymers.

42. The method of claim 1, further comprising detecting linked genetic variations present in said plurality of sample polynucleotide molecules.

43. The method of claim 1, further comprising differentiating between haplotypes present in said plurality of sample polynucleotide molecules.

44. The method of claim 1, wherein (b) comprises ligating said plurality of barcoded molecules to said sample plurality of polynucleotide molecules.

45. The method of claim 1, wherein in (b), said plurality of barcoded molecules are generated upon nucleic acid amplification of said plurality of sample polynucleotide molecules using said plurality of barcode molecules within said plurality of partitions.

46. The method of claim 1, wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises an immobilization sequence or an annealing sequence for a sequencing primer, or complement thereof.

47. The method of claim 1, wherein a barcode molecule of said plurality of barcode molecules comprises a sequence complementary to a sequence of a sample polynucleotide molecule of said plurality of sample polynucleotide molecules.

48. The method of claim 11, wherein said plurality of barcode molecules is attached to said plurality of beads via a linker.

49. The method of claim 13, wherein said plurality of barcode molecules is attached to said plurality of gel beads via a linker.

50. The method of claim 48, wherein said linker is a photolabile linker.

51. The method of claim 49, wherein said linker is a photolabile linker.

52. The method of claim 1, wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises a barcode sequence.

53. The method of claim 1, wherein said plurality of barcode molecules further comprises a plurality of primer sequences.

54. The method of claim 53, wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises a barcode sequence, or complement thereof, of said plurality of barcode sequences and a primer sequence, or complement thereof, of said plurality of primer sequences.

55. The method of claim 1, wherein said plurality of barcode molecules further comprises a plurality of primer sequences and wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises a barcode sequence, or complement thereof, of said plurality of barcode sequences and a primer sequence, or complement thereof, of said plurality of primer sequences.

56. The method of claim 1, wherein said plurality of partitions further comprises a plurality of primers.

57. The method of claim 1, wherein said plurality of barcode molecules further comprises a plurality of primer sequences, and wherein said plurality of partitions further comprises a plurality of primers.

58. The method of claim 1, wherein said plurality of sample polynucleotide molecules comprises genomic deoxyribonucleic acid (gDNA).

59. The method of claim 1, wherein said plurality of sample polynucleotide molecules comprises genomic deoxyribonucleic acid (gDNA), wherein said plurality of barcode molecules further comprises a plurality of primer sequences, and wherein a primer sequence of said plurality of primer sequences is a targeted primer sequence for nucleic acid amplification of gDNA.

60. The method of claim 59, wherein step (b) comprises targeted nucleic acid amplification of gDNA using said targeted primer sequence.

61. The method of claim 1, wherein in a partition of said plurality of partitions comprises an extract from a cell.

62. The method of claim 61, wherein said cell is a single cell.

63. The method of claim 1, wherein a partition of said plurality of partitions comprises a cell lysate.

64. The method of claim 63, wherein said cell lysate is from a single cell.

65. A method for nucleic acid sequence analysis, comprising:

(a) providing a plurality of partitions, a plurality of sample polynucleotide molecules comprising genomic deoxyribonucleic acid (gDNA), and a plurality of barcode molecules,

wherein said plurality of barcode molecules is coupled to a plurality of beads and a barcode molecule of said plurality of barcode molecules comprises a barcode sequence and a primer sequence,

wherein at least 1,000 partitions of said plurality of partitions each comprise (i) a sample polynucleotide molecule from said plurality of sample polynucleotide molecules, and (ii) a barcode molecule from said plurality of barcode molecules, and

wherein said plurality of barcode sequences are different across said at least 1,000 partitions; and

(b) using said plurality of sample polynucleotide molecules and said plurality of barcode molecules in said at least 1,000 partitions to generate a plurality of barcoded polynucleotide molecules,

wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises said barcode sequence, or a complement thereof, and said primer sequence, or a complement thereof, and

wherein said plurality of barcoded polynucleotide molecules comprises said plurality of barcode sequences that are different across said at least 1,000 partitions.

66. The method of claim 65, wherein said at least 1,000 partitions is 1,000 to 10,000 partitions.

67. The method of claim 65, wherein said at least 1,000 partitions is 1,000 to 100,000 partitions.

68. The method of claim 65, wherein said at least 1,000 partitions is 1,000 to 1,000,000 partitions.

69. The method of claim 65, wherein said at least 1,000 partitions is 1,000 to 10,000,000 partitions.

70. The method of claim 65, wherein said at least 1,000 partitions is 10,000 to 100,000 partitions.

71. The method of claim 65, wherein a partition of said at least 1,000 partitions comprises at least 1,000 barcode molecules of said plurality of barcode molecules.

72. The method of claim 71, wherein each of said at least 1,000 barcode molecules comprise a common barcode sequence.

73. The method of claim 71, wherein said partition comprises at least 20,000 barcode molecules of said plurality of barcode molecules.

74. The method of claim 73, wherein each of said at least 20,000 barcode molecules comprise a common barcode sequence.

75. The method of claim 65, wherein said plurality of beads is a plurality of solid particles.

76. The method of claim 65, wherein said plurality of beads is a plurality of gel beads.

77. The method of claim 65, further comprising, subsequent to (b), subjecting said plurality of barcoded polynucleotide molecules to release from said plurality of partitions.

78. The method of claim 65, wherein at least one partition of said plurality of partitions is free of barcode molecules.

79. The method of claim 65, wherein said at least 1,000 different barcodes molecules includes 1,000 to 10,000 different barcode molecules.

80. The method of claim 65, wherein said at least 1,000 different barcode molecules includes 1,000 to 100,000 different barcode molecules.

81. The method of claim 65, wherein said at least 1,000 different barcode molecules includes 10,000 to 100,000 different barcode molecules.

82. The method of claim 65, wherein said plurality of barcode molecules comprises at least 10,000 different barcode molecules.

83. The method of claim 65, wherein said plurality of barcode molecules comprises at least 100,000 different barcode molecules.

84. The method of claim 65, wherein a partition of said at least 1,000 partitions comprises a cell.

85. The method of claim 65, further comprising fragmenting a nucleic acid molecule into said plurality of sample polynucleotide molecules.

86. The method of claim 65, wherein said plurality of partitions is a plurality of droplets.

87. The method of claim 65, wherein said plurality of partitions is a plurality of wells.

88. The method of claim 65, wherein said at least 1,000 partitions are a subset of said plurality of partitions.

89. The method of claim 65, wherein said at least 1,000 partitions comprises at least 10,000 partitions, and wherein each of said at least 10,000 partitions has a different barcode sequence of said plurality of barcode sequences.

90. The method of claim 65, wherein said at least 1,000 partitions comprises at least 100,000 partitions, and wherein each of said at least 100,000 partitions has a different barcode sequence of said plurality of barcode sequences.

91. The method of claim 65, further comprising subjecting said plurality of sample polynucleotide molecules and said plurality of barcode molecules in said at least 1,000 partitions to nucleic acid amplification under conditions sufficient to generate said plurality of barcoded polynucleotide molecules.

92. The method of claim 65, wherein a bead of said plurality of beads comprises a bond that is cleavable upon application of a stimulus.

93. The method of claim 92, wherein said bond is a disulfide bond or a peptide bond.

94. The method of claim 65, further comprising exposing a bead of said beads to a stimulus to release barcode molecules of said plurality of barcode molecules.

95. The method of claim 94, wherein a partition of said at least 1,000 partitions comprises said stimulus.

96. The method of claim 94, wherein said stimulus initiates depolymerization of a component of said bead.

97. The method of claim 94, wherein said stimulus is selected from the group consisting of a reducing agent, a change in pH, an osmotic trigger, and a change in ion concentration.

98. The method of claim 65, wherein said beads comprise cross-linked polymers.

99. The method of claim 65, further comprising determining sequences of said plurality of barcoded polynucleotide molecules and detecting linked genetic variations present in said plurality of sample polynucleotide molecules.

100. The method of claim 65, further comprising determining sequences of said plurality of barcoded polynucleotide molecules and differentiating between haplotypes present in said plurality of sample polynucleotide molecules.

101. The method of claim 65, wherein (b) comprises ligating said plurality of barcoded molecules to said sample plurality of polynucleotide molecules.

102. The method of claim 65, wherein in (b), said plurality of barcoded molecules are generated upon nucleic acid amplification of said plurality of sample polynucleotide molecules using said plurality of barcode molecules within said plurality of partitions.

103. The method of claim 65, wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises an immobilization sequence or an annealing sequence for a sequencing primer, or complement thereof.

104. The method of claim 65, wherein a barcode molecule of said plurality of barcode molecules comprises a sequence complementary to a sequence of a sample polynucleotide molecule of said plurality of sample polynucleotide molecules.

105. The method of claim 65, wherein said plurality of barcode molecules is attached to said plurality of beads via a linker.

106. The method of claim 76, wherein said plurality of barcode molecules is attached to said plurality of gel beads via a linker.

107. The method of claim 105, wherein said linker is a photolabile linker.

108. The method of claim 106, wherein said linker is a photolabile linker.

109. The method of claim 65, wherein said plurality of barcode molecules further comprises a plurality of primer sequences.

110. The method of claim 109, wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises said primer sequence, or complement thereof, which is from said plurality of primer sequences.

111. The method of claim 65, wherein said plurality of barcode molecules further comprises a plurality of primer sequences and wherein a barcoded polynucleotide molecule of said plurality of barcoded polynucleotide molecules comprises said primer sequence, or complement thereof, which is from said plurality of primer sequences.

112. The method of claim 65, wherein said plurality of partitions further comprises a plurality of primers.

113. The method of claim 65, wherein said plurality of barcode molecules further comprises a plurality of primer sequences, and wherein said plurality of partitions further comprises a plurality of primers.

114. The method of claim 65, wherein said plurality of barcode molecules further comprises a plurality of primer sequences, and wherein said primer sequence is from said plurality of primer sequences and is a targeted primer sequence for nucleic acid amplification of said gDNA.

115. The method of claim 65, wherein step (b) comprises targeted nucleic acid amplification of said gDNA using said targeted primer sequence.

116. The method of claim 65, wherein a partition of said plurality of partitions comprises an extract from a cell.

117. The method of claim 116, wherein said cell is a single cell.

118. The method of claim 65, wherein a partition of said plurality of partitions comprises a cell lysate.

119. The method of claim 118, wherein said cell lysate is from a single cell.