Methods and systems for processing polynucleotides

• US 10,480,028 B2
• Filed: 12/19/2017
• Issued: 11/19/2019
• 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:
  US15/847,752
• Publication Number:
  US 20180112266A1
• Time in Patent Office: 700 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 analyzing cells comprising:

(a) contacting a plurality of cells with an antibody to provide a cell bound to said antibody, wherein said antibody is attached to a reporter oligonucleotide comprising a reporter sequence;

(b) co-partitioning at least a subset of said plurality of cells into a plurality of wells, wherein a well of said plurality of wells comprises (i) said cell bound to said antibody and (ii) a first nucleic acid molecule comprising a barcode sequence; and

(c) using said reporter oligonucleotide and said first nucleic acid molecule to generate a second nucleic acid molecule comprising (i) said reporter sequence, and (ii) said barcode sequence.

2. The method of claim 1, further comprising sequencing said second nucleic acid molecule or a derivative thereof to identify said reporter sequence and said barcode sequence.

3. The method of claim 1, wherein (c) comprises hybridizing said reporter oligonucleotide to said first nucleic acid molecule and performing a nucleic acid extension reaction to generate said second nucleic acid molecule.

4. The method of claim 1, wherein (c) is performed in said well of said plurality of wells.

5. The method of claim 1, wherein said first nucleic acid molecule is attached to a bead and wherein, in (b), said well comprises (i) said cell bound to said antibody and (ii) said bead.

6. The method of claim 5, wherein (c) comprises (i) hybridizing said reporter oligonucleotide to said first nucleic acid molecule to provide said bead comprising said reporter oligonucleotide attached thereto;

and (ii) performing a nucleic acid extension reaction to generate said second nucleic acid molecule.

7. The method of claim 6, wherein, prior to said nucleic acid extension reaction, said bead comprising said reporter oligonucleotide attached thereto is removed from said well.

8. The method of claim 6, wherein said bead is a magnetic bead.

9. The method of claim 8, wherein, prior to said nucleic acid extension reaction, said magnetic bead comprising said reporter oligonucleotide attached thereto is removed from said well.

10. The method of claim 9, wherein said magnetic bead is removed using a magnetic field.

11. The method of claim 1, wherein said first nucleic acid molecule comprises a sequence complementary to a sequence of said reporter oligonucleotide.

12. The method of claim 1, wherein said first nucleic acid molecule comprises a unique molecular sequence.

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

14. The method of claim 13, wherein said primer sequence is a sequencing primer sequence.

15. The method of claim 1, wherein said reporter oligonucleotide comprises a primer sequence.

16. The method of claim 15, wherein said primer sequence is a sequencing primer sequence.

17. The method of claim 1, wherein said reporter oligonucleotide comprises an adapter sequence configured to permit attachment to a flow cell of a sequencer.

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

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

20. The method of claim 5 wherein (b) comprises co-partitioning at least said subset of said plurality of cells and a plurality of beads into said plurality of wells to provide said well comprising (i) said cell attached to said antibody and (ii) said bead.

21. The method of claim 1, wherein (b) comprises co-partitioning at least said subset of said plurality of cells and a plurality of nucleic acid barcode molecules into said plurality of wells to provide said well comprising (i) said cell bound to said antibody and (ii) said first nucleic acid molecule.

22. The method of claim 1, wherein said plurality of cells comprises 100 cells.

23. The method of claim 1, wherein said plurality of cells comprises 1,000 cells.

24. The method of claim 1, wherein said plurality of cells comprises 10,000 cells.

25. The method of claim 1, wherein said reporter sequence is complementary to said sequence of said reporter oligonucleotide.

30. The method of claim 1, wherein (a) comprises contacting said plurality of cells with a plurality of antibodies comprising a plurality of reporter oligonucleotides, wherein a given antibody of said plurality of antibodies is attached to a given reporter oligonucleotide of said plurality of reporter oligonucleotides and wherein said given reporter oligonucleotide comprises a given reporter sequence that identifies said given antibody.

31. The method of claim 30, wherein, in (b), said microwell comprises (i) said cell bound to at least a subset of said plurality of antibodies, including said given antibody;

and (ii) a plurality of nucleic acid barcode molecules, including said first nucleic acid molecule;

wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a common barcode sequence.

32. The method of claim 31, wherein (c) comprises generating a plurality of barcoded nucleic acid molecules, including said second nucleic acid molecule, and wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises (i) said given reporter sequence corresponding to said given antibody of said subset of said plurality of antibodies;

and (ii) said common barcode sequence.

33. The method of claim 32, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a unique molecular sequence.

34. The method of claim 33, wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises said unique molecular sequence.

35. The method of claim 34, further comprising sequencing said plurality of barcoded nucleic acid molecules or a derivative thereof to measure the quantity of a protein in said cell.

36. The method of claim 6, wherein said reporter oligonucleotide comprises a poly-A sequence, said first nucleic acid molecule comprises a poly-T sequence, and said hybridizing occurs via hybridization between said poly-A sequence and said poly-T sequence.

37. The method of claim 34, further comprising (d) using a messenger ribonucleic acid RNA (mRNA) of said cell and a third nucleic acid molecule comprising said barcode sequence to generate a fourth nucleic acid molecule comprising (i) a sequence corresponding to said mRNA and (ii) said barcode sequence;

wherein said third nucleic acid molecule comprises a poly-T sequence, and wherein (d) comprises hybridizing a poly-A sequence of said mRNA to said poly-T sequence.

38. The method of claim 37, further comprising sequencing said fourth nucleic acid molecule or a derivative thereof to identify said sequence corresponding to said mRNA and said barcode sequence.

39. The method of claim 32, wherein each reporter oligonucleotide of said plurality of reporter oligonucleotides comprises a poly-A sequence, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a poly-T sequence, and wherein (c) comprises (i) hybridizing reporter oligonucleotides of said plurality of reporter oligonucleotides to nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules;

and (ii) performing a nucleic acid extension reaction to generate said plurality of barcoded nucleic acid molecules.

40. The method of claim 39, further comprising (d) hybridizing a poly-A sequence of a plurality of messenger RNA (mRNA) molecules of said cell to said poly-T sequence of nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules and performing a reverse transcription reaction to generate a plurality of barcoded complementary deoxyribonucleic acid (cDNA) molecules, wherein each barcoded cDNA molecule of said plurality of barcoded cDNA molecules comprises (i) a sequence corresponding to a mRNA molecule of said plurality of mRNA molecules and (ii) said common barcode sequence.

41. The method of claim 40, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a unique molecular sequence.

42. The method of claim 41, wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules and each barcoded cDNA molecule of said plurality of barcoded cDNA molecules comprises said unique molecular sequence.

43. The method of claim 42, further comprising (i) sequencing said plurality of barcoded nucleic acid molecules or a derivative thereof;

(ii) sequencing said plurality of barcoded cDNA molecules or a derivative thereof; and

(iii) using said unique molecular sequence to measure a quantity of a protein or a quantity of a mRNA in said cell.

44. The method of claim 1, wherein said plurality of nucleic acid barcode molecules are attached to a bead and wherein said microwell comprises said bead.

45. The method of claim 40, wherein said plurality of nucleic acid barcode molecules are attached to a bead and wherein said microwell comprises said bead.

46. The method of claim 1, wherein said antibody binds to a cell surface feature of said cell.

47. The method of claim 1, wherein said antibody binds to a protein of said cell.

48. The method of claim 47, wherein said protein comprises a cell surface protein.

49. The method of claim 47, wherein said protein comprises an intracellular protein.

50. The method of claim 48, wherein said cell surface protein is a cluster of differentiation (CD) protein.

51. The method of claim 30, wherein each antibody of said plurality of antibodies that has a binding affinity for a common antigen comprises a common reporter sequence.

52. The method of claim 51, wherein each antibody of said plurality of antibodies that has a binding affinity for a different antigen comprises a different reporter sequence.

53. The method of claim 30, wherein different antibodies are attached to reporter oligonucleotides having different reporter sequences corresponding to said different antibodies.

26. A method for analyzing cells comprising:

(a) contacting a first plurality of cells with a first antibody to provide a first cell bound to said first antibody, wherein said first antibody is attached to a first reporter oligonucleotide comprising a first reporter sequence;

(b) contacting a second plurality of cells with a second antibody to provide a second cell bound to said second antibody, wherein said second antibody is attached to a second reporter oligonucleotide comprising a second reporter sequence, wherein said first reporter sequence is different than said second reporter sequence;

(c) pooling at least a subset of said first plurality of cells and at least a subset of said second plurality of cells to provide a pooled plurality of cells;

(d) co-partitioning at least a subset of said pooled plurality of cells into a plurality of wells to provide (i) a first well comprising said first cell bound to said first antibody and a first nucleic acid molecule comprising a first barcode sequence; and

(ii) a second well comprising said second cell bound to said second antibody and a second nucleic acid molecule comprising a second barcode sequence; and

(e) generating (i) a third nucleic acid molecule comprising said first reporter sequence and said first barcode sequence; and

(ii) a fourth nucleic acid molecule comprising said second reporter sequence and said second barcode sequence.

27. The method of claim 26, further comprising

(f) sequencing (i) said third nucleic acid molecule or a derivative thereof to identify said first reporter sequence and said first barcode sequence and (ii) said fourth nucleic acid molecule or a derivative thereof to identify said second reporter sequence and said second barcode sequence;

and

(g) using (i) said first reporter sequence and said first barcode sequence to identify said first cell of said first plurality of cells; and

(ii) said second reporter sequence and said second barcode sequence to identify said second cell of said second plurality of cells.

28. The method of claim 26, wherein said first reporter oligonucleotide is complementary to said first nucleic acid molecule or said second reporter oligonucleotide is complementary to said second nucleic acid molecule.

54. The method of claim 26, wherein (a) comprises contacting said first plurality of cells with a plurality of antibodies comprising a plurality of reporter oligonucleotides, wherein each antibody of said plurality of antibodies is attached to a reporter oligonucleotide comprising a reporter sequence.

55. The method of claim 54, wherein, in (d), said first well comprises (i) said first cell bound to at least a subset of said plurality of antibodies, including said first antibody, and (ii) a plurality of nucleic acid barcode molecules, including said first nucleic acid molecule;

wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a common barcode sequence.

56. The method of claim 55, wherein (e) comprises generating a plurality of barcoded nucleic acid molecules, including said third nucleic acid molecule, and wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises (i) a reporter sequence corresponding to an antibody of said subset of said plurality of antibodies;

and (ii) said common barcode sequence.

57. The method of claim 56, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a unique molecular sequence.

58. The method of claim 57, wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises said unique molecular sequence.

59. The method of claim 58, further comprising sequencing said plurality of barcoded nucleic acid molecules or a derivative thereof to measure the quantity of a protein in said first cell.

60. The method of claim 27, wherein said first nucleic acid molecule is attached to a bead and wherein, in (d), said first well comprises (i) said first cell bound to said first antibody and (ii) said bead.

61. The method of claim 60, wherein said first reporter oligonucleotide comprises a poly-A sequence, wherein said first nucleic acid molecule comprises a poly-T sequence, and wherein (g) comprises (i) hybridizing said poly-A sequence of said first reporter oligonucleotide to said poly-T sequence of said first nucleic acid molecule to provide said bead comprising said first reporter oligonucleotide attached thereto, and (ii) performing a nucleic acid extension reaction to generate said third nucleic acid molecule.

62. The method of claim 61, further comprising (h) using a messenger ribonucleic acid RNA (mRNA) of said first cell and a fifth nucleic acid molecule comprising said first barcode sequence to generate a sixth nucleic acid molecule comprising (i) a sequence corresponding to said mRNA and (ii) said first barcode sequence;

wherein said fifth nucleic acid molecule comprises a poly-T sequence, and wherein (h) comprises hybridizing a poly-A sequence of said mRNA to said poly-T sequence.

63. The method of claim 62, further comprising sequencing said sixth nucleic acid molecule or a derivative thereof to identify said sequence corresponding to said mRNA and said first barcode sequence.

64. The method of claim 56, wherein each reporter oligonucleotide of said plurality of reporter oligonucleotides comprises a poly-A sequence, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a poly-T sequence, and wherein (e) comprises (i) hybridizing reporter oligonucleotides of said plurality of reporter oligonucleotides to nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules;

and (ii) performing a nucleic acid extension reaction to generate said plurality of barcoded nucleic acid molecules.

65. The method of claim 64, further comprising (f) hybridizing a poly-A sequence of a plurality of messenger RNA (mRNA) molecules of said first cell to said poly-T sequence of nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules to generate a plurality of barcoded complementary deoxyribonucleic acid (cDNA) molecules, wherein each barcoded cDNA molecule of said plurality of barcoded cDNA molecules comprises (i) a sequence corresponding to a mRNA molecule of said plurality of mRNA molecules and (ii) said common barcode sequence.

66. The method of claim 65, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a unique molecular sequence.

67. The method of claim 66, wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules and each barcoded cDNA molecule of said plurality of barcoded cDNA molecules comprises said unique molecular sequence.

68. The method of claim 67, further comprising (i) sequencing said plurality of barcoded nucleic acid molecules or a derivative thereof;

(ii) sequencing said plurality of barcoded cDNA molecules or a derivative thereof; and

(iii) using said unique molecular sequence to measure a quantity of a protein or a quantity of a mRNA in said first cell.

69. The method of claim 55, wherein said plurality of nucleic acid barcode molecules are attached to a bead and wherein said first well comprises said bead.

70. The method of claim 65, wherein said plurality of nucleic acid barcode molecules are attached to a bead and wherein said first well comprises said bead.

71. The method of claim 26, wherein said first antibody binds to a cell surface feature of said first cell.

72. The method of claim 26, wherein said first antibody binds to a protein of said first cell.

73. The method of claim 72, wherein said protein comprises a cell surface protein.

74. The method of claim 72, wherein said protein comprises an intracellular protein.

75. The method of claim 73, wherein said cell surface protein is a cluster of differentiation (CD) protein.

76. The method of claim 72, wherein each antibody of said plurality of antibodies that has a binding affinity for a common antigen comprises a common reporter sequence.

77. The method of claim 76, wherein each antibody of said plurality of antibodies that has a binding affinity for a different antigen comprises a different reporter sequence.

78. The method of claim 54, wherein different antibodies are attached to reporter oligonucleotides having different reporter sequences corresponding to said different.

29. A method for analyzing cells comprising:

(a) providing a microwell array comprising a plurality of microwells;

(b) providing a plurality of cells, wherein a cell of the plurality of cells is bound to an antibody attached to a reporter oligonucleotide comprising a reporter sequence;

(c) providing a plurality of beads, wherein a bead of the plurality of beads comprises a barcode sequence that differs across the plurality of beads;

(d) co-partitioning said plurality of cells and said plurality of beads into said microwell array, wherein a microwell of said plurality of microwells comprises (i) said cell bound to said antibody and (ii) said bead; and

(e) using said reporter oligonucleotide and said bead to generate a sequencing library.

79. The method of claim 29, wherein, in (b), said cell is bound to at least a subset of a plurality of antibodies comprising a plurality of reporter oligonucleotides, including said antibody, and wherein each antibody of said plurality of antibodies is attached to a given reporter oligonucleotide comprising a given reporter sequence.

80. The method of claim 79, wherein, in (d), said microwell comprises (i) said cell bound to said at least said subset of said plurality of antibodies, including said antibody;

and (ii) said bead, wherein said bead comprises a plurality of nucleic acid barcode molecules;

wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a common barcode sequence.

81. The method of claim 80, wherein (e) comprises generating a plurality of barcoded nucleic acid molecules, and wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises (i) said given reporter sequence corresponding to a given antibody of said subset of said plurality of antibodies;

and (ii) said common barcode sequence.

82. The method of claim 81, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a unique molecular sequence.

83. The method of claim 82, wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises said unique molecular sequence.

84. The method of claim 83, further comprising sequencing said plurality of barcoded nucleic acid molecules or a derivative thereof to measure the quantity of a protein in said cell.

85. The method of claim 29, wherein said reporter oligonucleotide comprises a poly-A sequence, wherein a first nucleic acid molecule is attached to said bead, wherein said first nucleic acid molecule comprises a poly-T sequence and said barcode sequence, and wherein (e) comprises (i) hybridizing said poly-A sequence of said reporter oligonucleotide to said poly-T sequence of said first nucleic acid molecule attached to said bead, to provide said bead comprising said reporter oligonucleotide attached thereto, and (ii) performing a nucleic acid extension reaction to generate a barcoded nucleic acid molecule.

86. The method of claim 85, wherein a second nucleic acid molecule is attached to said bead, wherein said second nucleic acid molecule comprises a poly-T sequence and said barcode sequence, and further comprising (f) hybridizing a poly-A sequence of a messenger ribonucleic acid RNA (mRNA) of said cell to said poly-T sequence of said second nucleic acid molecule to provide said bead comprising said mRNA attached thereto;

and (g) generating a third nucleic acid molecule comprising (i) a sequence corresponding to said mRNA and (ii) said barcode sequence.

87. The method of claim 86, further comprising sequencing said third nucleic acid molecule or a derivative thereof to identify said sequence corresponding to said mRNA and said barcode sequence.

88. The method of claim 81, wherein each reporter oligonucleotide of said plurality of reporter oligonucleotides comprises a poly-A sequence, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a poly-T sequence, and wherein (e) comprises (i) hybridizing reporter oligonucleotides of said plurality of reporter oligonucleotides to nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules;

and (ii) performing a nucleic acid extension reaction to generate said plurality of barcoded nucleic acid molecules.

89. The method of claim 88, further comprising (f) hybridizing a poly-A sequence of a plurality of messenger RNA (mRNA) molecules of said cell to said poly-T sequence of nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules and performing a reverse transcription reaction to generate a plurality of barcoded complementary deoxyribonucleic acid (cDNA) molecules, wherein each barcoded cDNA molecule of said plurality of barcoded cDNA molecules comprises (i) a sequence corresponding to a mRNA molecule of said plurality of mRNA molecules and (ii) said common barcode sequence.

90. The method of claim 89, wherein each nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a unique molecular sequence.

91. The method of claim 90, wherein each barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules and each barcoded cDNA molecule of said plurality of barcoded cDNA molecules comprises said unique molecular sequence.

92. The method of claim 91, further comprising (i) sequencing said plurality of barcoded nucleic acid molecules or a derivative thereof;

(ii) sequencing said plurality of barcoded cDNA molecules or a derivative thereof; and

(iii) using said unique molecular sequence to measure the quantity of a protein or the quantity of a mRNA in said cell.

93. The method of claim 29, wherein said antibody binds to a cell surface feature of said cell.

94. The method of claim 29, wherein said antibody binds to a protein of said cell.

95. The method of claim 94, wherein said protein comprises a cell surface protein.

96. The method of claim 94, wherein said protein comprises an intracellular protein.

97. The method of claim 95, wherein said cell surface protein is a cluster of differentiation (CD) protein.

98. The method of claim 79, wherein each antibody of said plurality of antibodies that has a binding affinity for a common antigen comprises a common reporter sequence.

99. The method of claim 98, wherein each antibody of said plurality of antibodies that has a binding affinity for a different antigen comprises a different reporter sequence.

100. The method of claim 79, wherein different antibodies are attached to reporter oligonucleotides having different reporter sequences corresponding to said different antibodies.