Methods and systems for processing polynucleotides
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.
1. A system for nucleic acid processing, comprising:
an array comprising 1,000 microwells configured to permit partitioning of a plurality of nucleic acid molecules, a plurality of supports, and a plurality of barcode molecules,
wherein said plurality of barcode molecules comprises a plurality of barcode sequences that are different across said 1,000 microwells, and
wherein a microwell of said array comprises: (i) a nucleic acid molecule of said plurality of nucleic acid molecules and (ii) barcode molecules of said plurality of barcode molecules, wherein said barcode molecules are attached to a support of said plurality of supports and, wherein said barcode molecules comprise a common barcode sequence.
2. The system of claim 1 , further comprising a sequencer configured to generate sequencing reads from said plurality of nucleic acid molecules or derivatives thereof.
3. The system of claim 1 , wherein said plurality of supports is a plurality of beads.
4. The system of claim 3 , wherein said plurality of beads is a plurality of gel beads.
5. The system of claim 1 , wherein said microwell comprises a cell comprising said nucleic acid molecule of said plurality of nucleic acid molecules.
6. The system of claim 1 , wherein said plurality of nucleic acid molecules comprise ribonucleic acid molecules.
7. The system of claim 1 , wherein said microwell comprises a reducing agent.
8. The system of claim 1 , wherein a barcode molecule of said barcode molecules comprises a sequence complementary to a sequence of said nucleic acid molecule.
9. The system of claim 1 , wherein said plurality of nucleic acid molecules comprises nucleic acid molecules with overlapping sequences.
10. The system of claim 1 , wherein said microwell comprises an antibody.
11. The system of claim 1 , wherein said 1,000 microwells are a subset of microwells of said array.
12. The system of claim 11 , wherein at least one microwell of said array does not include a barcode molecule of said plurality of barcode molecules or a nucleic acid molecule of said plurality of nucleic acid molecules.
13. The system of claim 1 , wherein said array comprises 5,000 microwells.
14. A system for cellular processing, comprising:
an array comprising a plurality of microwells configured to permit partitioning of a plurality of supports and a plurality of cells,
wherein said plurality of microwells comprises 1,000 microwells, and
wherein said system is configured to permit partitioning of said plurality of cells, said plurality of supports, and a plurality of barcode molecules in said plurality of microwells such that said plurality of barcode molecules comprises a plurality of barcode sequences that are different across microwells of said array, and
wherein a microwell of said plurality of microwells is configured to comprise: (a) a cell of said plurality of cells; and (b) a support of said plurality of supports, wherein said support comprises barcode molecules of said plurality of barcode molecules, wherein said barcode molecules comprise a common barcode sequence.
15. The system of claim 14 , wherein said plurality of barcode molecules is attached to said plurality of supports.
16. The system of claim 15 , wherein said support of (b) is attached to a barcode molecule of said plurality of barcode molecules, wherein said barcode molecule comprises a barcode sequence that is different from barcode sequences of other barcode molecules attached to other supports in said plurality of microwells.
17. The system of claim 14 , further comprising a sequencer configured to generate sequencing reads from said plurality of barcode molecules or derivatives thereof.
18. The system of claim 14 , wherein said plurality of supports is a plurality of beads.
19. The system of claim 18 , wherein said plurality of beads is a plurality of gel beads.
20. The system of claim 14 , wherein said microwell further comprises a reducing agent.
21. The system of claim 14 , wherein said microwell further comprises an antibody.
22. The system of claim 14 , wherein said array comprises 10,000 microwells.
23. The system of claim 14 , wherein at least one microwell of said plurality of microwells does not include a barcode molecule of said plurality of barcode molecules or a cell of said plurality of cells.
24. An array, comprising:
a plurality of microwells comprising a plurality of cells and a plurality of supports,
wherein said plurality of supports comprises a plurality of barcode molecules,
wherein said plurality of microwells comprises 1,000 microwells,
wherein said plurality of barcode molecules comprise a plurality of barcode sequences that are different across said 1,000 microwells, and
wherein a microwell of said plurality of microwells comprises: (a) a cell of said plurality of cells; and (b) a support of said plurality of supports, wherein said support is attached to barcode molecules of said plurality of barcode molecules, wherein said barcode molecules comprise a common barcode sequence.
25. The array of claim 24 , wherein said barcode molecules attached to said support of (b) comprise a barcode sequence that is different from barcode sequences of barcode molecules attached to other supports in said plurality of microwells.
26. The array of claim 24 , wherein said plurality of supports is a plurality of beads.
27. The array of claim 26 , wherein said plurality of beads is a plurality of gel beads.
28. The array of claim 24 , wherein said microwell comprises an antibody.
29. The system of claim 10 , wherein said antibody is coupled to an oligonucleotide reporter molecule.
30. The system of claim 29 , wherein said oligonucleotide reporter molecule comprises a reporter barcode sequence, which reporter barcode sequence identifies said antibody.
31. The system of claim 21 , wherein said antibody is coupled to an oligonucleotide reporter molecule.
32. The system of claim 31 , wherein said oligonucleotide reporter molecule comprises a reporter barcode sequence, which reporter barcode sequence identifies said antibody.
33. The array of claim 28 , wherein said antibody is coupled to an oligonucleotide reporter molecule.
34. The array of claim 33 , wherein said oligonucleotide reporter molecule comprises a reporter barcode sequence, which reporter barcode sequence identifies said antibody.
35. The system of claim 3 , wherein said plurality of beads is a plurality of degradable beads.
36. The system of claim 3 , wherein said barcode molecules are releasably attached to said support.
37. The system of claim 18 , wherein said plurality of beads is a plurality of degradable beads.
38. The system of claim 18 , wherein said barcode molecules are releasably attached to said support.
39. The array of claim 27 , wherein said plurality of beads is a plurality of degradable beads.
40. The array of claim 27 , wherein said barcode molecules are releasably attached to said support.
41. The system of claim 1 , wherein said array comprises said plurality of nucleic acid molecules, said plurality of supports, and said plurality of barcode molecules.
42. The system of claim 14 , wherein said plurality of microwells comprises said plurality of supports and said plurality of cells.
43. The system of claim 14 , wherein said microwell comprises said cell and said support.
44. The system of claim 1 , wherein said plurality of supports comprises a plurality of solid particles.
45. The system of claim 14 , wherein said plurality of supports comprises a plurality of solid particles.
46. The array of claim 24 , wherein said plurality of supports comprises a plurality of solid particles.
47. The array of claim 24 , wherein said microwell comprises a reducing agent.
48. The array of claim 24 , wherein at least one microwell of said plurality of microwells does not include a barcode molecule of the plurality of barcode molecules or any cell of said plurality of cells.
49. The system of claim 1 , wherein said nucleic acid molecule does not comprise a barcode sequence, and wherein said nucleic acid molecule is hybridized to a barcode molecule of said barcode molecules.
50. The system of claim 1 , wherein said support in said microwell is not attached to an amplification product of said nucleic acid molecule.
51. The system of claim 14 , wherein said cell comprises a plurality of nucleic acid molecules, and wherein a barcode molecule of said barcode molecules comprises a sequence that is complementary to a sequence of a nucleic acid molecule of said plurality of nucleic acid molecules.
52. The array of claim 24 , wherein said cell comprises a plurality of nucleic acid molecules, and wherein a barcode molecule of said barcode molecules comprises a sequence that is complementary to a sequence of a nucleic acid molecule of said plurality of nucleic acid molecules.
53. The system of claim 1 , wherein said barcode molecules further comprise a priming sequence.
54. The system of claim 53 , wherein said priming sequence is an RNA priming sequence.
55. The system of claim 54 , wherein said RNA priming sequence is a poly-T sequence.
56. The system of claim 14 , wherein said barcode molecules further comprise a priming sequence.
57. The system of claim 56 , wherein said priming sequence is an RNA priming sequence.
58. The system of claim 57 , wherein said RNA priming sequence is a poly-T sequence.
59. The array of claim 24 , wherein said barcode molecules further comprise a priming sequence.
60. The array of claim 59 , wherein the priming sequence is an RNA priming sequence.
61. The array of claim 60 , wherein the RNA priming sequence is a poly-T sequence.
62. The system of claim 1 , wherein said barcode molecules further comprise a unique molecular sequence segment.
63. The system of claim 14 , wherein said barcode molecules further comprise a unique molecular sequence segment.
64. The array of claim 24 , wherein said barcode molecules further comprise a unique molecular sequence segment.
65. The system of claim 1 , wherein said barcode molecules further comprises one or more functional sequences.
66. The system of claim 65 , wherein said one or more functional sequences comprise a sequencing primer sequence or a sequencing primer binding sequence.
67. The system of claim 66 , wherein said sequencing primer sequence or said sequencing primer binding sequence is a R1 sequence, a R2 sequence, a partial R1 sequence, or a partial R2 sequence.
68. The system of claim 65 , wherein said one or more functional sequences comprise a sequence configured to attach to a flow cell of a sequencer.
69. The system of claim 68 , wherein said sequence configured to attach to said flow cell of a sequencer is a P5 sequence, a P7 sequence, a partial P5 sequence, or a partial P7 sequence.
70. The system of claim 14 , wherein said barcode molecules further comprises one or more functional sequences.
71. The system of claim 70 , wherein said one or more functional sequences comprise a sequencing primer sequence or a sequencing primer binding sequence.
72. The system of claim 71 , wherein said sequencing primer sequence or said sequencing primer binding sequence is a R1 sequence, a R2 sequence, a partial R1 sequence, or a partial R2 sequence.
73. The system of claim 70 , wherein said one or more functional sequences comprise a sequence configured to attach to a flow cell of a sequencer.
74. The system of claim 73 , wherein said sequence configured to attach to said flow cell of a sequencer is a P5 sequence, a P7 sequence, a partial P5 sequence, or a partial P7 sequence.
75. The array of claim 24 , wherein said barcode molecules further comprises one or more functional sequences.
76. The array of claim 75 , wherein said one or more functional sequences comprise a sequencing primer sequence or a sequencing primer binding sequence.
77. The array of claim 76 , wherein said sequencing primer sequence or said sequencing primer binding sequence is a R1 sequence, a R2 sequence, a partial R1 sequence, or a partial R2 sequence.
78. The array of claim 75 , wherein said one or more functional sequences comprise a sequence configured to attach to a flow cell of a sequencer.
79. The array of claim 78 , wherein said sequence configured to attach to said flow cell of a sequencer is a P5 sequence, a P7 sequence, a partial P5 sequence, or a partial P7 sequence.
80. The system of claim 1 , wherein said microwell comprises an endonuclease.
81. The system of claim 14 , wherein said microwell comprises an endonuclease.
82. The array of claim 24 , wherein said microwell comprises an endonuclease.
83. The system of claim 1 , wherein said microwell comprises a polymerase.
84. The system of claim 14 , wherein said microwell comprises a polymerase.
85. The array of claim 24 , wherein said microwell comprises a polymerase.
86. The system of claim 1 , wherein said microwell comprises a reverse transcriptase.
87. The system of claim 14 , wherein said microwell comprises a reverse transcriptase.
88. The array of claim 24 , wherein said microwell comprises a reverse transcriptase.
89. The system of claim 1 , wherein said microwell comprises an enzyme with terminal transferase activity.
90. The system of claim 14 , wherein said microwell comprises an enzyme with terminal transferase activity.
91. The array of claim 24 , wherein said microwell comprises an enzyme with terminal transferase activity.
92. The system of claim 1 , wherein said microwell comprises a switch oligonucleotide.
93. The system of claim 14 , wherein said microwell comprises a switch oligonucleotide.
94. The array of claim 24 , wherein said microwell comprises a switch oligonucleotide.
95. The system of claim 1 , wherein said 1 , 000 microwells comprise sealed microwells.
96. The system of claim 95 , wherein said sealed microwells are sealed with an oil.
97. The system of claim 95 , wherein said sealed microwells are sealed with a wax.
98. The system of claim 14 , wherein said 1 , 000 microwells comprise sealed microwells.
99. The system of claim 98 , wherein said sealed microwells are sealed with an oil.
100. The system of claim 98 , wherein said sealed microwells are sealed with a wax.
101. The array of claim 24 , wherein said 1 , 000 microwells comprise sealed microwells.
102. The array of claim 101 , wherein said sealed microwells are sealed with an oil.
103. The array of claim 101 , wherein said sealed microwells are sealed with a wax.
104. The system of claim 1 , wherein a barcode molecule of said barcode molecules is attached to said support via at least one linker.
105. The system of claim 104 , wherein said at least one linker comprises a disulfide linker.
106. The system of claim 14 , wherein a barcode molecule of said barcode molecules is attached to said support via at least one linker.
107. The system of claim 106 , wherein said at least one linker comprises a disulfide linker.
108. The array of claim 24 , wherein a barcode molecule of said barcode molecules is attached to said support via at least one linker.
109. The array of claim 108 , wherein said at least one linker comprises a disulfide linker.