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 method for cellular processing, comprising: providing an array comprising (1) a plurality of supports, (2) a plurality of cells, and (3) a plurality of barcode molecules, wherein said array comprises 1,000 microwells, 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 1,000 microwells comprises: (i) a cell of said plurality of cells; and (ii) a support of said plurality of supports, wherein said support comprises a common barcode sequence that is common to said support.
2. The method of claim 1 , wherein said plurality of barcode molecules is attached to said plurality of supports.
3. The method of claim 2 , wherein said support in (ii) is attached to a barcode molecule of said plurality of barcode molecules and wherein said barcode molecule comprises a barcode sequence that is different from barcode sequences of other barcode molecules attached to other supports in microwells of said 1,000 microwells.
4. The method of claim 1 , further comprising using barcode molecules of said plurality of barcode molecules to barcode a plurality of nucleic acid molecules to thereby generate a plurality of barcoded nucleic acid molecules, wherein said plurality of cells comprises said plurality of nucleic acid molecules.
5. The method of claim 4 , further comprising using a sequencer to identify sequences of said plurality of nucleic acid molecules or derivatives thereof.
6. The method of claim 4 , wherein said barcode molecules comprise a sequence that is complementary to a sequence of a nucleic acid molecule of said plurality of nucleic acid molecules.
7. The method of claim 4 , wherein said plurality of nucleic acid molecules comprises nucleic acid molecules with overlapping sequences.
8. The method of claim 4 , wherein said plurality of nucleic acid molecules does not include a barcode sequence, and wherein a nucleic acid molecule of said plurality of nucleic acid molecules is hybridized to a barcode molecule of said plurality of barcode molecules.
9. The method of claim 4 , wherein said support is not attached to an amplification product of said plurality of nucleic acid molecules.
10. The method of claim 4 , wherein said plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA).
11. The method of claim 4 , wherein said plurality of nucleic acid molecules comprises ribonucleic acid (RNA).
12. The method of claim 4 , wherein said plurality of barcoded nucleic acid molecules comprises a plurality of double-stranded barcoded nucleic acid molecules, and wherein said method further comprises denaturing a double-stranded barcoded nucleic acid molecule of said plurality of double-stranded barcoded nucleic acid molecules to yield a single-stranded barcoded nucleic acid molecule.
13. The method of claim 12 , further comprising providing a primer molecule comprising a primer sequence that is complementary to a sequence of said single-stranded barcoded nucleic acid molecule, annealing said primer sequence to said sequence, and extending said primer molecule using said single-stranded barcoded nucleic acid molecule.
14. The method of claim 1 , wherein said plurality of supports is a plurality of beads.
15. The method of claim 14 , wherein said plurality of beads is a plurality of gel beads.
16. The method of claim 1 , wherein said microwell further comprises a reducing agent.
17. The method of claim 1 , wherein said microwell further comprises an antibody.
18. The method of claim 1 , wherein said array comprises 5,000 microwells.
19. The method of claim 1 , wherein said 1,000 microwells are a subset of microwells of said array.
20. The method of claim 1 , wherein at least one microwell of said 1,000 microwells does not include (i) a barcode molecule of said plurality of barcode molecules or (ii) any cell of said plurality of cells.
21. The method of claim 1 , wherein said providing comprises partitioning said plurality of cells into said array.
22. The method of claim 1 , wherein said microwell comprises multiple barcode molecules of said plurality of barcode molecules.
23. The method of claim 22 , wherein a barcode molecule of said multiple barcode molecules comprises a barcode sequence of said plurality of barcode sequences.
24. The method of claim 22 , wherein each of said multiple barcode molecules comprises said common barcode sequence.
25. The method of claim 1 , wherein said array comprises 2,500 microwells.
26. The method of claim 1 , wherein said array comprises 5,000 microwells.
27. The method of claim 1 , wherein said array comprises 7,500 microwells.
28. The method of claim 1 , wherein said array comprises 10,000 microwells.
29. The method of claim 1 , wherein said microwell further comprises an enzyme.
30. The method of claim 29 , wherein said enzyme is a polymerase.
31. The method of claim 1 , wherein said microwell is sealed with a sealing fluid.
32. The method of claim 31 , wherein said sealing fluid is a non-aqueous fluid.
33. The method of claim 32 , wherein said non-aqueous fluid is oil.
34. The method of claim 1 , wherein said microwell is sealed.
35. The method of claim 34 , wherein said microwell is sealed using a laminate, tape, plastic cover, or wax.
36. The method of claim 17 , wherein said antibody is coupled to an oligonucleotide reporter molecule.
37. The method of claim 36 , wherein said oligonucleotide reporter molecule comprises a reporter barcode sequence, which reporter barcode sequence identifies said antibody.
38. The method of claim 14 , wherein said plurality of beads is a plurality of degradable beads.
39. The method of claim 1 , wherein said plurality of supports comprises a plurality of solid particles.
40. The method of claim 1 , wherein said microwell comprises a reducing agent.
41. The method of claim 1 , wherein at least one microwell of said 1,000 microwells does not include a barcode molecule of the plurality of barcode molecules or any cell of said plurality of cells.
42. The method of claim 1 , 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.
43. The method of claim 1 , wherein barcode molecules of said plurality of barcode molecules comprise a priming sequence.
44. The method of claim 43 , wherein said priming sequence is an RNA priming sequence.
45. The method of claim 44 , wherein said RNA priming sequence is a poly-T sequence.
46. The method of claim 1 , wherein barcode molecules of said plurality of barcode molecules further comprise a unique molecular sequence segment.
47. The method of claim 1 , wherein barcode molecules of said plurality of barcode molecules further comprise one or more functional sequences.
48. The method of claim 47 , wherein said one or more functional sequences comprise a sequencing primer sequence or a sequencing primer binding sequence.
49. The method of claim 48 , 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.
50. The method of claim 47 , wherein said one or more functional sequences comprise a sequence configured to attach to a flow cell of a sequencer.
51. The method of claim 50 , 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.
52. The method of claim 1 , wherein said microwell comprises an endonuclease.
53. The method of claim 1 , wherein said microwell comprises a polymerase.
54. The method of claim 1 , wherein said microwell comprises a reverse transcriptase.
55. The method of claim 1 , wherein said microwell comprises an enzyme with terminal transferase activity.
56. The method of claim 1 , wherein said microwell comprises a switch oligonucleotide.
57. The method of claim 1 , wherein barcode molecules of said plurality of barcode molecules comprise a random sequence.
58. The method of claim 1 , further comprising, performing a nucleic acid reaction in said microwell.
59. The method of claim 58 , wherein said nucleic acid reaction is a nucleic acid extension reaction.