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 of nucleic acid sequence analysis, comprising:
(a) providing a plurality of partitions comprising a plurality of polynucleotide molecules and a plurality of barcode molecules, wherein:
i. said plurality of partitions comprises at least 1,000 partitions;
ii. said plurality of barcode molecules comprises at least 1,000 different nucleic acid barcode molecules having different barcode sequences;
iii. said at least 1,000 different nucleic acid barcode molecules are disposed within separate partitions of said at least 1,000 partitions, such that each of said at least 1,000 partitions has a different barcode sequence of said different barcode sequences; and
iv. said at least 1,000 partitions are at least 1% of said plurality of partitions;
(b) subsequent to (a), generating a plurality of barcoded molecules from said plurality of polynucleotide molecules and said plurality of barcode molecules within said plurality of partitions; and
(c) directing said plurality of barcoded molecules generated in (b) to nucleic acid sequencing, wherein a sequence of a given polynucleotide of said plurality of polynucleotide molecules is determined.
2. The method of claim 1 , wherein said plurality of partitions is a plurality of droplets.
3. The method of claim 1 , wherein a given partition of said at least 1,000 partitions comprises at least 1,000 nucleic acid barcode molecules, which at least 1,000 nucleic acid barcode molecules have a common barcode sequence from a given one of said at least 1,000 different nucleic acid barcode molecules.
4. The method of claim 3 , wherein a given partition of said at least 1,000 partitions comprises at least 20,000 nucleic acid barcode molecules, which at least 20,000 nucleic acid barcode molecules have a common barcode sequence from a given one of said at least 1,000 different nucleic acid barcode molecules.
5. The method of claim 1 , wherein said plurality of barcode molecules are coupled to beads.
6. The method of claim 5 , wherein said beads are gel beads.
7. The method of claim 5 , where each of said beads comprises a bond that is cleavable upon application of a stimulus.
8. The method of claim 5 , further comprising exposing said beads to a stimulus to release said plurality of barcode molecules.
9. The method of claim 8 , wherein said stimulus is a chemical stimulus.
10. The method of claim 1 , further comprising, subsequent to (b), subjecting said plurality of barcoded molecules to release from said plurality of partitions.
11. The method of claim 1 , further comprising, prior to (c), subjecting said plurality of barcoded molecules to nucleic acid amplification.
12. The method of claim 1 , wherein at least one partition of said plurality of partitions does not include a barcode molecule.
13. The method of claim 1 , wherein a given partition of said at least 1,000 partitions comprises multiple copies of a given barcode molecule of said plurality of barcode molecules.
14. The method of claim 1 , wherein said at least 1,000 partitions are at least 50% of said plurality of partitions.
15. The method of claim 1 , wherein said plurality of barcode molecules comprises at least 10,000 different nucleic acid barcode molecules.
16. The method of claim 1 , wherein said plurality of barcode molecules comprises at least 100,000 different nucleic acid barcode molecules.
17. The method of claim 1 , wherein said plurality of partitions comprises at least 5,000 partitions.
18. The method of claim 1 , wherein said plurality of partitions includes reagents necessary for nucleic acid amplification.
19. The method of claim 1 , further comprising fragmenting a nucleic acid molecule into said plurality of polynucleotide molecules.
20. The method of claim 19 , wherein (c) comprises subjecting said plurality of barcoded molecules or derivatives thereof to nucleic acid sequencing, thereby determining a sequence of said nucleic acid molecule.
21. The method of claim 1 , wherein said plurality of partitions is a plurality of wells.
22. The method of claim 1 , wherein said at least 1,000 partitions are at least 10% of said plurality of partitions.
23. 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 different barcode sequences.
24. 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 different barcode sequences.
25. The method of claim 1 , wherein a given partition of said at least 1,000 partitions comprises at least 1,000 nucleic acid barcode molecules.
26. The method of claim 25 , wherein said given partition of said at least 1,000 partitions comprises at least 10,000 nucleic acid barcode molecules.
27. The method of claim 26 , wherein said given partition of said at least 1,000 partitions comprises at least 100,000 nucleic acid barcode molecules.
28. The method of claim 27 , wherein said given partition of said at least 1,000 partitions comprises at least 1,000,000 nucleic acid barcode molecules.
29. The method of claim 1 , wherein a given partition of said at least 1,000 partitions is a droplet in an emulsion, and wherein said droplet comprises a bead associated with a given one of said at least 1,000 different nucleic acid barcode molecules.
30. The method of claim 1 , wherein a given partition of said at least 1,000 partitions is a well, and wherein said well comprises a surface associated with a given one of said at least 1,000 different nucleic acid barcode molecules.
31. The method of claim 1 , wherein a given partition of said at least 1,000 partitions is a capsule, and wherein said capsule comprises a surface associated with a given one of said at least 1,000 different nucleic acid barcode molecules.
32. The method of claim 7 , wherein said bond is a disulfide bond.
33. The method of claim 7 , wherein said bond is a peptide bond.
34. The method of claim 9 , wherein said chemical stimulus comprises a reducing agent.
35. The method of claim 8 , wherein, for a given partition of said at least 1,000 partitions, said stimulus comprises an agent disposed in said given partition.
36. The method of claim 8 , wherein said stimulus initiates depolymerization of a component of said bead.
37. The method of claim 8 , wherein said stimulus is selected from the group consisting of a change in pH, an osmotic trigger and a change in ion concentration.
38. The method of claim 5 , wherein said beads comprise cross-linked polymers.
39. The method of claim 1 , wherein a given one of said plurality of barcode molecules comprises a priming sequence.
40. The method of claim 1 , wherein said at least 1,000 partitions do not contain components necessary for polymerase chain reaction (PCR).
41. The method of claim 1 , further comprising determining polynucleotide phasing.
42. The method of claim 1 , further comprising detection of linked genetic variations present in said plurality of polynucleotide molecules.
43. The method of claim 1 , further comprising differentiating between haplotypes present in said plurality of polynucleotide molecules.
44. The method of claim 1 , wherein a given one of said plurality of barcoded molecules or derivative thereof comprises a sequencing adaptor.
45. The method of claim 1 , wherein (b) comprises ligating said plurality of barcode molecules to said plurality of polynucleotide molecules.
46. The method of claim 1 , wherein in (b), said plurality of barcoded molecules are generated upon nucleic acid amplification of said plurality of polynucleotide molecules using said plurality of barcode molecules within said plurality of partitions.
47. The method of claim 46 , wherein said nucleic acid amplification is reverse transcription amplification.
48. The method of claim 10 , further comprising subjecting said plurality of barcoded molecules to nucleic acid amplification prior to (c).
49. The method of claim 10 , wherein (b) comprises ligating a given barcode molecule of said plurality of barcode molecules to a given polynucleotide molecule of said plurality of polynucleotide molecules.
50. The method of claim 1 , wherein a given one of said plurality of barcoded molecules comprises an immobilization sequence or an annealing sequence for a sequencing primer.
51. The method of claim 1 , wherein a given one of said plurality of barcode molecules comprises a sequence complementary to a sequence of a given one of said plurality of polynucleotide molecules.
52. The method of claim 1 , wherein said plurality of polynucleotide molecules comprises deoxyribonucleic acid (DNA) molecules.
53. The method of claim 52 , wherein said DNA molecules are genomic DNA molecules.
54. The method of claim 1 , wherein said plurality of polynucleotide molecules comprises ribonucleic acid (RNA) molecules.
55. The method of claim 54 , wherein said RNA molecules are messenger RNA molecules.
56. The method of claim 1 , wherein a given one of said plurality of polynucleotide molecules is derived from a cell.
57. The method of claim 1 , wherein a given one of said at least 1,000 partitions comprises a cell.
58. The method of claim 1 , wherein a given one of said at least 1,000 partitions comprises a cell lysate.
59. The method of claim 1 , wherein a given one of said plurality of partitions comprises a single cell.
60. The method of claim 59 , wherein said given one of said plurality of partitions comprises a plurality of cells.
61. The method of claim 60 , wherein said plurality of cells comprises said plurality of polynucleotide molecules.
62. The method of claim 1 , wherein a given one of said plurality of barcode molecules is coupled to a bead.
63. The method of claim 62 , wherein said bead is a gel bead.
64. The method of claim 62 , wherein said bead comprises a bond that is cleavable upon application of a stimulus.
65. The method of claim 62 , further comprising exposing said bead to a stimulus to release said plurality of barcode molecules.
66. The method of claim 62 , wherein a given one of said plurality of barcode molecules is encapsulated within said bead.
67. The method of claim 62 , wherein a given one of said plurality of barcode molecules is attached to a surface of said bead.
68. The method of claim 62 , wherein said bead is a solid particle.
69. The method of claim 5 , wherein said beads are solid particles.
70. The method of claim 62 , wherein said bead is not a gel bead.
71. The method of claim 5 , wherein said beads are not gel beads.
72. The method of claim 1 , wherein (c) comprises subjecting said plurality of barcoded molecules or derivatives thereof to nucleic acid sequencing, thereby determining said sequence of said given polynucleotide.