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 comprising:
(a) providing a target polynucleotide;
(b) fragmenting said target polynucleotide to generate a plurality of non-overlapping first polynucleotide fragments;
(c) partitioning said first polynucleotide fragments to generate partitioned first polynucleotide fragments, wherein at least one partition of said partitioned first polynucleotide fragments comprises a first polynucleotide fragment with a unique sequence within said at least one partition; and
(d) fragmenting said partitioned first polynucleotide fragments to generate a plurality of second polynucleotide fragments,
wherein in (b)-(c), the plurality of non-overlapping first polynucleotide fragments is not quantified.
2. The method of claim 1 , further comprising:
fragmenting said target polynucleotide to generate a plurality of non-overlapping third polynucleotide fragments;
partitioning said third polynucleotide fragments to generate partitioned third polynucleotide fragments, wherein at least one partition of said partitioned third polynucleotide fragments comprises a third polynucleotide fragment with a unique sequence within said at least one partition; and
fragmenting said partitioned third polynucleotide fragments to generate a plurality of fourth polynucleotide fragments.
3. The method of claim 2 , wherein said third polynucleotide fragments overlap with said first polynucleotide fragments.
4. The method of claim 2 , wherein said fourth polynucleotide fragments overlap with said second polynucleotide fragments.
5. The method of claim 1 , wherein said target polynucleotide is selected from the group consisting of DNA, RNA, and cDNA.
6. The method of claim 2 , wherein at least one of said first, second, third, and fourth polynucleotide fragments are generated by an enzyme.
7. The method of claim 6 , wherein said enzyme is a restriction enzyme.
8. The method of claim 7 , wherein said restriction enzyme used to generate said first polynucleotide fragments is different from said restriction enzyme used to generate said third polynucleotide fragments.
9. The method of claim 7 , wherein said restriction enzyme used to generate said second polynucleotide fragments is different from said restriction enzyme used to generate said fourth polynucleotide fragments.
10. The method of claim 7 , wherein said restriction enzyme has a recognition site of at least about six nucleotides in length.
11. The method of claim 2 , wherein said first or third polynucleotide fragments have a median length of at least 10,000 nucleotides.
12. The method of claim 2 , wherein said second or fourth polynucleotide fragments have a median length of less than 200 nucleotides.
13. The method of claim 1 , further comprising attaching said second polynucleotide fragments to barcodes to generate barcoded second polynucleotide fragments.
14. The method of claim 2 , further comprising attaching said fourth polynucleotide fragments to barcodes to generate barcoded fourth polynucleotide fragments.
15. The method of claim 13 , wherein said barcodes are polynucleotide barcodes.
16. The method of claim 15 wherein said attaching is performed using an enzyme.
17. The method of claim 16 , wherein said enzyme is a ligase.
18. The method of claim 13 , further comprising pooling said barcoded polynucleotide fragments to generate pooled barcoded polynucleotide fragments.
19. The method of claim 18 , further comprising sequencing said pooled barcoded polynucleotide fragments.
20. The method of claim 1 , wherein at least one step is performed in a device.
21. The method of claim 20 , wherein said device comprises a well.
22. The method of claim 21 , wherein said well is a microwell.
23. The method of claim 22 , wherein said partitioning is performed by dispensing said first or third polynucleotide fragments into said microwell.
24. The method of claim 22 , wherein said microwell comprises reagents.
25. The method of claim 24 , wherein said reagents are selected from the group consisting of barcodes, enzymes, adapters, and combinations thereof.
26. The method of claim 24 , wherein said reagents are physically separated from a polynucleotide placed in said microwell.
27. The method of claim 26 , wherein said physical separation is performed by containing said reagents within a microcapsule.
28. The method of claim 26 , wherein said physical separation is performed by overlaying said microwell with a layer.
29. The method of claim 28 , wherein said layer is selected from the group consisting of an oil, a wax, and a membrane.
30. The method of claim 26 , further comprising sealing said microwell after addition of said polynucleotide.
31. The method of claim 20 , wherein said device further comprises a microfluidic channel.
32. The method of claim 31 , wherein said partitioning is performed by fluid flow in said microfluidic channel.
33. The method of claim 1 , wherein said partitioning is performed using a method selected from the group consisting of emulsification, spotted arrays, surface acoustic waves, and piezoelectric droplet generation.
34. The method of claim 1 , wherein said fragmenting said partitioned first or third polynucleotide fragments is performed by a method selected from the group consisting of mechanical disruption, sonication, chemical fragmentation, treatment with ultraviolet light, and heating, and combinations thereof.
35. The method of claim 1 , further comprising dividing said first polynucleotide fragments or said third polynucleotide fragments into two or more aliquots and partitioning each aliquot separately.
36. A method comprising:
(a) providing a target polynucleotide;
(b) fragmenting said target polynucleotide to generate a plurality of non-overlapping first polynucleotide fragments;
(c) partitioning said first polynucleotide fragments to generate partitioned first polynucleotide fragments, such that at least one partition comprises a first polynucleotide fragment with a unique sequence within said at least one partition; and
(d) fragmenting said partitioned first polynucleotide fragments with at least one restriction enzyme in at least one partition, and at least two restriction enzymes across all partitions, to generate a plurality of second polynucleotide fragments,
wherein in (b)-(c), the plurality of non-overlapping first polynucleotide fragments is not quantified.
37. The method of claim 2 , wherein said target polynucleotide is selected from the group consisting of DNA, RNA, and cDNA.
38. The method of claim 14 , wherein said barcodes are polynucleotide barcodes.
39. The method of claim 14 , further comprising pooling said barcoded polynucleotide fragments to generate pooled barcoded polynucleotide fragments.
40. The method of claim 2 , wherein at least one step is performed in a device.
41. The method of claim 2 , wherein said partitioning is performed using a method selected from the group consisting of emulsification, spotted arrays, surface acoustic waves, and piezoelectric droplet generation.
42. The method of claim 2 , wherein said fragmenting said partitioned first or third polynucleotide fragments is performed by a method selected from the group consisting of mechanical disruption, sonication, chemical fragmentation, treatment with ultraviolet light, and heating, and combinations thereof.
43. The method of claim 2 , further comprising dividing said first polynucleotide fragments or said third polynucleotide fragments into two or more aliquots and partitioning each aliquot separately.