Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-9567631-B2
Application number	US-201314104650-A
Country	US
Kind code	B2
Filing date	Dec 12, 2013
Priority date	Dec 14, 2012
Publication date	Feb 14, 2017
Grant date	Feb 14, 2017

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.

First claim

What is claimed is:

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 th

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Saxonov Serge
• Ness Kevin
• Hardenbol Paul

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/683 Primary

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title