Methods and systems for processing polynucleotides

• US 9,410,201 B2
• Filed: 04/07/2015
• Issued: 08/09/2016
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Saxonov, Serge, Ness, Kevin, Hardenbol, Paul
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/680,808
• Publication Number:
  US 20150218633A1
• Time in Patent Office: 490 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

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.

Claims

1. A method of nucleic acid sequence analysis, comprising:

(a) fragmenting a first nucleic acid molecule to provide a plurality of polynucleotide molecules;

(b) partitioning the plurality of polynucleotide molecules of the first nucleic acid molecule into a plurality of separate partitions, wherein each of the plurality of separate partitions comprises;

i. at least 20 polynucleotide molecules from the plurality of polynucleotide molecules, the at least 20 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules; and

ii. a plurality of barcode molecules, wherein each barcode molecule within a given partition shares a common barcode sequence and is associated with a bead within the partition;

(c) after (b), generating barcoded fragment molecules from the at least 20 polynucleotide molecules and plurality of barcode molecules within each of the separate partitions; and

(d) analyzing the barcoded fragment molecules generated in (c), thereby analyzing a nucleic acid sequence of the first nucleic acid molecule.

2. The method of claim 1, wherein each of the plurality of separate partitions comprises at least 50 polynucleotide molecules, the at least 50 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules.

3. The method of claim 1, wherein each of the plurality of separate partitions comprises at least 100 polynucleotide molecules the at least 100 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules.

4. The method of claim 1, wherein each of the plurality of separate partitions comprises at least 1000 polynucleotide molecules, the at least 1000 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules.

5. The method of claim 1, wherein each of the plurality of separate partitions comprises a different barcode sequence.

6. The method of claim 5, wherein each of at least 100 different partitions comprises a different barcode sequence.

7. The method of claim 5, wherein each of at least 1,000 different partitions comprises a different barcode sequence.

8. The method of claim 5, wherein each of at least 5,000 different partitions comprises a different barcode sequence.

9. The method of claim 5, wherein each of at least 10,000 different partitions comprises a different barcode sequence.

10. The method of claim 5, wherein each of at least 100,000 different partitions comprises a different barcode sequence.

11. The method of claim 5, wherein each of at least 1,000,000 different partitions comprises a different barcode sequence.

12. The method of claim 1, wherein (c) comprises providing the barcode molecules with a primer sequence, and amplifying at least a portion of the at least 20 polynucleotides within a given partition by extending the primer sequence and barcode molecule.

13. The method of claim 1, wherein the plurality of separate partitions comprises droplets in an emulsion.

14. The method of claim 13, wherein the droplets comprise aqueous droplets in an oil phase.

15. The method of claim 1, wherein the plurality of separate partitions further comprises one or more reagents selected from the group of ligases, polymerases, primers, dNTPs, and restriction enzymes.

16. The method of claim 1, wherein (a) comprises treating the first nucleic acid molecule with a rare cutter enzyme.

17. The method of claim 1, wherein an individual polynucleotide molecule of the plurality of polynucleotide molecules comprises a length of greater than 5000 nucleotides.

18. The method of claim 1, wherein the at least 20 polynucleotide molecules within a given partition comprise polynucleotide molecules having a length of greater than 5000 nucleotides and less than 500,000 nucleotides.

19. The method of claim 1, wherein the at least 20 polynucleotide molecules within a given partition comprise polynucleotide molecules having a length of 5000 to 10,000 nucleotides.

20. The method of claim 1, wherein the at least 20 polynucleotide molecules within a given partition comprise polynucleotide molecules having a length of 10,000 to 100,000 nucleotides.

21. The method of claim 1, wherein the at least 20 polynucleotide molecules within a given partition comprise polynucleotide molecules having a length of 100,000 to 500,000 nucleotides.

22. A method of nucleic acid sequence analysis, comprising:

(a) partitioning a plurality of polynucleotide molecules into a plurality of separate partitions, where each of the plurality of separate partitions comprises;

i. at least 20 polynucleotide molecules, the at least 20 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules of a nucleic acid; and

ii. a plurality of barcode molecules, where each barcode molecule within a given partition shares a common barcode sequence that is different from a barcode sequence in each of a plurality of other partitions and is associated with a bead within the partition;

(b) after (a), generating barcoded polynucleotide molecules from the at least 20 polynucleotide molecules and plurality of barcode molecules within each partition; and

(c) analyzing the barcoded polynucleotide molecules, thereby analyzing the sequence of the nucleic acid.

23. The method of claim 22, wherein each of the plurality of separate partitions comprises at least 50 polynucleotide molecules, the at least 50 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules.

24. The method of claim 22, wherein each of the plurality of partitions comprises at least 100 polynucleotide molecules, the at least 100 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules.

25. The method of claim 22, wherein each of the plurality of partitions comprises at least 1000 polynucleotide molecules, the at least 1000 polynucleotide molecules comprising non-overlapping fragment polynucleotide molecules.

26. The method of claim 22, wherein each of at least 1,000 different partitions comprises a different barcode sequence.

27. The method of claim 22, wherein each of at least 5,000 different partitions comprises a different barcode sequence.

28. The method of claim 22, wherein each of at least 10,000 different partitions comprises a different barcode sequence.

29. The method of claim 22, wherein each of at least 100,000 different partitions comprises a different barcode sequence.