METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180274027A1
• Filed: 12/21/2017
• Published: 09/27/2018
• 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, Schnall-Levin, Michael, Jarosz, Mirna
• Granted Patent: US 10,676,789 B2
• Time in Patent Office: Days
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

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 for nucleic acid sequence analysis, comprising:

(a) providing a plurality of partitions, a plurality of polynucleotide molecules and a plurality of barcode molecules, wherein said plurality of barcode molecules comprise a plurality of barcode sequences, wherein at least 1,000 partitions of said plurality of partitions each comprise (i) a polynucleotide molecule from said plurality of polynucleotide molecules, and (ii) a barcode molecule from said plurality of barcode molecules, wherein said plurality of barcode sequences are different across said at least 1,000 partitions;

(b) using said plurality of polynucleotide molecules and said plurality of barcode molecules in said at least 1,000 partitions to generate a plurality of barcoded polynucleotide molecules, which plurality of barcoded polynucleotide molecules comprises said plurality of barcode sequences that are different across said at least 1,000 partitions; and

(c) determining sequences of said plurality of barcoded polynucleotide molecules.

2. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 10,000 partitions.

3. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 100,000 partitions.

4. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 1,000,000 partitions.

5. The method of claim 1, wherein said at least 1,000 partitions is 1,000 to 10,000,000 partitions.

6. The method of claim 1, wherein said at least 1,000 partitions is 10,000 to 100,000 partitions.

7. The method of claim 1, wherein a given partition of said at least 1,000 partitions comprises at least 1,000 barcode molecules of said plurality of barcode molecules.

8. The method of claim 7, wherein said at least 1,000 barcode molecules have a common barcode sequence from a given one of said plurality barcode molecules.

9. The method of claim 7, wherein said given partition comprises at least 20,000 barcode molecules of said plurality of barcode molecules.

10. The method of claim 9, wherein said at least 20,000 barcode molecules have a common barcode sequence from a given one of said plurality of barcode molecules.

11. The method of claim 1, wherein said plurality of barcode molecules are coupled to beads.

12. The method of claim 11, wherein said beads are solid particles.

13. The method of claim 11, wherein said beads are gel beads.

14. The method of claim 1, further comprising, subsequent to (b), subjecting said plurality of barcoded polynucleotide molecules to release from said plurality of partitions.

15. The method of claim 1, further comprising, prior to (c), subjecting said plurality of barcoded polynucleotide molecules to nucleic acid amplification.

16. The method of claim 1, wherein at least one partition of said plurality of partitions does not include a barcode molecule.

17. The method of claim 1, wherein said at least 1,000 different barcodes molecules includes 1,000 to 10,000 different barcode molecules.

18. The method of claim 1, wherein said at least 1,000 different barcode molecules includes 1,000 to 100,000 different barcode molecules.

19. The method of claim 1, wherein said at least 1,000 different barcode molecules includes 10,000 to 100,000 different barcode molecules.

20. The method of claim 1, wherein said plurality of barcode molecules comprises at least 10,000 different barcode molecules.

21. The method of claim 1, wherein said plurality of barcode molecules comprises at least 100,000 different barcode molecules.

22. The method of claim 1, wherein a given one of said at least 1,000 partitions comprises a cell.

23. The method of claim 1, further comprising fragmenting a nucleic acid molecule into said plurality of polynucleotide molecules.

24. The method of claim 1, wherein said plurality of partitions is a plurality of droplets.

25. The method of claim 1, wherein said plurality of partitions is a plurality of wells.

26. The method of claim 1, wherein said at least 1,000 partitions are a subset of said plurality of partitions.

27. 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 plurality of barcode sequences.

28. 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 plurality of barcode sequences.

29. The method of claim 1, further comprising subjecting said plurality of polynucleotide molecules and said plurality of barcode molecules in said at least 1,000 partitions to nucleic acid amplification under conditions sufficient to generate said plurality of barcoded polynucleotide molecules.

30. The method of claim 1, wherein (c) comprises subjecting said plurality of barcoded polynucleotide molecules or derivatives thereof to nucleic acid sequencing.