Methods and systems for processing polynucleotides

• US 10,612,090 B2
• Filed: 01/16/2019
• Issued: 04/07/2020
• 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:
  US16/249,688
• Publication Number:
  US 20190136319A1
• Time in Patent Office: 447 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 1/683 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 for biological material processing, comprising:

providing a microwell array comprising (1) a plurality of beads, (2) a plurality of cells, and (3) a plurality of barcode molecules, wherein said microwell array comprises 1,000 microwells, wherein said plurality of barcode molecules comprises a plurality of barcode sequences that are different across said 1,000 microwells, and

wherein a microwell of said microwell array accommodates a volume of less than 5 nanoliters and comprises;

(i) a cell of said plurality of cells; and

(ii) a bead of said plurality of beads.

2. The method of claim 1, wherein said plurality of barcode molecules is attached to said plurality of beads.

3. The method of claim 1, further comprising using barcode molecules of said plurality of barcode molecules to barcode a plurality of polynucleotide molecules, wherein said plurality of cells comprises said plurality of polynucleotide molecules.

4. The method of claim 3, further comprising using a sequencer to identify sequences of said plurality of polynucleotide molecules or derivatives thereof.

5. The method of claim 1, wherein said plurality of beads is a plurality of solid particles.

6. The method of claim 1, wherein said plurality of beads is a plurality of gel beads.

7. The method of claim 1, wherein said microwell further comprises a reducing agent.

8. The method of claim 1, wherein said microwell further comprises an antibody.

9. The method of claim 1, wherein said microwell array comprises at least 5,000 microwells.

10. The method of claim 1, wherein said 1,000 microwells are a subset of microwells of said microwell array.

11. The method of claim 10, wherein at least one microwell of said microwell array does not include (i) a barcode molecule of said plurality of barcode molecules or (ii) a cell of said plurality of cells.

12. The method of claim 1, wherein said providing comprises partitioning said plurality of cells into said microwell array.

13. The method of claim 2, wherein said bead in (ii) is attached to a barcode molecule of the plurality of barcode molecules and wherein said barcode molecule comprises a barcode sequence that is different from barcode sequences of other barcode molecules attached to other beads in microwells of said plurality of microwells.

14. The method of claim 3, wherein said barcode molecules comprise a sequence that is complementary to a sequence of a polynucleotide molecule of said plurality of polynucleotide molecules.

15. The method of claim 3, wherein said plurality of polynucleotide molecules comprises polynucleotide molecules with overlapping sequences.

16. The method of claim 3, wherein said plurality of polynucleotide molecules does not include a barcode sequence, and wherein a polynucleotide molecule of said plurality of polynucleotide molecules is hybridized to a barcode molecule of said plurality of barcode molecules.

17. The method of claim 3, wherein said bead is not attached to an amplification product of said plurality of polynucleotide molecules.

18. The method of claim 3, wherein said plurality of polynucleotide molecules comprises deoxyribonucleic acid (DNA).

19. The method of claim 3, wherein said plurality of polynucleotide molecules comprises ribonucleic acid (RNA).

20. The method of claim 1, wherein said microwell comprises multiple barcode molecules of said plurality of barcode molecules, wherein said multiple barcode molecules comprise a barcode molecule of said plurality of barcode molecules.

21. The method of claim 20, wherein each of said multiple barcode molecules comprises a barcode sequence of said plurality of barcode sequences.

22. The method of claim 1, wherein said microwell array comprises 2,500 microwells.

23. The method of claim 1, wherein said microwell array comprises 5,000 microwells.

24. The method of claim 1, wherein said microwell array comprises 7,500 microwells.

25. The method of claim 1, wherein said microwell array comprises 10,000 microwells.

26. The method of claim 1, wherein said microwell further comprises an enzyme.

27. The method of claim 26, wherein said enzyme is a polymerase.