Methods and systems for processing polynucleotides

• US 10,227,648 B2
• Filed: 12/28/2016
• Issued: 03/12/2019
• 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:
  US15/392,557
• Publication Number:
  US 20170335385A1
• Time in Patent Office: 804 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 system for nucleic acid processing, comprising:

a microwell array comprising 1,000 microwells configured to partition a plurality of polynucleotide molecules, a plurality of beads, and a plurality of barcode molecules in the 1,000 microwells of the microwell array,

wherein the plurality of barcode molecules comprises a plurality of barcode sequences that are different across the 1,000 microwells, and

wherein a microwell of the microwell array comprises;

(i) a polynucleotide molecule from the plurality of polynucleotide molecules; and

(ii) a barcode molecule from the plurality of barcode molecules, wherein the barcode molecule is attached to a bead from the plurality of beads and comprises a barcode sequence from the plurality of barcode sequences.

2. The system of claim 1, further comprising a sequencer that generates sequences of the plurality of polynucleotide molecules or derivatives thereof.

3. The system of claim 1, wherein the plurality of beads is a plurality of solid particles.

4. The system of claim 1, wherein the plurality of beads is a plurality of gel beads.

5. The system of claim 1, wherein the microwell comprises a cell comprising the polynucleotide molecule from the plurality of polynucleotide molecules.

6. The system of claim 1, wherein the microwell comprises a reducing agent.

7. The system of claim 1, wherein the barcode molecule comprises a sequence complementary to a sequence of the polynucleotide molecule.

8. The system of claim 1, wherein the plurality of polynucleotide molecules comprises polynucleotide molecules with overlapping sequences.

9. The system of claim 1, wherein the microwell comprises an antibody.

10. The system of claim 1, wherein the microwell comprises multiple barcode molecules from the plurality of barcode molecules, wherein the multiple barcode molecules comprise the barcode molecule.

11. The system of claim 10, wherein each of the multiple barcode molecules comprises the barcode sequence.

12. The system of claim 1, wherein the 1,000 microwells are a subset of microwells of the microwell array.

13. The system of claim 12, wherein at least one microwell of the microwell array does not include a barcode molecule from the plurality of barcode molecules or a polynucleotide molecule from the plurality of polynucleotide molecules.

14. The system of claim 1, wherein the microwell of the microwell array is configured to accommodate a volume of less than about 500 nanoliters (nL).

15. The system of claim 1, wherein the microwell array comprises at least 5,000 microwells.

34. The system of claim 1, wherein the polynucleotide molecule does not include a barcode sequence, and wherein the polynucleotide molecule is hybridized to the barcode molecule.

35. The system of claim 1, wherein the bead in the microwell is not attached to an amplification product of the polynucleotide molecule.

37. The system of claim 14, wherein the microwell is configured to accommodate a volume of less than about 200 nL.

38. The system of claim 37, wherein the microwell is configured to accommodate a volume of less than about 100 nL.

39. The system of claim 38, wherein the microwell is configured to accommodate a volume of less than about 50 nL.

40. The system of claim 39, wherein the microwell is configured to accommodate a volume of less than about 25 nL.

41. The system of claim 40, wherein the microwell is configured to accommodate a volume of less than about 10 nL.

42. The system of claim 41, wherein the microwell is configured to accommodate a volume of less than about 5 nL.

43. The system of claim 15, wherein the microwell array comprises 2,500 microwells.

44. The system of claim 43, wherein the microwell array comprises 5,000 microwells.

45. The system of claim 44, wherein the microwell array comprises 7,500 microwells.

46. The system of claim 45, wherein the microwell array comprises 10,000 microwells.

16. A system for nucleic acid processing, comprising:

a microwell array comprising a plurality of microwells configured to partition a plurality of beads and a plurality of cells;

wherein the plurality of microwells comprises 1,000 microwells;

wherein a microwell of the plurality of microwells is configured to accommodate a volume of less than about 5 nanoliters (nL); and

wherein the system is configured to partition the plurality of cells, the plurality of beads, and a plurality of barcode molecules into the plurality of microwells such that the plurality of barcode molecules comprises a plurality of barcode sequences that are different across microwells of the microwell array, and wherein the microwell is configured to comprise;

(a) a cell of the plurality of cells; and

(b) a bead of the plurality of beads.

17. The system of claim 16, wherein the plurality of barcode molecules is attached to the plurality of beads.

18. The system of claim 17, wherein the bead of (b) is attached to a barcode molecule of the plurality of barcode molecules and comprises a barcode sequence that is different from barcode sequences of other barcode molecules attached to other beads in the microwells.

19. The system of claim 16, further comprising a sequencer that generates sequences of the plurality of barcode molecules or derivatives thereof.

20. The system of claim 16, wherein the plurality of beads is a plurality of solid particles.

21. The system of claim 16, wherein the plurality of beads is a plurality of gel beads.

22. The system of claim 16, wherein the microwell further comprises a reducing agent.

23. The system of claim 16, wherein the microwell of the microwell array comprises an antibody.

24. The system of claim 16, wherein the microwell array comprises 10,000 microwells and wherein the microwell is configured to comprise:

(a) the cell of the plurality of cells; and

(b) the bead of the plurality of beads.

25. The assay of claim 16, wherein at least one microwell of the plurality of microwells does not include a barcode molecule from the plurality of barcode molecules or a cell from the plurality of cells.

36. The system of claim 18, wherein the cell comprises a plurality of polynucleotide molecules, and wherein the barcode sequence comprises a sequence that is complementary to a sequence of a polynucleotide of the plurality of polynucleotide molecules.

47. The system of claim 16, wherein the microwell comprises the cell of the plurality of cells and the bead of the plurality of beads.

26. A microwell array, comprising:

a plurality of microwells comprising a plurality of cells and a plurality of beads;

wherein the plurality of beads comprises a plurality of barcode molecules;

wherein the plurality of microwells comprises 1,000 microwells;

wherein the plurality of barcode molecules comprise a plurality of barcode sequences that are different across the 1,000 microwells;

wherein a microwell of the plurality of microwells is configured to accommodate a volume of less than about 5 nanoliters; and

wherein the microwell comprises;

(a) a cell of the plurality of cells; and

(b) a bead of the plurality of beads, wherein the bead is attached to a barcode molecule of the plurality of barcode molecules.

27. The microwell array of claim 26, wherein the barcode molecule attached to the bead of (b) comprises a barcode sequence that is different from barcode sequences of barcode molecules attached to other beads in microwells of the plurality of microwells.

28. The microwell array of claim 26, wherein the plurality of beads is a plurality of solid particles.

29. The microwell array of claim 26, wherein the plurality of beads is a plurality of gel beads.

30. The microwell array of claim 26, wherein the microwell comprises a reducing agent.

31. The microwell array of claim 26, wherein the microwell comprises an antibody.

32. The microwell array of claim 26, wherein at least one microwell does not include a barcode molecule from the plurality of barcode molecules or a cell from the plurality of cells.

33. The microwell array of claim 26, wherein the plurality of microwells comprises 10,000 microwells and wherein the microwell comprises:

(a) a cell of the plurality of cells; and

(b) a bead of the plurality of beads.