Methods and systems for processing polynucleotides

• US 10,450,607 B2
• Filed: 03/06/2019
• Issued: 10/22/2019
• Est. Priority Date: 08/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mirna, Saxonov, Serge
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/294,769
• Publication Number:
  US 20190203291A1
• Time in Patent Office: 230 Days
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• 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 1/6816 characterised by the detection means 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 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/10 the purpose being amplify/increase the copy number of target nucleic acid
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• 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 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

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 or analysis, comprising:

(a) a plurality of antibody-oligonucleotide conjugates, wherein an antibody-oligonucleotide conjugate of said plurality of antibody-oligonucleotide conjugates comprises an antibody and an oligonucleotide reporter molecule comprising a reporter barcode sequence, and wherein said reporter barcode sequence identifies said antibody;

(b) a plurality of beads comprising a plurality of barcode sequences, wherein a bead of said plurality of beads comprises a plurality of nucleic acid barcode molecules comprising a common barcode sequence; and

(c) a microwell array comprising 1,000 microwells, wherein said microwell array is configured to partition a plurality of cells and said plurality of beads into microwells of said microwell array, wherein a microwell of said microwell array comprises;

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

(ii) said bead, and wherein said single cell is bound to said antibody-oligonucleotide conjugate.

2. The system of claim 1, wherein said plurality of nucleic acid barcode molecules is releasably attached to said bead.

3. The system of claim 1, wherein said plurality of nucleic acid barcode molecules is attached to said bead via a disulfide bond.

4. The system of claim 1, wherein, in said microwell, said bead comprises said oligonucleotide reporter molecule attached thereto.

5. The system of claim 1, wherein said bead is a magnetic bead.

6. The system of claim 5, further comprising a magnetic source configured to produce a magnetic field, wherein said magnetic bead is removable from said microwell using said magnetic field.

7. The system of claim 1, wherein said bead is a gel bead.

8. The system of claim 1, wherein said plurality of nucleic acid barcode molecules comprises a unique molecular sequence.

9. The system of claim 8, wherein said unique molecular sequence is a random sequence.

10. The system of claim 1, wherein said microwell array comprises 10,000 microwell.

11. The system of claim 1, wherein said microwell array comprises 100,000 microwell.

12. The system of claim 1, wherein said microwell array comprises 1,000,000 microwell.

13. The system of claim 1, wherein antibody-oligonucleotide conjugates of said plurality of antibody-oligonucleotide conjugates are configured to bind to a common antigen and comprise a common reporter barcode sequence.

14. The system of claim 1, wherein antibody-oligonucleotide conjugates of said plurality of antibody-oligonucleotide conjugates are configured to bind to different antigens and comprise different reporter barcode sequences.

15. The system of claim 1, wherein said antibody-oligonucleotide conjugate is bound to a surface feature of said single cell.

16. The system of claim 1, wherein said antibody-oligonucleotide conjugate is bound to a protein of said single cell.

17. The system of claim 1, wherein said plurality of nucleic acid barcode molecules comprise one or more functional sequences selected from the group consisting of a first primer sequence, a first primer binding sequence, a capture sequence configured to attach to said reporter molecule, and a first adapter sequence configured to permit attachment to a flow cell of a sequencer.

18. The system of claim 17, wherein said first primer sequence comprises a sequencing primer sequence, or wherein said first primer binding sequence comprises a sequencing primer binding sequence.

19. The system of claim 17, wherein said reporter molecule comprises one or more functional sequences selected from the group consisting of a second primer sequence, a second primer binding sequence, a sequence complementary to said capture sequence, and a second adapter sequence configured to permit attachment to said flow cell of said sequencer.

20. The system of claim 1, further comprising reagents configured to generate a barcoded nucleic acid molecule comprising said reporter barcode sequence and said common barcode sequence.

21. The system of claim 20, wherein said reagents comprise one or more components selected from the group consisting of a buffer, a polymerase, a ligase, and a primer.

22. The system of claim 20, further comprising a sequencing component comprising a sequencing instrument, wherein said sequencing component is configured to generate sequencing reads corresponding to said reporter barcode sequence and said common barcode sequence.

23. The system of claim 22, further comprising a computing component comprising a processor, a user interface, and an electronic display, wherein said computing component is configured to analyze said sequencing reads and display an analysis of nucleic acid sequencing data on said electronic display.

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

25. The system of claim 1, wherein said common barcode sequence is different from barcode sequences of other barcode molecules attached to other beads in other microwells of said microwell array.

26. A system for nucleic acid processing, comprising:

a microwell array comprising a plurality of microwells configured to partition (i) a plurality of beads comprising nucleic acid molecules comprising barcode sequences, and (ii) a plurality of cells;

wherein said plurality of microwells comprises 1,000 microwells;

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

wherein said system is configured to partition said plurality of beads such that said barcode sequences are different across microwells of said microwell array;

wherein said microwell is configured to comprise;

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

(ii) a bead of said plurality of beads, wherein said cell is bound to an antibody-oligonucleotide conjugate comprising an antibody and an oligonucleotide reporter molecule comprising a reporter barcode sequence, and wherein said reporter barcode sequence identifies said antibody.

31. The system of claim 26, wherein said microwell comprises said cell of said plurality of cells.

32. The system of claim 26, wherein said microwell comprises said bead of said plurality of beads.

33. The system of claim 26, wherein said microwell comprises said cell of said plurality of cells and said bead of said plurality of beads.

34. The system of claim 26, wherein said bead is a gel bead.

35. The system of claim 26, wherein said bead comprises a disulfide bond.

36. The system of claim 26, wherein a nucleic acid molecule comprising a barcode sequence of said barcode sequences is attached to said bead via a covalent linkage.

37. The system of claim 36, wherein said covalent linkage is a disulfide bond.

38. The system of claim 26, wherein a nucleic acid molecule comprising a barcode sequence of said barcode sequences is releasably attached to said bead.

39. The system of claim 38, wherein said microwell comprises an agent configured to release said nucleic acid molecule from said bead.

40. The system of claim 26, wherein said antibody is configured to bind to a tumor cell.

41. The system of claim 26, wherein said antibody is configured to bind to a neural cell.

42. The system of claim 26, wherein said antibody is configured to bind to a cell surface feature of said cell.

43. The system of claim 42, wherein said cell surface feature is a cell surface protein.

44. The system of claim 26, wherein said antibody is configured to bind to a protein of said cell.

45. The system of claim 26, wherein said nucleic acid molecules comprising barcode sequences comprise a poly-thymine (poly-T) sequence.

46. The system of claim 26, wherein said nucleic acid molecules comprising barcode sequences comprise one or more functional sequences selected from the group consisting of a first primer sequence, a first primer binding sequence, a capture sequence configured to attach to said oligonucleotide reporter molecule, and a first adapter sequence configured to permit attachment to a flow cell of a sequencer.

47. The system of claim 46, wherein said oligonucleotide reporter molecule comprises one or more functional sequences selected from the group consisting of a second primer sequence, a second primer binding sequence, a sequence complementary to said capture sequence, and a second adapter sequence configured to permit attachment to said flow cell of said sequencer.

48. The system of claim 26, wherein said bead is a degradable bead.

49. The system of claim 48, wherein said microwell comprises an agent configured to degrade said bead.

50. The system of claim 49, wherein said agent is a reducing agent.

51. The system of claim 26, wherein said microwell comprises a template switching oligonucleotide.

52. The system of claim 26, wherein said nucleic acid molecules comprising barcode sequences comprise a unique molecular identifier.

53. The system of claim 26, wherein said microwell comprises a polymerase.

54. The system of claim 26, wherein said microwell comprises a ligase.

55. The system of claim 26, wherein said bead comprises a plurality of nucleic acid molecules comprising a common barcode sequence.

56. The system of claim 55, further comprising a sequencing module configured to generate sequencing reads corresponding to said reporter barcode sequence and said common barcode sequence.

57. The system of claim 56, further comprising a computing module comprising a processor, a user interface, and an electronic display, wherein said computing module is configured to analyze said sequencing reads and display an analysis of nucleic acid sequencing data on said electronic display.

27. A microfluidic system for nucleic acid processing or analysis, comprising:

(a) a plurality of antibody-oligonucleotide conjugates, wherein an antibody-oligonucleotide conjugate of said plurality of antibody-oligonucleotide conjugates comprises an antibody and an oligonucleotide reporter molecule comprising a reporter barcode sequence, and wherein said reporter barcode sequence identifies said antibody;

(b) a plurality of beads comprising a plurality of barcode sequences, wherein a bead of said plurality of beads comprises a plurality of nucleic acid barcode molecules comprising a common barcode sequence; and

(c) a microfluidic device comprising a channel network configured to partition a plurality of cells and said plurality of beads into a plurality of droplets, wherein a droplet of said plurality of droplets comprises;

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

(ii) said bead, and wherein said single cell is bound to said antibody-oligonucleotide conjugate.

29. The microfluidic system of claim 27, wherein a channel of said channel network is configured to receive an input fluid comprising said plurality of cells.

30. The microfluidic system of claim 27, wherein a channel of said channel network is fluidly coupled to an outlet reservoir that is configured to receive said droplet.

28. A microfluidic system for nucleic acid processing, comprising:

a microfluidic device comprising a channel network configured to partition (i) a plurality of cells (ii) a plurality of beads, and (iii) a plurality of nucleic acid barcode molecules into a plurality of droplets,

wherein said plurality of nucleic acid barcode molecules comprises a plurality of nucleic acid barcode sequences that are different across said plurality of droplets,

wherein a droplet of said plurality of droplets comprises;

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

(ii) a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules, wherein said cell is bound to an antibody-oligonucleotide conjugate comprising an antibody and an oligonucleotide reporter molecule comprising a reporter barcode sequence, wherein said reporter barcode sequence identifies said antibody, and wherein said nucleic acid barcode molecule is attached to a bead of said plurality of beads and comprises a barcode sequence of said plurality of barcode sequences.