METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20190136316A1
• Filed: 12/06/2018
• Published: 05/09/2019
• 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, Hardenbol, Paul, Bharadwaj, Rajiv, Zheng, Xinying, Belgrader, Phillip
• Granted Patent: US 10,752,949 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 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

1-29. -29. (canceled)

30. A method for nucleic acid processing, comprising:

(a) co-partitioning a cell and a bead in a partition, wherein said bead comprises a plurality of barcode oligonucleotide molecules each comprising a barcode sequence;

(b) lysing said cell in said partition to release a nucleic acid molecule from said cell;

(c) capturing said nucleic acid molecule using said bead via a barcode oligonucleotide molecule of said plurality of barcode oligonucleotide molecules;

(d) removing said bead from said partition; and

(e) external to said partition, conducting a nucleic acid reaction with said nucleic acid molecule, or a derivative thereof, to yield a plurality of barcoded nucleic acid products.

31. The method of claim 30, wherein said plurality of barcode oligonucleotide molecules each comprises a common barcode sequence.

32. The method of claim 30, wherein said nucleic acid reaction comprises a reverse transcription reaction.

33. The method of claim 32, wherein (e) comprises introducing a reverse transcription mixture to said nucleic acid molecule.

34. The method of claim 30, wherein said nucleic acid reaction comprises exonuclease treatment.

35. The method of claim 34, wherein (e) comprises introducing an exonuclease to said nucleic acid molecule.

36. The method of claim 30, wherein (e) comprises conducting said nucleic acid reaction with a plurality of nucleic acid molecules to yield said plurality of barcoded nucleic acid products, wherein said plurality of nucleic acid molecules comprises said nucleic acid molecule and at least one other nucleic acid molecule from another cell.

37. The method of claim 36, wherein said plurality of barcoded nucleic acid products comprises a first subset of products each comprising a common barcode sequence, or a complement thereof, that is common to said cell, and a second subset of products each comprising another common barcode sequence, or a complement thereof, that is common to said another cell, wherein said another common barcode is different from said common barcode sequence.

38. The method of claim 30, wherein (e) comprises contacting said plurality of nucleic acid molecules with a bulk reaction mixture.

39. The method of claim 30, further comprising, subsequent to (e), subjecting said barcoded nucleic acid products to exonuclease treatment by introducing exonucleases.

40. The method of claim 39, further comprising, subsequent to said exonuclease treatment, inhibiting exonuclease activity of said exonucleases.

41. The method of claim 30, further comprising, prior to (a), sorting a sample comprising a plurality of cells to identify said cell.

42. The method of claim 41, wherein said sorting comprises filtering said plurality of cells to yield a subset of cells, wherein said subset of cells comprises said cell.

43. The method of claim 41, wherein said sample comprises 1,000 cells.

44. The method of claim 43, wherein said sample comprises 10,000 cells.

45. The method of claim 30, wherein said plurality of barcoded nucleic acid products comprise complementary deoxyribonucleic acid (cDNA) molecules.

46. The method of claim 45, wherein each of said plurality of barcode oligonucleotide molecules comprises a sequence for priming the synthesis of cDNA.

47. The method of claim 46, wherein said sequence for priming is a poly-T sequence

48. The method of claim 30, wherein said nucleic acid molecule is a messenger RNA (mRNA) molecule.

49. The method of claim 30, further comprising, subsequent to (e), subjecting said barcoded nucleic acid products to one or more reactions to generate a set of nucleic acid molecules, and sequencing said set of nucleic acid molecules.

50. The method of claim 49, wherein said set of nucleic acid molecules are amplified products from said barcoded nucleic acid products.

51. The method of claim 30, wherein each of said plurality of barcode oligonucleotide molecules comprises a universal primer sequence.

52. The method of claim 30, wherein each of said plurality of barcode oligonucleotide molecules comprises a unique molecular sequence that varies across said plurality of barcode oligonucleotide molecules.

53. The method of claim 30, wherein said bead is a magnetic bead.

54. The method of claim 53, wherein (d) comprises applying a magnetic field to said magnetic bead to remove said magnetic bead from said partition.

55. The method of claim 30, wherein said plurality of barcode oligonucleotide molecules are attached to said bead.

56. The method of claim 30, wherein said nucleic acid molecule attaches to said barcode oligonucleotide molecule by hybridization.

57. The method of claim 30, wherein said partition comprises not more than a single cell and not more than a single bead.

58. A method for nucleic acid processing, comprising:

(a) co-partitioning a cell and a bead in a well, wherein said bead comprises a plurality of barcode oligonucleotide molecules each comprising a barcode sequence;

(b) lysing said cell in said well to release a nucleic acid molecule from said cell;

(c) capturing said nucleic acid molecule using said bead via a barcode oligonucleotide molecule of said plurality of barcode oligonucleotide molecules;

(d) removing said bead from said well; and

(e) external to said well, conducting a nucleic acid reaction with said nucleic acid molecule, or a derivative thereof, to yield a plurality of barcoded nucleic acid products.

59. A method for nucleic acid processing, comprising:

(a) co-partitioning a cell and a bead in a droplet, wherein said bead comprises a plurality of barcode oligonucleotide molecules each comprising a barcode sequence;

(b) lysing said cell in said droplet to release a nucleic acid molecule from said cell;

(c) capturing said nucleic acid molecule using said bead via a barcode oligonucleotide molecule of said plurality of barcode oligonucleotide molecules;

(d) removing said bead from said droplet; and

(e) external to said droplet, conducting a nucleic acid reaction with said nucleic acid molecule, or a derivative thereof, to yield a plurality of barcoded nucleic acid products.