Methods and Systems for Processing Polynucleotides

Patent metadata

Field	Value
Publication number	US-2020199669-A1
Application number	US-201916570898-A
Country	US
Kind code	A1
Filing date	Sep 13, 2019
Priority date	Jun 26, 2014
Publication date	Jun 25, 2020
Grant date	—

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.

First claim

1 .- 30 . (canceled)

31 . A method, comprising:
(a) providing a support, a first analyte, and a second analyte, wherein said support comprises a plurality of nucleic acid barcode molecules coupled thereto, wherein said plurality of nucleic acid barcode molecules comprise a first nucleic acid barcode molecule and a second nucleic acid barcode molecule, wherein said first nucleic acid barcode molecule comprises a barcode sequence and a template switching sequence, wherein said second nucleic acid barcode molecule comprises said barcode sequence and said template switching sequence; and (b) generating (i) a first barcoded nucleic acid molecule using said first nucleic acid barcode molecule and said first analyte by coupling said template switching sequence of said first nucleic acid barcode molecule to said first analyte, and (ii) a second barcoded nucleic acid molecule using said second nucleic acid barcode molecule and said second analyte by coupling said template switching sequence of said second nucleic acid barcode molecule to said second analyte.

32 . The method of claim 31 , wherein said first analyte is a different type of analyte than said second analyte.

33 . The method of claim 31 , wherein said first analyte comprises an oligonucleotide molecule.

34 . The method of claim 33 , wherein said oligonucleotide molecule comprises a complementary deoxyribonucleic acid (cDNA) molecule derived from a ribonucleic acid (RNA) molecule, wherein said cDNA molecule comprises a sequence configured to couple to said template switching sequence.

35 . The method of claim 34 , further comprising, prior to (a), generating said cDNA molecule from said RNA molecule.

36 . The method of claim 33 , wherein said oligonucleotide molecule comprises a reporter oligonucleotide molecule comprising a reporter barcode sequence, or derivative of said reporter oligonucleotide molecule.

37 . The method of claim 36 , wherein said first analyte is a cell feature binding group comprising said reporter oligonucleotide molecule, or derivative thereof, and wherein said reporter barcode sequence is associated with said cell feature binding group.

38 . The method of claim 37 , wherein said cell feature binding group comprises one or more members selected from the group consisting of an antibody, an antibody fragment, a cell surface receptor binding molecule, and an antigen binding molecule.

39 . The method of claim 37 , wherein said second analyte is a second cell feature binding group comprising a second reporter oligonucleotide molecule, or derivative thereof, wherein said second reporter oligonucleotide molecule comprises a second reporter barcode sequence, wherein said second reporter barcode sequence is associated with said second cell feature binding group, and wherein said cell feature binding group and said second cell feature binding group are different.

40 . The method of claim 31 , wherein said template switching sequence of said first nucleic acid barcode molecule comprises a poly-nucleotide sequence at a 3′ terminus of said first nucleic acid barcode molecule.

41 . The method of claim 31 , wherein said template switching sequence of said first nucleic acid barcode molecule comprises a poly-guanine (polyG) sequence at said 3′ terminus of said first nucleic acid barcode molecule.

42 . The method of claim 41 , wherein said polyG sequence comprises a ribonucleic acid base.

43 . The method of claim 31 , wherein said template switching sequence is from about 6 nucleotides to about 20 nucleotides in length.

44 . The method of claim 31 , wherein said first nucleic acid barcode molecule further comprises a first unique molecular sequence, wherein said second nucleic acid barcode molecule comprises a second unique molecular sequence, and wherein said first unique molecular sequence is different than said second unique molecular sequence.

45 . The method of claim 31 , wherein said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are covalently linked to said support.

46 . The method of claim 45 , wherein said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are each covalently linked to said support through a cleavable linkage.

47 . The method of claim 46 , wherein said cleavable linkage comprises one or more members selected form the group consisting of a chemically cleavable linkage, a photocleavable linkage, and a thermally cleavable linkage.

48 . The method of claim 31 , wherein said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are coupled to said support through a disulfide linker.

49 . The method of claim 31 , wherein said support comprises a cystamine.

50 . The method of claim 31 , wherein said support is a bead.

51 . The method of claim 50 , wherein said bead is a gel bead.

52 . The method of claim 51 , further comprising degrading said gel bead upon application of a stimulus.

53 . The method of claim 50 , wherein said plurality of nucleic acid barcode molecules is releasably attached to said bead.

54 . The method of claim 31 , wherein said plurality of nucleic acid barcode molecules is releasably attached to said support.

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Hindson Christopher
• Schnall-Levin Michael
• Ness Kevin
• Jarosz Mirna
• Saxonov Serge
• Hardenbol Paul
• Bharadwaj Rajiv
• Zheng Xinying
• Belgrader Phillip

Classifications

• C12Q1/683 Primary

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title

• C12Q2563/159

  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 title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title