Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-2018016634-A1
Application number	US-201715717871-A
Country	US
Kind code	A1
Filing date	Sep 27, 2017
Priority date	Jun 26, 2014
Publication date	Jan 18, 2018
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

What is claimed is:

1 . A method for single cell processing or analysis, comprising:
(a) bringing said single cell from a plurality of cells in contact with one or more antibodies, wherein an antibody from said one or more antibodies comprises a first nucleic acid molecule comprising a first barcode sequence; (b) co-partitioning said single cell with a single bead from a plurality of beads in a droplet, wherein said single bead comprises a second nucleic acid molecule comprising a second barcode sequence; and (c) generating from said first nucleic acid molecule and said second nucleic acid molecule, a third nucleic acid molecule comprising (i) said first barcode sequence or a complement thereof, and (ii) said second barcode sequence or a complement thereof.

2 . The method of claim 1 , wherein said single bead comprises a plurality of nucleic acid molecules comprising said second nucleic acid molecule, wherein each of said plurality of nucleic acid molecules comprises said second barcode sequence.

3 . The method of claim 2 , wherein said plurality of nucleic acid molecules comprises at least 1,000 nucleic acid molecules each comprising said second barcode sequence.

4 . The method of claim 2 , wherein said plurality of nucleic acid molecules comprises at least 1,000,000 nucleic acid molecules each comprising said second barcode sequence.

5 . The method of claim 1 , wherein (c) is performed in said droplet.

6 . The method of claim 5 , further comprising disrupting said droplet to release said third nucleic acid molecule from said droplet.

7 . The method of claim 6 , further comprising subjecting said third nucleic acid molecule to one or more reactions subsequent to releasing said third nucleic acid molecule from said droplet.

8 . The method of claim 7 , wherein said one or more reactions comprise polymerase chain reaction.

9 . The method of claim 7 , wherein said one or more reactions comprise addition of one or more functional sequences to said third nucleic acid molecule or a derivative thereof, wherein said one or more functional sequences are configured to permit attachment to a flow cell of a sequencer.

10 . The method of claim 1 , wherein said plurality of beads comprises a plurality of nucleic acid molecules having barcodes sequences that are different across said plurality of beads.

11 . The method of claim 1 , wherein said single bead is a gel bead.

12 . The method of claim 11 , further comprising, prior to (c), releasing said second nucleic acid molecule from said single bead.

13 . The method of claim 12 , wherein said second nucleic acid molecule is released from said single bead upon exposure to a chemical stimulus in said droplet.

14 . The method of claim 1 , further comprising using a sequencer to sequence said third nucleic acid molecule or a derivative thereof, to identify said first barcode sequence and said second barcode sequence, wherein said first barcode sequence identifies a protein to which said antibody is specific and said second barcode sequence identifies said single cell.

15 . The method of claim 14 , wherein said protein is a cell surface protein.

16 . The method of claim 1 , wherein said second nucleic acid molecule further comprises a unique molecular sequence.

17 . The method of claim 1 , wherein said second nucleic acid molecule further comprises a sequence that is complementary to a portion of said first nucleic acid molecule.

18 . The method of claim 17 , wherein said sequence is a poly-T sequence.

19 . The method of claim 18 , wherein (c) comprises hybridizing said poly-T sequence to said portion of said first nucleic acid molecule comprising a poly-A sequence.

20 . The method of claim 1 , wherein (c) comprises performing a nucleic acid amplification reaction to generate said third nucleic acid molecule.

21 . The method of claim 1 , wherein (c) comprises performing a reverse transcription reaction to generate said third nucleic acid molecule.

22 . The method of claim 1 , wherein (c) comprises performing a template switching reaction to generate said third nucleic acid molecule.

23 . The method of claim 1 , wherein said third nucleic acid molecule comprises a functional sequence that is configured to permit attachment to a flow cell of a sequencer.

24 . The method of claim 1 , further comprising, subsequent to (c), adding one or more priming sequences for priming extension of said third nucleic acid molecule or a derivative thereof in a sequencer.

25 . The method of claim 1 , wherein said antibody is covalently attached to said first nucleic acid molecule.

26 . The method of claim 1 , wherein said antibody is non-covalently attached to said first nucleic acid molecule.

27 . A method for single cell processing or analysis, comprising:
(a) co-partitioning said single cell from a plurality of cells with a single bead from a plurality of beads in a partition, wherein said single cell is coupled to an antibody comprising a first nucleic acid molecule comprising a first barcode sequence, wherein said single bead comprises a second nucleic acid molecule comprising a second barcode sequence; and (b) in said partition, generating from said first nucleic acid molecule and said second nucleic acid molecule, a third nucleic acid molecule comprising (i) said first barcode sequence or a complement thereof, and (ii) said second barcode sequence or a complement thereof.

28 . The method of claim 27 , wherein said partition is a droplet or a well.

29 . The method of claim 27 , wherein said antibody is coupled to a protein of said single cell.

30 . The method of claim 27 , wherein said partition is among a plurality of partitions.

Assignees

• 10X Genomics Inc

Inventors

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

Classifications

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2563/149

  Particles, e.g. beads · 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

• C12Q1/6806

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

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title