Methods and Systems for Processing Polynucleotides

• US 20180094314A1
• Filed: 12/05/2017
• Published: 04/05/2018
• Est. Priority Date: 06/26/2014
• 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 10,030,267 B2
• Time in Patent Office: 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 1/683 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 2535/122 Massive parallel sequencing
• 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 2537/149 Sequential reactions
• 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 2565/629 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 method for identifying a protein and a messenger ribonucleic acid (mRNA) molecule as originating from a single cell, comprising:

(a) bringing said single cell in contact with one or more antibodies, wherein a given antibody of said one or more antibodies is specific to said protein, wherein said given antibody is conjugated to a first nucleic acid molecule comprising an antibody barcode sequence;

(b) co-partitioning said single cell with a single bead in a partition, wherein said single bead comprises a plurality of nucleic acid barcode molecules, wherein said plurality of nucleic acid barcode molecules comprises a second nucleic acid molecule and a third nucleic acid molecule each comprising a cell barcode sequence;

(c) generating, from said first nucleic acid molecule and said second nucleic acid molecule, a fourth nucleic acid molecule comprising (i) said antibody barcode sequence or a complement thereof, and (ii) said cell barcode sequence or a complement thereof;

(d) generating, from said messenger mRNA molecule of said single cell and said third nucleic acid molecule, a fifth nucleic acid molecule comprising (i) a sequence derived from said mRNA molecule and (ii) said cell barcode sequence; and

(e) using a sequencer to sequence said fourth nucleic acid molecule, said fifth nucleic acid molecule, or a derivative(s) thereof, to identify said antibody barcode sequence, said cell barcode sequence and said sequence derived from said mRNA molecule, wherein said antibody barcode sequence identifies said protein, said sequence derived from said mRNA molecule identifies said mRNA molecule, and said cell barcode sequence identifies said single cell, thereby identifying said protein and said mRNA as originating from said single cell.

2. The method of claim 1, wherein said partition is a droplet among a plurality of droplets.

3. The method of claim 1, wherein said partition is a well among a plurality of wells.

4. The method of claim 1, further comprising subjecting said fourth nucleic acid molecule or said fifth nucleic acid molecule to one or more reactions subsequent to release from said partition.

5. A method for identifying a protein as originating from a single cell, comprising:

(a) bringing said single cell in contact with one or more antibodies, wherein a given antibody of said one or more antibodies is specific to said protein, wherein said given antibody is conjugated to a first nucleic acid molecule comprising an antibody barcode sequence;

(b) co-partitioning said single cell with a single bead in a partition, wherein said single bead comprises a plurality of nucleic acid barcode molecules each comprising a cell barcode sequence, wherein said plurality of nucleic acid barcode molecules are configured for barcoding of constituents of said single cell, and wherein said plurality of nucleic acid barcode molecules comprises a second nucleic acid molecule comprising said cell barcode sequence;

(c) performing one or more reactions to generate, from said first nucleic acid molecule and said second nucleic acid molecule, a third nucleic acid molecule comprising (i) said antibody barcode sequence or a complement thereof, and (ii) said cell barcode sequence or a complement thereof; and

(d) using a sequencer to sequence said third nucleic acid molecule or a derivative thereof, to identify said antibody barcode sequence and said cell barcode sequence, wherein said antibody barcode sequence identifies said protein and said cell barcode sequence identifies said single cell, thereby identifying said protein as originating from said single cell.

6. The method of claim 5, wherein said plurality of nucleic acid barcode molecules comprises at least 100,000 nucleic acid molecules comprising said cell barcode sequence.

7. The method of claim 5, wherein (c) is performed in said partition.

8. The method of claim 5, further comprising releasing said third nucleic acid molecule from said partition.

9. The method of claim 8, further comprising subjecting said third nucleic acid molecule to one or more additional reactions subsequent to releasing said third nucleic acid molecule from said partition.

10. The method of claim 9, wherein said one or more additional reactions comprise polymerase chain reaction.

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

12. The method of claim 5, wherein said single bead is from a plurality of beads comprising a plurality of nucleic acid molecules having barcodes sequences that are different across said plurality of beads.

13. The method of claim 5, wherein said single bead is a gel bead.

14. The method of claim 13, further comprising, prior to (c), releasing said second nucleic acid molecule from said single bead.

15. The method of claim 14, wherein said second nucleic acid molecule is released from said single bead upon exposure to a chemical stimulus in said partition.

16. The method of claim 5, wherein said protein is a cell surface protein.

17. The method of claim 5, wherein said second nucleic acid molecule further comprises a unique molecular sequence.

18. The method of claim 5, wherein said second nucleic acid molecule further comprises a sequence that is complementary to a portion of said first nucleic acid molecule.

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

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

21. The method of claim 5, wherein said one or more reactions comprise a nucleic acid amplification reaction.

22. The method of claim 5, wherein said one or more reactions comprise a reverse transcription reaction.

23. The method of claim 5, wherein said one or more reactions comprise a template switching reaction.

24. The method of claim 5, wherein said third nucleic acid molecule comprises a functional sequence that is configured to permit attachment to a flow cell of said sequencer.

25. The method of claim 5, 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 said sequencer.

26. The method of claim 5, wherein said antibody is covalently attached to said first nucleic acid molecule.

27. The method of claim 5, further comprising generating from a messenger ribonucleic acid (mRNA) molecule of said single cell and a fourth nucleic acid molecule of said plurality of nucleic barcode molecules, a fifth nucleic acid molecule comprising (i) a sequence derived from said mRNA molecule and (ii) said cell barcode sequence.

28. The method of claim 27, wherein (d) comprises using said sequencer to sequence said fifth nucleic acid molecule or a derivative thereof, to associate with said single cell both (i) said protein and (ii) said mRNA molecule.

29. The method of claim 5, wherein said partition is a droplet among a plurality of droplets.

30. The method of claim 5, wherein said partition is a well among a plurality of wells.