Methods and systems for processing polynucleotides

• US 10,457,986 B2
• Filed: 09/27/2018
• Issued: 10/29/2019
• 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
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/144,832
• Publication Number:
  US 20190032129A1
• Time in Patent Office: 397 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 analyzing analytes comprising:

(a) providing a first analyte, a second analyte, a cell feature binding group comprising a reporter molecule comprising a reporter barcode sequence associated with said cell feature binding group, and a support having coupled thereto a plurality of nucleic acid barcode molecules,

wherein said second analyte is a different type of analyte than said first analyte,

wherein said cell feature binding group is configured to couple to said first analyte, and

wherein said plurality of nucleic acid barcode molecules comprise (i) a common barcode sequence and (ii) a common capture sequence; and

(b) generating;

(i) a first barcoded nucleic acid molecule comprising;

(1) a first sequence corresponding to said reporter barcode sequence and (2) a second sequence corresponding to said common barcode sequence, and

(ii) a second barcoded nucleic acid molecule comprising;

(1) a third sequence corresponding to said second analyte and (2) a fourth sequence corresponding to said common barcode sequence.

2. The method of claim 1, wherein said support is a bead.

3. The method of claim 2, wherein said bead is a degradable bead.

4. The method of claim 2, wherein said bead is a gel bead.

5. The method of claim 1, wherein said second analyte is a ribonucleic acid (RNA) molecule.

6. The method of claim 5, wherein said RNA molecule is a messenger RNA (mRNA) molecule derived from a cell.

7. The method of claim 6, wherein (b) comprises:

(1) hybridizing said common capture sequence of a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to said mRNA molecule; and

(2) extending said nucleic acid barcode molecule in a reverse transcription reaction to generate said second barcoded nucleic acid molecule.

8. The method of claim 6, wherein (b) comprises generating a complementary deoxyribonucleic acid (cDNA) molecule derived from said mRNA molecule and using said cDNA molecule and a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to generate said second barcoded nucleic acid molecule.

9. The method of claim 6, wherein (a) comprises providing a cell comprising said mRNA.

10. The method of claim 1, wherein, in (a), said first analyte, said second analyte, and said support are provided in a partition among a plurality of partitions.

11. The method of claim 10, wherein said partition is a droplet among a plurality of droplets.

12. The method of claim 10, wherein said partition is a well among a plurality of wells.

13. The method of claim 1, wherein said common capture sequence comprises a poly-thymine (poly-T) sequence.

14. The method of claim 1, wherein said common capture sequence comprises a template switching oligonucleotide (TSO) sequence.

15. The method of claim 1, wherein said first analyte is a protein.

16. The method of claim 15, wherein, prior to (b), said protein is bound to said cell feature binding group comprising said reporter molecule, and wherein (b) comprises using said reporter molecule and a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to generate said first barcoded nucleic acid molecule.

17. The method of claim 16, wherein said cell feature binding group comprises an antibody, an antibody fragment, or a cell surface receptor binding molecule.

18. The method of claim 16, wherein (b) comprises:

(1) hybridizing said common capture sequence of said nucleic acid barcode molecule to said reporter molecule; and

(2) extending said nucleic acid barcode molecule in a nucleic acid extension reaction to generate said first barcoded nucleic acid molecule.

19. The method of claim 15, wherein (a) comprises providing a cell comprising said protein.

20. The method of claim 19, wherein said protein is a cell surface protein.

21. The method of claim 1, further comprising performing one or more reactions to add one or more additional nucleic acid sequences to said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule.

22. The method of claim 1, wherein (b) is performed with the aid of a template switch oligonucleotide.

23. The method of claim 1, wherein said plurality of nucleic acid barcode molecules are releasably attached to said support and wherein, after (a), nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules are released from said support.

24. The method of claim 1, further comprising sequencing (1) said first barcoded nucleic acid molecule or a derivative thereof and (2) said second barcoded nucleic acid molecule or a derivative thereof to generate first sequencing reads corresponding to said first barcoded nucleic acid molecule or said derivative thereof and second sequencing reads corresponding to said second barcoded nucleic acid molecule or said derivative thereof.

25. The method of claim 24, further comprising using said first sequencing reads and said second sequencing reads to identify said first analyte and said second analyte, respectively.

26. A method for analyzing analytes, comprising:

(a) providing a messenger ribonucleic acid (mRNA) molecule, a protein molecule bound to a cell-binding ligand, and a support having coupled thereto a plurality of nucleic acid barcode molecules,

wherein said cell-binding ligand comprises a reporter barcode sequence associated with said cell-binding ligand, and

wherein said plurality of nucleic acid barcode molecules comprise;

(i) a common barcode sequence and (ii) a common capture sequence; and

(b) generating

(i) a first barcoded nucleic acid molecule comprising (1) a first sequence corresponding to said mRNA molecule or a derivative thereof and (2) a second sequence corresponding to said common barcode sequence, and

(ii) a second barcoded nucleic acid molecule comprising (1) a third sequence corresponding to said reporter barcode sequence and (2) a fourth sequence corresponding to said common barcode sequence.

27. The method of claim 26, wherein said cell-binding ligand is an antibody, an antibody fragment, or a cell-surface receptor binding molecule.

28. The method of claim 26, wherein (a) further comprises providing a cell comprising said mRNA molecule and said protein molecule bound to said cell-binding ligand.

29. The method of claim 26, wherein, in (a), said mRNA molecule, said protein molecule bound to said cell-binding ligand, and said support are provided in a partition among a plurality of partitions.

30. A method for analyzing cells comprising:

(a) contacting a plurality of cells with a plurality of antibodies to provide a cell of said plurality of cells bound to an antibody of said plurality of antibodies,

wherein said antibody is coupled to a reporter oligonucleotide comprising a reporter barcode sequence and a poly-adenine (poly-A) sequence,

wherein said reporter barcode sequence identifies said antibody, and

wherein said cell comprises a plurality of messenger ribonucleic acid (mRNA) molecules;

(b) partitioning said plurality of cells and a plurality of beads into a plurality of partitions, wherein a partition of said plurality of partitions comprises said cell bound to said antibody and a bead of said plurality of beads,

wherein said bead comprises a plurality of nucleic acid barcode molecules each comprising a common barcode sequence and a poly-thymine (poly-T) sequence;

(c) in said partition, contacting said bead comprising said plurality of nucleic acid barcode molecules with (i) said reporter oligonucleotide and (ii) mRNA molecules of said plurality of mRNA molecules; and

(d) generating;

(i) a first barcoded nucleic acid molecule comprising (1) a first sequence corresponding to said reporter barcode sequence and (2) a second sequence corresponding to said common barcode sequence, and

(ii) a second barcoded nucleic acid molecule comprising (1) a third sequence corresponding to a sequence of an mRNA molecule of said plurality of mRNA molecules and (2) a fourth sequence corresponding to said common barcode sequence.