Methods and systems for processing polynucleotides

• US 9,856,530 B2
• Filed: 05/05/2017
• Issued: 01/02/2018
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Saxonov, Serge, Ness, Kevin, Hardenbol, Paul, Schnall-Levin, Michael, Jarosz, Mirna
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/588,519
• Publication Number:
  US 20170247757A1
• Time in Patent Office: 242 Days
• Field of Search: None
• 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 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

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 nucleic acid preparation or analysis, comprising:

(a) providing;

(i) at least 1,000 gel beads;

(ii) releasably attached to each of said at least 1,000 gel beads, at least 1,000 barcode molecules comprising identical barcode sequences that are distinct from barcode sequences of at least 1,000 barcode molecules releasably attached to any other gel bead of said at least 1,000 gel beads; and

(iii) a plurality of cells each comprising a plurality of polynucleotide molecules;

(b) generating a plurality of droplets, wherein at least 1,000 droplets of said plurality of droplets each comprise;

(i) a single gel bead from said at least 1,000 gel beads; and

(ii) a single cell from said plurality of cells; and

(c) in each of said at least 1,000 droplets, using said plurality of polynucleotide molecules from said single cell and barcode molecules of said at least 1,000 barcode molecules from said single gel bead to generate a plurality of barcoded polynucleotide molecules,

wherein said barcode molecules become detached from said gel bead.

2. The method of claim 1, wherein, prior to (c), said plurality of polynucleotide molecules are released from said single cell in each of said at least 1,000 droplets.

3. The method of claim 1, wherein said plurality of polynucleotide molecules are a plurality of messenger ribonucleic acid (mRNA) molecules.

4. The method of claim 3, wherein (c) comprises reverse transcribing said plurality of mRNA molecules in presence of said barcode molecules to generate said plurality of barcoded polynucleotide molecules.

5. The method of claim 1, further comprising causing said plurality of barcoded polynucleotide molecules to be released from said at least 1,000 droplets.

6. The method of claim 5, further comprising amplifying said plurality of barcoded polynucleotide molecules by nucleic acid amplification subsequent to releasing said plurality of barcoded polynucleotide molecules from said at least 1,000 droplets.

7. The method of claim 1, further comprising amplifying said plurality of barcoded polynucleotide molecules by nucleic acid amplification in each of said at least 1,000 droplets.

8. The method of claim 1, further comprising causing said plurality of barcoded polynucleotide molecules or derivatives thereof to be sequenced.

9. The method of claim 1, wherein a subset of said plurality of droplets does not include a cell.

10. The method of claim 1, wherein a subset of said plurality of droplets does not include a gel bead.

11. The method of claim 1, wherein in each of said at least 1,000 droplets, said barcode molecules are released from said single gel bead.

12. The method of claim 11, wherein each of said at least 1,000 droplets further comprise a reducing agent that depolymerizes said single gel bead, thereby releasing said barcode molecules.

13. The method of claim 1, wherein said at least 1,000 gel beads are formed by polymerization of droplets comprising said at least 1,000 barcode molecules.

14. The method of claim 1, wherein said at least 1,000 barcode molecules are disposed within said single gel bead.

15. The method of claim 1, wherein, in (a), said at least 1,000 gel beads are a subset of a plurality of gel beads.

16. The method of claim 15, wherein said plurality of gel beads comprises at least 10,000 gel beads.

17. The method of claim 1, wherein said at least 1,000 barcode molecules comprise combinatorial assemblies of sequences from sequence modules.

18. The method of claim 17, wherein each of said combinatorial assemblies comprises a combinatorial assembly of a first sequence and a second sequence.

19. The method of claim 17, wherein each of said combinatorial assemblies comprises a combinatorial assembly of a first sequence, a said second sequence, and a third sequence.

20. The method of claim 1, wherein said plurality of droplets comprises at least 10,000 droplets.

21. The method of claim 1, wherein said plurality of droplets comprises at least 100,000 droplets.

22. The method of claim 1, wherein said at least 1,000 gel beads comprises at least 10,000 gel beads, and wherein said at least 1,000 droplets comprises at least 10,000 droplets.

23. The method of claim 1, wherein said at least 1,000 gel beads comprises at least 100,000 gel beads, and wherein said at least 1,000 droplets comprises at least 100,000 droplets.

24. The method of claim 1, wherein said at least 1,000 barcode molecules are at least 10,000 barcode molecules.

25. The method of claim 1, wherein said at least 1,000 barcode molecules are at least 100,000 barcode molecules.

26. The method of claim 1, wherein said at least 1,000 barcode molecules are at least 1,000,000 barcode molecules.

27. The method of claim 1, wherein said plurality of polynucleotide molecules are from 10,000-100,000 polynucleotide molecules.

28. The method of claim 1, wherein said barcode molecules become detached from said gel bead before generation of said barcoded polynucleotide molecules.

29. The method of claim 1, wherein said barcode molecules become detached from said gel bead following generation of said barcoded polynucleotide molecules.

30. The method of claim 1, wherein said barcode molecules become detached from said gel bead during generation of said barcoded polynucleotide molecules.