Methods and systems for processing polynucleotides

• US 10,208,343 B2
• Filed: 12/05/2017
• Issued: 02/19/2019
• 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, Hardenbol, Paul, Zheng, Xinying, Belgrader, Phillip
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/831,726
• Publication Number:
  US 20180094312A1
• Time in Patent Office: 441 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 library of partitions for barcoding nucleic acid molecules, comprising:

a plurality of partitions comprising a plurality of gel beads that are degradable upon application of a stimulus, wherein said plurality of gel beads comprises a plurality of barcode sequences that are different across said plurality of gel beads,

wherein a given partition of said plurality of partitions comprises a given gel bead of said plurality of gel beads and an agent configured to degrade said given gel bead,

wherein said given gel bead of said plurality of gel beads comprises a plurality of nucleic acid molecules, and

wherein each of said plurality of nucleic acid molecules comprises a barcode sequence from said plurality of barcode sequences and a unique molecular sequence, wherein (i) said barcode sequence is constant across said plurality of nucleic acid molecules, and (ii) said unique molecular sequence varies across said plurality of nucleic acid molecules.

2. The library of claim 1, wherein said plurality of gel beads comprises at least 1,000 gel beads.

3. The library of claim 2, wherein said plurality of gel beads comprises at least 10,000 gel beads.

4. The library of claim 3, wherein said plurality of gel beads comprises at least 100,000 gel beads.

5. The library of claim 1, wherein said plurality of nucleic acid molecules comprises at least 1,000 nucleic acid molecules.

6. The library of claim 5, wherein said plurality of nucleic acid molecules comprises at least 10,000 nucleic acid molecules.

7. The library of claim 6, wherein said plurality of nucleic acid molecules comprises at least 100,000 nucleic acid molecules.

8. The library of claim 7, wherein said plurality of nucleic acid molecules comprises at least 1,000,000 nucleic acid molecules.

9. The library of claim 1, wherein each of said plurality of nucleic acid molecules comprises a functional sequence for coupling to a flow cell of a sequencer.

10. The library of claim 1, wherein each of said plurality of nucleic acid molecules comprises a priming sequence for a target nucleic acid molecule.

11. The library of claim 10, wherein said priming sequence is a poly-thymine (poly-T) sequence.

12. The library of claim 11, wherein said target nucleic acid molecule is a ribonucleic acid molecule, and wherein said ribonucleic acid molecule comprises an anchoring sequence configured to permit said poly-T sequence to hybridize at a sequence end of said ribonucleic acid molecule.

13. The library of claim 1, wherein each of said plurality of nucleic acid molecules comprises a random priming sequence.

14. The library of claim 1, wherein said given gel bead is porous.

15. The library of claim 1, wherein said plurality of gel beads is substantially monodisperse.

16. The library of claim 1, wherein said plurality of nucleic acid molecules is coupled to said given gel bead.

17. The library of claim 16, wherein said plurality of nucleic acid molecules is releasably coupled to said given gel bead.

31. The library of claim 1, wherein said agent is a reducing agent.

18. A composition for barcoding a nucleic acid molecule, comprising:

a partition comprising a bead having a plurality of nucleic acid molecules, wherein each of said plurality of nucleic acid molecules comprises a sequence having a barcode sequence and a unique molecular sequence, wherein (i) said barcode sequence is constant across said plurality of nucleic acid molecules, and (ii) said unique molecular sequence varies across said plurality of nucleic acid molecules, wherein said partition comprises an agent configured to release said sequence into said partition.

19. The composition of claim 18, wherein said plurality of nucleic acid molecules comprises at least 1,000 nucleic acid molecules.

20. The composition of claim 19, wherein said plurality of nucleic acid molecules comprises at least 10,000 nucleic acid molecules.

21. The composition of claim 20, wherein said plurality of nucleic acid molecules comprises at least 100,000 nucleic acid molecules.

22. The composition of claim 21, wherein said plurality of nucleic acid molecules comprises at least 1,000,000 nucleic acid molecules.

23. The composition of claim 18, wherein each of said plurality of nucleic acid molecules comprises a functional sequence for coupling to a flow cell of a sequencer.

24. The composition of claim 18, wherein each of said plurality of nucleic acid molecules comprises a priming sequence for a target nucleic acid molecule.

25. The composition of claim 24, wherein said priming sequence is a poly-thymine (poly-T) sequence.

26. The composition of claim 25, wherein said target nucleic acid molecule is a ribonucleic acid molecule, and wherein said ribonucleic acid molecule comprises an anchoring sequence configured to permit said poly-T sequence to hybridize at a sequence end of said ribonucleic acid molecule.

27. The composition of claim 18, wherein each of said plurality of nucleic acid molecules comprises a random priming sequence.

28. The composition of claim 18, wherein said bead is porous.

29. The composition of claim 18, wherein said plurality of nucleic acid molecules is coupled to said bead.

30. The composition of claim 29, wherein said plurality of nucleic acid molecules is releasably coupled to said bead.

32. The composition of claim 18, wherein said agent is a reducing agent.