Methods and systems for processing polynucleotides

• US 10,533,221 B2
• Filed: 10/19/2018
• Issued: 01/14/2020
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Jarosz, Mirna, Hardenbol, Paul, Schnall-Levin, Michael, Ness, Kevin, Saxonov, Serge
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/165,389
• Publication Number:
  US 20190085391A1
• Time in Patent Office: 452 Days
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 2531/113 PCR
• 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 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

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. In some cases, this disclosure provides methods for the generation of polynucleotide barcode libraries, and for the attachment of such polynucleotides to target polynucleotides.

Claims

1. A method for generating a plurality of nucleic acid molecules comprising barcode sequences, comprising:

synthesizing a plurality of linear nucleic acid molecules comprising a plurality of barcode sequences;

separating said plurality of linear nucleic acid molecules into a plurality of partitions comprising a plurality of beads, wherein a partition of said plurality of partitions comprises a single linear nucleic acid molecule of said plurality of linear nucleic acid molecules and a single bead of said plurality of beads;

in said partition, subjecting said single linear nucleic acid molecule to nucleic acid amplification to generate at least 1,000 nucleic acid molecules attached to said single bead, wherein said at least 1,000 nucleic acid molecules comprise a barcode sequence of said plurality of barcode sequences, which barcode sequence is identical for said at least 1,000 nucleic acid molecules; and

recovering said at least 1,000 nucleic acid molecules attached to said single bead from said partition.

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

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

4. The method of claim 1, wherein said at least 1,000 nucleic acid molecules are releasably attached to said single bead.

5. The method of claim 1, wherein said plurality of barcode sequences comprise 1,000 different barcode sequences.

6. The method of claim 1, wherein said plurality of barcode sequences comprise 10,000 different barcode sequences.

7. The method of claim 1, wherein said plurality of barcode sequences comprise 100,000 different barcode sequences.

8. The method of claim 1, wherein said plurality of barcode sequences comprise 1000,000 different barcode sequences.

9. The method of claim 1, wherein said at least 1,000 nucleic acid molecules attached to said single bead are at least 10,000 nucleic acid molecules.

10. The method of claim 1, wherein said at least 1,000 nucleic acid molecules attached to said single bead are at least 100,000 nucleic acid molecules.

11. The method of claim 1, wherein said at least 1,000 nucleic acid molecules attached to said single bead are at least 1000,000 nucleic acid molecules.

12. The method of claim 1, wherein said at least 1,000 nucleic acid molecules attached to said single bead are at least 10,000,000 nucleic acid molecules.

13. The method of claim 1, wherein each linear nucleic acid molecule of said plurality of linear nucleic acid molecules further comprises an immobilization sequence and wherein said plurality of beads are coupled to a nucleic acid molecule comprising a sequence complementary to said immobilization sequence.

14. The method of claim 13, wherein each linear nucleic acid molecule of said plurality of linear nucleic acid molecules further comprises an adapter sequence capable of coupling to a target nucleic acid molecule.

15. The method of claim 13, wherein each linear nucleic acid molecule of said plurality of linear nucleic acid molecules further comprises a sequencing primer sequence.

16. The method of claim 1, wherein said plurality of partitions is a plurality of wells.

17. The method of claim 1, wherein said plurality of partitions is a plurality of aqueous droplets in an emulsion.

18. The method of claim 1, wherein said separating comprises performing a limiting dilution, thereby generating diluted polynucleotides.

19. The method of claim 18, wherein said separating further comprises partitioning said diluted polynucleotides.

20. The method of claim 1, wherein greater than 5% of said plurality of partitions comprise a single linear nucleic acid molecule of said plurality of linear nucleic acid molecules that is unique within that partition.

21. The method of claim 1, wherein said single linear nucleic acid molecule comprises less than 100 nucleotides.

22. The method of claim 1, wherein said single linear nucleic acid molecule comprises less than 50 nucleotides.

23. The method of claim 1, wherein said single linear nucleic acid molecule comprises less than 200 nucleotides.

24. The method of claim 1, wherein said single bead comprises crosslink bonds that are cleavable upon application of stimuli.

25. The method of claim 24, wherein said stimuli comprises one or more members selected from the group consisting of chemical stimuli, biological stimuli, light stimuli, thermal stimuli, magnetic stimuli, and addition of a medium to a well.

26. The method of claim 1, wherein said nucleic acid amplification reaction comprises one or more members selected from the group consisting of polymerase chain reaction, asymmetric polymerase chain reaction, emulsion PCR (ePCR), multiple annealing and looping-based amplification cycles (MALBAC), and single primer isothermal amplification.

27. The method of claim 1, wherein said nucleic acid amplification reaction comprises a nucleic acid extension reaction.

28. The method of claim 1, wherein said plurality of linear nucleic acid molecules comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

29. The method of claim 1, wherein said synthesizing comprises random DNA synthesis.

30. The method of claim 14, further comprising attaching a linear nucleic molecule of said plurality of linear nucleic acid molecules to said target nucleic acid molecule.

31. The method of claim 30, wherein said attaching comprises ligation or amplification.

32. The method of claim 1, wherein said barcode sequence comprises at least 5 nucleotides.

33. The method of claim 1, wherein said barcode sequence comprises at least 10 nucleotides.

34. The method of claim 1, wherein said plurality of barcode sequences comprise random barcode sequences.