Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same

• US 10,557,158 B2
• Filed: 12/20/2018
• Issued: 02/11/2020
• Est. Priority Date: 01/12/2015
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hardenbol, Paul, Patel, Pranav, Hindson, Benjamin, Wyatt, Paul William, Bjornson, Keith, Wu, Indira, Belhocine, Kamila
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US16/228,362
• Publication Number:
  US 20190169666A1
• Time in Patent Office: 418 Days
• Field of Search: None
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes

Abstract

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

Claims

1. A method for nucleic acid processing, comprising:

(a) synthesizing a double-stranded nucleic acid molecule comprising at least one uracil;

(b) at one or more sites corresponding to said at least one uracil, generating a plurality of single-stranded nucleic acid molecules from said double-stranded nucleic acid molecule; and

(c) using a nucleic acid barcode molecule comprising a barcode sequence to generate a barcoded nucleic acid molecule comprising (i) a sequence of a single-stranded nucleic acid molecule of said plurality of single-stranded nucleic acid molecules or reverse complement thereof; and

(ii) said barcode sequence or reverse complement thereof.

2. The method of claim 1, further comprising, subsequent to (b), (1) using a single-stranded nucleic acid molecule of said plurality of single-stranded nucleic acid molecules to generate an additional double-stranded nucleic acid molecule, and (2) using a ligating enzyme to ligate said nucleic acid barcode molecule to said additional double-stranded nucleic acid molecule.

3. The method of claim 2, wherein (1) comprises hybridizing a primer to said single-stranded nucleic acid molecule and performing a nucleic acid extension reaction to generate said additional double-stranded nucleic acid molecule.

4. The method of claim 3, wherein said primer comprises a random N-mer sequence of 5 to 25 nucleotides in length.

5. The method of claim 2, wherein said ligating enzyme is a deoxyribonucleic acid (DNA) ligase.

6. The method of claim 5, wherein said ligating enzyme is T4 DNA ligase.

7. The method of claim 1, wherein said nucleic acid barcode molecule is double stranded.

8. The method of claim 1, wherein (b) comprises excising said at least one uracil to yield one or more initiation sites corresponding to said one or more sites, and performing one or more nucleic acid extension reactions from said one or more initiation sites to generate said plurality of single-stranded nucleic acid molecules.

9. The method of claim 8, wherein said at least one uracil is excised using an excising enzyme.

10. The method of claim 9, wherein said excising enzyme comprises uracil deoxyribonucleic acid (DNA) glycosylase.

11. The method of claim 8, wherein in (b), a polymerizing enzyme having strand displacement activity engages said double-stranded nucleic acid molecule at an initiation site of said one or more initiation sites to perform a nucleic acid extension reaction of said one or more nucleic acid extension reactions.

12. The method of claim 11, wherein said nucleic acid extension reaction is performed in a primer-independent manner.

13. The method of claim 11, wherein said one or more nucleic acid extension reactions are isothermal.

14. The method of claim 1, wherein said nucleic acid barcode molecule is of a plurality of nucleic acid barcode molecules.

15. The method of claim 1, wherein (a), (b), or (c) are performed in a partition.

16. The method of claim 15, wherein said partition is a droplet of a plurality of droplets in an emulsion.

17. The method of claim 15, wherein said partition is a well of a plurality of wells.

18. The method of claim 1, wherein said nucleic acid barcode molecule further comprises one or more functional sequences.

19. The method of claim 18, wherein said one or more functional sequences are selected from the group consisting of an adapter sequence, a primer sequence, a primer annealing sequence, an attachment sequence, a sequencing primer sequence, and a partial sequencing primer sequence.

20. The method of claim 1, further comprising sequencing said barcoded nucleic acid molecule or a derivative thereof.

21. The method of claim 1, wherein (a) comprises (i) providing a template nucleic acid molecule, a primer, uracil-containing nucleotides, and a polymerizing enzyme;

(ii) hybridizing said primer to said template nucleic acid molecule and performing a nucleic acid extension reaction to generate said double-stranded nucleic acid molecule comprising said at least one uracil.

22. The method of claim 21, wherein said polymerizing enzyme is a phi29 polymerase.

23. The method of claim 21, wherein said primer comprises a random N-mer sequence of 5 to 25 nucleotides in length.

24. The method of claim 1, wherein said nucleic acid barcode molecule is attached to a bead.

25. The method of claim 24, wherein said nucleic acid barcode molecule is releasably attached to said bead.

26. The method of claim 25, wherein said bead is a gel bead.

27. The method of claim 26, wherein said gel bead is a degradable gel bead.

28. The method of claim 1, further comprising performing one or more reactions to append one or more functional sequences to said barcoded nucleic acid molecule.

29. The method of claim 28, wherein said one or more functional sequences are selected from the group consisting of an adapter sequence, a primer sequence, a primer annealing sequence, an attachment sequence, a sequencing primer sequence, and a partial sequencing primer sequence.

30. The method of claim 1, wherein said single-stranded nucleic acid molecule does not comprise uracil.