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

Patent metadata

Field	Value
Publication number	US-12553074-B2
Application number	US-202318388735-A
Country	US
Kind code	B2
Filing date	Nov 10, 2023
Priority date	Jan 12, 2015
Publication date	Feb 17, 2026
Grant date	Feb 17, 2026

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.

First claim

What is claimed is:

1 . A method for nucleic acid processing, comprising:
(a) providing a double-stranded nucleic acid molecule comprising at least one uracil; (b) in presence of barcode molecules each comprising a barcode sequence, generating a plurality of nucleic acid fragments from said double-stranded nucleic acid molecule at one or more sites corresponding to said at least one uracil, wherein said plurality of nucleic acid fragments comprise different sequences,
wherein said barcode molecules and said double-stranded nucleic acid molecule are separate molecules; and
(c) generating nucleic acid products using said plurality of nucleic acid fragments and said barcode molecules, wherein each nucleic acid product comprises (i) a sequence corresponding to a nucleic acid fragment of said plurality of nucleic acid fragments; and (ii) said barcode sequence or reverse complement thereof.

2 . The method of claim 1 , wherein (b) and (c) are performed in a partition among a plurality of partitions.

3 . The method of claim 2 , wherein said partition is a droplet among a plurality of droplets in an emulsion.

4 . The method of claim 2 , wherein said partition is a well among a plurality of wells.

5 . The method of claim 1 , wherein (b) comprises excising said at least one uracil from said double-stranded nucleic acid molecule.

6 . The method of claim 1 , further comprising nicking said double-stranded nucleic acid molecule at said one or more sites corresponding to said at least one uracil and performing one or more nucleic acid extension reactions from said one or more sites.

7 . The method of claim 6 , further comprising performing said one or more nucleic acid extension reactions with a polymerase having strand displacement activity.

8 . The method of claim 7 , wherein said polymerase is a phi29 polymerase.

9 . The method of claim 6 , further comprising performing said one or more nucleic acid extension reactions isothermally.

10 . The method of claim 1 , wherein said double-stranded nucleic acid molecule comprises genomic DNA.

11 . The method of claim 1 , wherein, in (b) nucleic acid fragments of said plurality of nucleic acid fragments are each from 1 kb to 100 kb in length.

12 . The method of claim 1 , further comprising sequencing said nucleic acid products or derivatives thereof.

13 . The method of claim 1 , further comprising performing (b) in presence of a random primer.

14 . The method of claim 13 , wherein said random primer comprises a random N-mer sequence of 5 to 25 nucleotides in length.

15 . The method of claim 1 , further comprising performing (b) in presence of a reducing agent.

16 . The method of claim 15 , wherein said reducing agent comprises DTT.

17 . The method of claim 1 , further comprising performing (b) in presence of a single stranded binding protein (SSB).

18 . The method of claim 1 , further comprising performing (b) in presence of deoxyuridine triphosphates (dUTPs).

19 . The method of claim 1 , wherein (c) further comprises ligating a barcode molecule of said barcode molecules to a nucleic acid fragment of said plurality of nucleic acid fragments.

20 . The method of claim 1 , wherein (c) further comprises generating said nucleic acid products via a nucleic acid extension reaction.

Assignees

• 10X Genomics Inc

Inventors

• Hardenbol Paul
• Patel Pranav
• Hindson Benjamin
• Wyatt Paul William
• Bjornson Keith
• Wu Indira
• Belhocine Zahra Kamila

Classifications

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01J2219/00722

  Nucleotides · CPC title

• B01J2219/00572

  Chemical means · CPC title

• B01J2219/00547

  Bar codes · CPC title