Processes and Systems for Preparation of Nucleic Acid Sequencing Libraries and Libraries Prepared Using Same

Patent metadata

Field	Value
Publication number	US-2016257984-A1
Application number	US-201614990276-A
Country	US
Kind code	A1
Filing date	Jan 7, 2016
Priority date	Jan 12, 2015
Publication date	Sep 8, 2016
Grant date	—

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

1 . A method of preparing a sequencing library comprising the steps of:
(a) providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads comprising oligonucleotide adapter sequence segments; (b) amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence comprising occasional dUTPs; and (c) excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

2 . The method of claim 1 , further comprising a step (d) of amplifying the nicked complementary nucleic acid sequence, and a step (e) of extending the sequence of the amplified nucleic acid sequence using a nucleic acid extension means.

3 . The method of claim 1 , wherein the steps are performed in a single reaction.

4 . The method of claim 1 , wherein the plurality of beads is a pooled bead population.

5 . The method of claim 4 , wherein the beads of the pooled bead population are co-partitioned with one or more of the components listed in step (a), and wherein the partition optionally comprises a droplet in an emulsion.

6 . The method of claim 1 , wherein the beads comprise degradable beads selected from chemically degradable beads, photodegradable beads and thermally degradable beads.

7 . The method of claim 6 , wherein the beads comprise chemically reducible cross-linkers.

8 . The method of claim 7 , wherein the chemically reducible cross-linkers comprise disulfide linkages.

9 . The method of claim 1 , wherein the amplification in step (b) is isothermal.

10 . The method of claim 1 , wherein the polymerase is phi29 DNA polymerase.

11 . The method of claim 2 , wherein the nucleic acid extension means is selected from the group consisting of a ligating enzyme, a nucleic acid extension enzyme and a transposase.

12 . The method of claim 11 wherein the library of amplified nucleic acid sequences comprises single stranded DNA and the ligating enzyme comprises an ATP independent enzyme.

13 . The method of claim 12 , wherein the ATP independent enzyme comprises thermostable 5′ App DNA/RNA ligase.

14 . The method of claim 11 , wherein the ligating enzyme comprises a topoisomerase.

15 . The method of claim 14 , wherein the topoisomerase is topoisomerase I.

16 . The method of claim 11 , wherein the ligating enzyme comprises T4 DNA ligase.

17 . A method of preparing a barcode sequencing library, comprising:
(a) providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, a nucleic acid extension means and a plurality of beads comprising oligonucleotide barcode sequence segments; (b) amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence comprising occasional dUTPs; and (c) excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence; (d) amplifying the nicked complementary nucleic acid sequence to provide a library of amplified nucleic acid sequences; and (e) releasing the barcode sequence segments from the pooled bead population; and (f) extending the sequence of the amplified nucleic acid sequences using the barcode sequence segments and the nucleic acid extension means to provide a barcode library or alternatively, ligating the barcode sequence segments, using a nucleic acid ligating enzyme, to the library of amplified nucleic acid sequences to provide a barcode library.

18 . The method of claim 17 , wherein the steps are performed in a single reaction.

19 . The method of claim 17 , wherein the plurality of beads is a pooled bead population.

20 . The method of claim 19 , wherein the beads of the pooled bead population are co-partitioned with one or more of the components listed in step (a), and wherein the partition optionally comprises a droplet in an emulsion.

21 . The method of any of claim 17 , wherein the beads comprise degradable beads selected from chemically degradable beads, photodegradable beads and thermally degradable beads.

22 . The method of claim 21 , wherein the beads comprise chemically reducible cross-linkers.

23 . The method of claim 22 , wherein the chemically reducible cross-linkers comprise disulfide linkages.

24 . The method of claim 17 , wherein the amplification in step (b) is isothermal.

25 . The method of claim 17 , wherein the polymerase is phi29 DNA polymerase.

26 . The method of claim 17 , wherein the nucleic acid extension means is selected from the group consisting of a ligating enzyme, a nucleic acid extension enzyme and a transposase.

27 . The method of claim 26 wherein the library of amplified nucleic acid sequences comprises single stranded DNA and the ligating enzyme comprises an ATP independent enzyme.

28 . The method of claim 27 , wherein the ATP independent enzyme comprises thermostable 5′ App DNA/RNA ligase.

29 . The method of claim 26 , wherein the ligating enzyme comprises a topoisomerase.

30 . The method of claim 29 , wherein the topoisomerase is topoisomerase I.

31 . The method of claim 26 , wherein the ligating enzyme comprises T4 DNA ligase.

32 . The method of claim 17 , wherein the barcode sequence segments comprise at least 4 nucleotides at least 10 nucleotides or at least 20 nucleotides.

33 . The method of claim 17 , wherein the barcode sequence segments comprise at least 1000 different barcode sequence segments.

34 . The method of claim 17 , wherein at least 1,000,000 oligonucleotide molecules are attached to each bead.

35 . The method of claim 19 , wherein the pooled bead population comprises at least 10 different bead populations.

36 . The method of claim 19 , wherein the pooled bead population comprises at least 100 different bead populations.

37 . The method of claim 19 , wherein the pooled bead population comprises at least 500 different bead populations.

38 . The method of claim 17 , wherein the oligonucleotide barcode sequence segments comprise at least one functional sequence.

39 . The method of claim 38 , wherein the functional sequence is selected from an adapter, a primer sequence, a primer annealing sequence, an attachment sequence, and a sequencing primer sequence.

40 . The method of claim 38 , wherein the functional sequence is sequestered and releasable in a releasing step comprising a stimulus selected from the list consisting of thermal increase and chemical cleavage.

41 . The method of claim 40 , wherein the releasing step comprises degrading at least a portion the beads of the bead population comprising oligonucleotide barcode sequence segments.

42 . The method of claim 41 , wherein degrading the beads comprises cleaving a chemical linkage comprising a disulfide bridge linkage between the barcode sequence segments and the bead, and the releasing step comprises exposing the beads to a reducing agent.

43 . The method of claim 42 , wherein the reducing

Assignees

• 10X Genomics Inc

Inventors

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

Classifications

• B01J2219/00722

  Nucleotides · CPC title

• B01J2219/00572

  Chemical means · CPC title

• B01J2219/00547

  Bar codes · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title