Compositions and methods for reducing base call errors by removing deaminated nucleotides from a nucleic acid library

Patent metadata

Field	Value
Publication number	US-2023392144-A1
Application number	US-202318328650-A
Country	US
Kind code	A1
Filing date	Jun 2, 2023
Priority date	Jun 3, 2022
Publication date	Dec 7, 2023
Grant date	—

Abstract

The present disclosure provides compositions comprising reagents employed in a nucleic acid library preparation workflow for removing deaminated bases, and methods for using the reagents. The compositions and methods described herein reduce base call errors, such as C:G to T:A transitions, in nucleic acid sequencing workflows.

First claim

What is claimed is:

1 . A method for reducing deaminated nucleotide bases in a nucleic acid library, comprising:
a) providing a plurality of linear nucleic acid library molecules, wherein individual library molecules in the plurality comprise a sequence of interest joined to at least one universal adaptor sequence having a binding sequence for a surface capture primer and one universal adaptor sequence having a binding sequence for a sequencing primer, and wherein at least one of the library molecule carries one or more deaminated nucleotide bases; b) contacting the plurality of nucleic acid library molecules with a reagent that removes deaminated nucleotide bases, thereby generating at least one library molecule carrying an abasic site; c) circularizing the plurality of nucleic acid library molecules to generate a plurality of covalently closed circular library molecules; d) distributing the plurality of covalently closed circular library molecules onto a support having a plurality of immobilized surface capture primers, under a condition suitable for hybridizing individual covalently closed circular library molecules to a surface capture primer; e) conducting a rolling circle amplification reaction to generate a plurality of nucleic acid concatemer template molecules immobilized to the support; and f) sequencing the plurality of nucleic acid concatemer template molecules to determine the sequence of at least a portion of the concatemer template molecules.

2 . The method of claim 1 , further comprising: (g) contacting the plurality of covalently closed circular library molecules with a reagent the removes deaminated nucleotide bases, thereby generating at least one circular library molecule carrying an abasic site.

3 . The method of claim 1 , wherein the reagent that removes deaminated nucleotide bases of step (b) comprises DNA glycosylase (UDG) and (i) AP lyase, (ii) Endo IV endonuclease, (iii) FPG glycosylase/AP lyase, and/or (iv) Endo VIII glycosylase/AP lyase, or a combination thereof.

4 . The method of claim 1 , wherein the plurality of immobilized surface capture primers is tethered to a polymer coating on the support.

5 . The method of claim 1 , wherein the rolling circle amplification reaction of step (e) comprises a strand displacing polymerase and a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and/or dUTP.

6 . The method of claim 1 , wherein the plurality of immobilized surface capture primers is located at pre-determined locations on the support.

7 . The method of claim 1 , wherein the plurality of immobilized surface capture primers is located at random locations on the support.

8 . The method of claim 1 , wherein the plurality of immobilized concatemer template molecules on the support is in fluid communication with each other to permit flowing a solution of reagents onto the support.

9 . The method of claim 8 , wherein the solution of reagents comprises enzymes, nucleotides, and divalent cations.

10 . The method of claim 8 , wherein the plurality of immobilized concatemer template molecules is essentially simultaneously reacted with the reagents in a massively parallel manner.

11 . The method of claim 1 , wherein the density of the plurality of immobilized concatemer template molecules on the polymer-coated support is 10 2 -10 12 per mm 2 .

12 . The method of claim 1 , wherein sequencing the plurality of immobilized concatemers comprises:
a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contacting is conducted under a condition suitable to form a plurality of complexed polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to a soluble sequencing primer; b) contacting the plurality of complexed sequencing polymerases with a plurality of nucleotides under a condition suitable for binding at least one nucleotide to a complexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the sugar 3′ position; c) incorporating at least one nucleotide into the 3′ end of the hybridized sequencing primers, thereby generating a plurality of nascent extended sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleo-base of the incorporated nucleotide.

13 . The method of claim 12 , wherein the plurality of nucleotides comprises:
a) a removable chain terminating moiety at the 3′ sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group; b) a removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3′OH moiety on the sugar group; and/or c) one type of nucleotide or a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

14 . The method of claim 13 , wherein sequencing the plurality of immobilized concatemers comprises:
a) contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of the soluble sequencing primers, wherein the contacting is conducted under a condition suitable to form a plurality of first complexed polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to a soluble sequencing primer; b) contacting the plurality of complexed sequencing polymerases with a plurality of detectably labeled multivalent molecules to form a plurality of multivalent-complexed polymerases, under a condition suitable for binding complementary nucleotide units of the multivalent molecules to at least two of the plurality of first complexed polymerases thereby forming a plurality of multivalent-complexed polymerases, and the condition inhibits incorporation of the complementary nucleotide units into the sequencing primers of the plurality of multivalent-complexed polymerases, wherein individual multivalent molecules in the plurality of multivalent molecules comprise a core attached to multiple nucleotide arms and each nucleotide arm is attached to a nucleotide unit; c) detecting the plurality of multivalent-complexed polymerases; and d) identifying the nucleo-base of the complementary nucleotide units that are bound to the plurality of first complexed polymerases in the plurality of multivalent-complexed polymerases, thereby determining the sequence of the nucleic acid template.

15 . The method of claim 14 , further comprising:
e) dissociating the plurality of multivalent-complexed polymerases, removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; f) contacting the plurality of the retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contacting is conducted under a condition suitable for binding the plurality of second sequencing polymerases to the plurality of the retained nucleic acid duplexes, thereby forming a plurality of second complexed polymerases each comprising a secon

Assignees

• Element Biosciences Inc

Inventors

• Price Andrew
• Zhao Junhua
• Kruglyak Semyon
• Blease Kelly
• Yu Hua
• Altomare Andrew
• Kelley Ryan
• Moreno Juan

Classifications

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6806 Primary

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

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2521/301

  Endonuclease · CPC title