Compositions and methods for pairwise sequencing

Patent metadata

Field	Value
Publication number	US-11891651-B2
Application number	US-202117521239-A
Country	US
Kind code	B2
Filing date	Nov 8, 2021
Priority date	Jun 17, 2021
Publication date	Feb 6, 2024
Grant date	Feb 6, 2024

Abstract

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

First claim

What is claimed:

1. A method for pairwise sequencing, comprising:
a) providing a plurality of immobilized single stranded nucleic acid concatemer template molecules, wherein individual concatemer template molecules in the plurality comprise at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the concatemer template molecule, wherein each individual concatemer template molecule in the plurality is immobilized to a first surface primer that is immobilized to a support, and wherein the immobilized first surface primers lack nucleotides having a scissile moiety;
b) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands, wherein individual immobilized concatemer template molecules have two or more extended forward sequencing primer strands hybridized thereon;
c) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands that are hybridized to the retained immobilized concatemer template molecules by conducting a primer extension reaction;
d) removing the retained immobilized concatemer template molecules by generating abasic sites in the immobilized concatemer template molecules at the nucleotide(s) having the scissile moiety and generating gaps at the abasic sites to generate a plurality of gap-containing single stranded nucleic acid concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized surface primers; and
e) sequencing the plurality of retained forward extension strands, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual retained forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon.

2. The method of claim 1 , wherein each individual concatemer template molecule in the plurality is covalently joined to an immobilized first surface primer.

3. The method of claim 1 , wherein each individual concatemer template molecule in the plurality is hybridized to an immobilized first surface primer.

4. The method of claim 1 , wherein individual immobilized concatemer template molecules in the plurality comprise two or more copies of a sequence of interest and any one or any combination of two or more of:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of a universal binding sequence for a soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of a universal binding sequence for a first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide,
(viii) two or more copies of a sample barcode sequence and/or
(ix) two or more copies of a unique molecular index sequence.

5. The method of claim 1 , wherein the sequencing of step (b) comprises hybridizing a plurality of soluble forward sequencing primers to the plurality of immobilized concatemer template molecules and conducting one or more sequencing reactions.

6. The method of claim 1 , wherein the sequencing of step (e) comprises hybridizing a plurality of soluble reverse sequencing primers to the plurality of retained forward extension strands and conducting one or more sequencing reactions.

7. The method of claim 4 , wherein the support further comprises a plurality of immobilized second surface primers that lack a nucleotide having a scissile moiety.

8. The method of claim 7 , wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatemer template molecules is hybridized to an immobilized second surface primer.

9. The method of claim 7 , wherein the plurality of immobilized second surface primers have 3′ OH extendible ends.

10. The method of claim 7 , wherein the plurality of immobilized second surface primers have 3′ non-extendible ends.

11. The method of claim 10 , wherein the 3′ non-extendible end comprises a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group.

12. The method of claim 1 , comprising at (a):
i) providing a support having a plurality of a first surface primers immobilized thereon, wherein the first surface primers have 3′ extendible ends; and
ii) generating a plurality of immobilized single stranded nucleic acid concatemer template molecules by hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized first surface primers and conducting a rolling circle amplification reaction with a plurality of strand displacing polymerases, and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP and nucleotides having a scissile moiety that can be cleaved to generate an abasic site, thereby generating a plurality of immobilized single stranded nucleic acid concatemer template molecules, wherein one or more individual immobilized single stranded nucleic acid concatemer template molecules within the plurality have at least one nucleotide with a scissile moiety, and wherein individual single stranded nucleic acid concatemer template molecules are covalently joined to immobilized first surface primers.

13. The method of claim 12 , wherein individual single-stranded circular nucleic acid library molecules in the plurality comprise a sequence of interest and any one or any combination of two or more of:
(i) a universal binding sequence for a soluble forward sequencing primer,
(ii) a universal binding sequence for a soluble reverse sequencing primer,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface primer,
(v) a universal binding sequence for a first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification primer,
(vii) a universal binding sequence for a soluble compaction oligonucleotide,
(viii) a sample barcode sequence and/or
(ix) a unique molecular index sequence.

14. The method of claim 1 , comprising prior to (a):
i) contacting in-solution a plurality of single-stranded circular nucleic acid library molecules with a plurality of first soluble amplification primers, a plurality of a strand displacing polymerases, and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP and nucleotides having a scissile moiety that can be cleaved to generate an abasic site, under conditions suitable to form a plurality of library-primer duplexes and conduct a rolling circle amplification reaction, thereby generating a plurality of single stranded nucleic acid concatemers, wherein one or more individual single stranded nucleic acid concatemers within the plurality have at least one nucleotide with a scissile moiety;
ii) distributing the rolling circle amplification reaction onto a support having a plurality of the first surface primers immobilized thereon, under conditions suitable for hybridizing one or more portions of individual single stranded concatemers to one or more immobilized first surface primers, wherein the first surface primers lack a nucleotide having a scissile moiety; and
iii) continuing the rolling circ

Assignees

• Element Biosciences Inc

Inventors

• Arslan Sinan
• Zhao Junhua
• He Molly
• Snow Samantha
• Light William
• Kellinger Matthew
• Previte Michael
• Kim Michael
• Yu Hua
• Hwang-Fu Yu-Hsien
• Tjioe Marco
• Boddicker Andrew

Classifications

• C12Q1/6806 Primary

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

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title