Compositions and methods for pairwise sequencing

Patent metadata

Field	Value
Publication number	US-11236388-B1
Application number	US-202117377285-A
Country	US
Kind code	B1
Filing date	Jul 15, 2021
Priority date	Jun 17, 2021
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

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 support having a plurality of surface primers immobilized thereon wherein the surface primers comprise a 3′ extendible end and lack a nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the surface primers;
b) 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 surface primers and conducting a rolling circle amplification reaction with a plurality of strand displacing polymerases and a plurality of nucleotides, thereby generating a plurality of immobilized single stranded nucleic acid concatemer template molecules, wherein individual single stranded nucleic acid concatemer template molecules are covalently joined to an immobilized surface primer, and wherein the plurality of nucleotides lacks a nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the concatemer template molecules;
c) sequencing the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers, 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;
d) 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 by conducting a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand-displacing polymerases to generate a plurality of forward extension strands and a plurality of partially displaced forward extension strands wherein the forward extension strands and the partially displaced forward extension strands are hybridized to the immobilized concatemer template molecules to form a plurality of immobilized amplicons; and
e) sequencing the plurality of immobilized partially displaced forward extension strands with a plurality of soluble reverse sequencing primers, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual immobilized partially displaced forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon, and wherein the immobilized partially displaced forward extension strands remain hybridized to the retained immobilized concatemer template molecules during sequencing of step e).

2. The method of claim 1 , wherein each of the single stranded circular nucleic acid library molecules in the plurality comprises a sequence of interest, and wherein the individual library molecules further comprise 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 surface primer,
(iv) a universal binding sequence for a first soluble amplification primer,
(v) a universal binding sequence for a second soluble amplification primer,
(vi) a universal binding sequence for a soluble compaction oligonucleotide,
(vii) a sample barcode sequence and/or
(viii) a unique molecular index sequence.

3. The method of claim 1 , wherein individual immobilized single stranded nucleic acid concatemer template molecules generated by the rolling circle amplification reaction comprise two or more copies of a sequence of interest, wherein the individual immobilized concatemer template molecules further comprise 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 surface primer,
(iv) two or more copies of a universal binding sequence for a first soluble amplification primer,
(v) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a soluble compaction oligonucleotide,
(vii) two or more copies of a sample barcode sequence and/or
(viii) two or more copies of a unique molecular index sequence.

4. The method of claim 1 , wherein replacing the plurality of extended forward sequencing primer strands of step d) comprises:
(i) removing the plurality of extended forward sequencing primer strands while retaining the immobilized concatemer template molecules; and
(ii) contacting the plurality of retained immobilized concatemer molecules with the plurality of soluble amplification primers, a plurality of nucleotides and a plurality of strand displacing polymerases, under a condition suitable to hybridize the plurality of soluble amplification primers to the plurality of retained immobilized concatemer template molecules and suitable for conducting polymerase-catalyzed strand displacing reactions thereby generating a plurality of forward extension strands and a plurality of partially displaced extended forward sequencing strands that are hybridized to the immobilized concatemer template molecules to form a plurality of immobilized amplicons.

5. The method of claim 4 , wherein the strand displacing polymerase comprises phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.

6. The method of claim 1 wherein the forward sequencing of step c) comprises:
c1) contacting a plurality of sequencing polymerases and a plurality of the soluble forward sequencing primers to a plurality of immobilized concatemer template molecules, 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 an immobilized concatemer template molecule hybridized to a soluble forward sequencing primer;
c2) 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;
c3) incorporating at least one nucleotide into the 3′ end of the hybridized forward sequencing primers thereby generating a plurality of nascent extended forward sequencing primers; and
c4) detecting the incorporated nucleotide and identifying the nucleo-base of the incorporated nucleotide.

7. The method of claim 6 , wherein at least one of the nucleotides in the plurality of nucleotides of step c2) comprises a removable chain terminating moiety attached to the 3′ carbon position of the 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, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3′OH moiety on the sugar group.

Assignees

• Element Biosciences Inc

Inventors

• Arslan Sinan
• Zhao Junhua
• He Molly
• Snow Samantha
• Light William
• Kellinger Matthew
• Previte Michael

Classifications

• C12Q1/6853

  using modified primers or templates · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · 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

• C12Q2600/158

  Expression markers · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title