Targeted depletion of non-target library molecules using poison primers during target capture of next-generation sequencing libraries

Patent metadata

Field	Value
Publication number	US-12590328-B2
Application number	US-202118004365-A
Country	US
Kind code	B2
Filing date	Jul 7, 2021
Priority date	Jul 8, 2020
Publication date	Mar 31, 2026
Grant date	Mar 31, 2026

Abstract

The present disclosure is directed to compositions, kits, and methods of target enrichment by unidirectional primer extension, whereby the compositions, kits, and methods utilize both poison primers and target capture primers.

First claim

The invention claimed is:

1 . A method of enriching at least one target nucleic acid molecule in a library of nucleic acid molecules, wherein the at least one target nucleic acid molecule originates from genomic nucleic acid molecules, wherein the method comprises the following steps:
(a) hybridizing a first target capture primer to a first target nucleic acid molecule in the library of nucleic acid molecules, wherein the first target capture primer comprises a capture moiety; (b) hybridizing a first poison primer to a first non-target nucleic acid molecule in the library of nucleic acid molecules, wherein the first non-target nucleic acid molecule is ribosomal RNA (rRNA), wherein the first poison primer does not include any capture moiety; (c) extending both the first hybridized target capture primer and the first hybridized poison primer, wherein the extension of the first hybridized target capture primer provides a first target capture primer extension complex comprising the first target nucleic acid molecule and the extended first target capture primer, wherein (i) the extension of the first hybridized poison primer prevents the first target capture primer from hybridizing to the first non-target nucleic acid molecule, or (ii) the extension of the first hybridized poison primer displaces a first target primer hybridized to the first non-target nucleic acid molecule; and (d) enriching the first target nucleic acid molecule relative to the library of nucleic acid molecules in the library of nucleic acid molecules.

2 . The method of claim 1 , wherein the first target capture primer and the first poison primer are added as a pool of primers to the library of nucleic acid molecules.

3 . The method of claim 1 , wherein each of the nucleic acid molecules in the library of nucleic acid molecules comprises a first end including a first adapter and a second end including a second adapter.

4 . The method of claim 3 , further comprising the step of amplifying the first target nucleic acid molecule with a first amplification primer and a second amplification primer, wherein the first amplification primer comprises a 3′ end complementary to the first adapter, and wherein the second amplification primer comprises a 3′ end complementary to the second adapter.

5 . The method of claim 4 , further comprising the step of sequencing the amplified target nucleic acid molecule.

6 . The method of claim 1 , wherein the enriching of the first target nucleic acid molecule relative to the library of nucleic acid molecules comprises: (i) capturing the first target capture primer extension complex; (i) removing un-captured non-target nucleic acid molecules; and (ii) releasing the first target nucleic acid molecule from the captured first target capture primer extension complex.

7 . The method of claim 6 , wherein the capturing of the first target capture primer extension complex comprises: contacting the first target capture primer extension complex with a functionalized substrate.

8 . The method of claim 7 , wherein the capture moiety of the first target capture primer comprises a first member of a pair of specific binding entities, and wherein the functionalized substrate comprises a second member of the pair of specific binding entities.

9 . The method of claim 8 , wherein the first member of the pair of specific binding entities is selected from the group consisting of biotin, an antigenic molecule, an enzyme substrate, a receptor ligand, a polysaccharide, a thiolated molecule, and an amine-terminated molecule.

10 . The method of claim 8 , wherein the second member of the pair of specific binding entities is selected from the group consisting of streptavidin, an antibody, an enzyme, a receptor, a lectin, a gold particle, and an NHS-activated moiety.

11 . The method of claim 7 , wherein the first target capture primer is coupled to a substrate through the capture moiety prior to the hybridizing of the first target capture primer to the first target nucleic acid molecule in the library of nucleic acid molecules, and wherein the hybridizing of the first target capture primer to the first target nucleic acid molecule and the extending of the first hybridized target capture primer thereby captures the first target nucleic acid molecule to the substrate.

12 . The method of claim 6 , wherein the capturing of the first target capture primer extension complex comprises: (i) hybridizing a universal capture oligonucleotide to the capture moiety of the first target capture primer extension complex to form a universal capture oligonucleotide complex, wherein the universal capture oligonucleotide comprises (1) a first member of a pair of specific binding entities, and (2) a nucleotide sequence complementary to at least a portion of a capture sequence of the capture moiety; (ii) contacting the universal capture complex with a functionalized substrate, wherein the functionalized substrate comprises a second member of the pair of specific binding entities.

13 . The method of claim 6 , wherein the removing of the un-captured nucleic acid molecules comprises washing away the un-captured non-target nucleic acid molecules.

14 . The method of claim 6 , wherein the releasing of the captured first target capture primer extension complex comprises: (i) hybridizing a release primer to the first target nucleic acid molecule; and (ii) extending the hybridized release primer.

15 . The method of claim 14 , wherein the hybridized first target capture primer is extended with a first polymerase, and wherein the hybridized release primer is extended with a second polymerase.

16 . The method of claim 15 , wherein the first and second polymerases are different.

17 . The method of claim 1 , wherein the first target capture primer has a melting temperature which is greater than a melting temperature of the first poison primer.

18 . The method of claim 1 , wherein the library of nucleic acid molecules comprises a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules, wherein the plurality of target nucleic acid molecules are in low abundance as compared with the plurality of non-target nucleic acid molecules.

19 . The method of claim 18 , wherein the plurality of target nucleic acid molecules comprise less than about 10% of the nucleic acid molecules in the library of nucleic acid molecules.

20 . The method of claim 18 , wherein the plurality of target nucleic acid molecules comprise less than about 5% of the nucleic acid molecules in the library of nucleic acid molecules.

21 . The method of claim 18 , wherein the plurality of target nucleic acid molecules comprise fusion transcripts.

Assignees

• Roche Sequencing Solutions Inc
• Kapa Biosystems Inc

Inventors

• Dannebaum Richard
• Godwin Brian Christopher
• Penkler David L
• Slabbert Etienne
• Van Der Merwe Ruben

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title