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

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.