Linked duplex target capture
The invention generally relates to sequencing library preparation methods. In certain embodiments, two or more template nucleic acids are joined together by a linking molecule, such as a PEG derivative. Identical copies of a nucleic acid fragment or both strands of a duplex fragment may be linked together. The linked nucleic acids are amplified, creating linked amplicons. Emulsion PCR with linked primers creates linked template nucleic acids for seeding sequencing clusters and errors can be readily identified by their presence on only one of the linked fragments.
1. A method for sequencing a region of interest, the method comprising:
providing y-adapters comprising a double-stranded stem portion, the stem portion comprising a universal priming site and its compliment;
ligating the y-adapters onto a plurality of duplex nucleic acid fragments wherein the plurality of duplex nucleic acid fragments comprise a sense strand, an antisense strand, and at least one genomic region of interest;
denaturing the plurality of ligated duplex nucleic acid fragments to create single-stranded nucleic acid fragments each comprising one of the universal priming site or its compliment;
amplifying the single-stranded nucleic acid fragments using universal primers, each comprising one of the universal priming site or its compliment to generate a plurality of amplified fragments comprising copies of the sense and antisense strands, wherein the universal primers are physically linked to a target probe complimentary to at least a portion of the region of interest or its compliment; and
sequencing the plurality of amplified copies, to determine a sequence of the region of interest.
2. The method of claim 1 , wherein the amplifying step occurs under conditions that require binding of the target probe to the region of interest or its compliment to permit binding of the universal primer to the universal priming site, and wherein the target probe is blocked from extension, wherein amplifying the single-stranded nucleic acid fragments comprises extending the universal primer using a strand displacing polymerase to produce the plurality of amplified fragments.
3. The method of claim 1 , wherein the denaturing and amplifying steps are performed in an emulsion droplet.
4. The method of claim 1 , wherein the y-adapters further comprise barcodes.
5. The method of claim 4 , wherein the barcodes comprise unique molecular identifiers.
6. The method of claim 1 , wherein the amplifying step comprises extending the universal primers using a strand displacing enzyme.