Linked 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 capturing regions of interest for sequencing, the method comprising:
providing adapters comprising a universal priming site;
adding the adapters onto a plurality of nucleic acid fragments wherein the plurality of nucleic acid fragments comprise at least one region of interest;
amplifying the nucleic acid fragments using universal primers, each universal primer comprising one of the universal priming sites or its compliment, 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, to generate a plurality of amplified fragments comprising copies of the region of interest; and
sequencing the plurality of amplified fragments, 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.
3. The method of claim 1 , wherein the target probe is blocked from extension, and wherein amplifying the nucleic acid fragments comprises extending the universal primer using a strand displacing polymerase to produce the plurality of amplified fragments.
4. The method of claim 1 , wherein the nucleic acid fragments comprise DNA fragments.
5. The method of claim 4 , further comprising denaturing DNA fragments prior to amplifying the nucleic acid fragments.
6. The method of claim 5 , wherein the denaturing and amplifying steps are performed in an emulsion droplet.
7. The method of claim 1 , wherein the addition of adapters is performed using a transposase.
8. The method of claim 1 , wherein the nucleic acids originate from a single cell.
9. The method of claim 1 , wherein the adapters further comprise barcodes.
10. The method of claim 9 , wherein the barcodes comprise unique molecular identifiers.
11. The method of claim 4 , wherein the probes target a forward DNA sense, a reverse DNA sense, or both the forward DNA sense and the reverse DNA sense.