Linked target capture and ligation
AssigneeBoreal Genomics, Inc.
InventorsJoel Pel, Andrea Marziali
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Abstract
The invention generally relates to capturing, amplifying, and sequencing nucleic acids. In certain embodiments, linked capture probes and multiple binding and extension steps improve specificity over traditional single binding target capture techniques. Methods of seeding sequencing clusters with captured target nucleic acids are also disclosed. Linked adapters may be used to increase adapter ligation selectively or efficiency and yield. Ligation adapters and primers can be linked to various sequence-specific or feature-specific molecules to selectively bind targets for ligation or amplification with universal adapters or primers.
Claims33
A method of selectively ligating adapters to a target nucleic acid, the method comprising:
- providing a first linked ligation adapter comprising a protein probe having affinity to a first portion of a target nucleic acid, the probe linked to a first adapter comprising a first universal priming site;
- exposing a sample comprising the target nucleic acid to the first linked ligation adapter;
- ligating the target nucleic acid to the first linked adapter;
- and
- amplifying the ligated target nucleic acid by PCR using a first universal primer complimentary to the first universal priming site.
A method for capturing genomic regions of interest for targeted DNA sequencing, the method comprising:
- ligating universal probe sites onto a plurality of duplex nucleic acid fragments wherein the plurality of duplex nucleic acid fragments comprise at least one genomic region of interest;
- denaturing the plurality of ligated duplex nucleic acid fragments to create single stranded nucleic acid fragments comprising universal probe sites;
- exposing the single stranded nucleic acid fragments to a plurality of linked capture probes comprising a target protein probe having affinity to at least a portion of the genomic region of interest, the target protein probe linked to a universal probe and a universal priming site, wherein the exposing step occurs under conditions that require binding of the target protein probe to the target nucleic acid sequence to permit binding of the universal probe to the universal probe site;
- extending the universal probe to produce a copy of the genomic region of interest;
- and
- sequencing the genomic region of interest.
Dependencies are inferred from the claim text.