Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers
(a) Custom adapters (i) are ligated to template DNA and the resulting product is amplified with universal primers. (b) Target regions are selectively amplified using custom probe-dependent primers (PDPs) (ii) which contain a recognition sequence (dark grey) with a 3’ blocker (black diamond) and are linked to an oligo containing a universal priming sequence for the first target capture PCR reaction (tcPCR1). (c) A second set of PDPs (iii), which contain Illumina adapters (red and black) between the probe and linked universal primer, are then added and a second target capture PCR reaction (tcPCR2) is completed prior to (d), clean up and QC and (e) loading on a sequencer. The inset shows detail of forward and reverse PDPs.