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Target enrichment technologies utilize single-stranded oligonucleotide probes to capture candidate genomic regions from a DNA sample before sequencing. We describe target capture using double-stranded probes, which consist of single-stranded, complementary long padlock probes (cLPPs), each selectively capturing one strand of a genomic target through circularization. Using two probes per target increases sensitivity for variant detection and cLPPs are easily produced by PCR at low cost. Additionally, we introduce an approach for generating capture libraries with uniformly randomized template orientations. This facilitates bidirectional sequencing of both the sense and antisense template strands during one paired-end read, which maximizes target coverage.

Multiplex target capture with double-stranded DNA probes