Multiplex target capture with double-stranded DNA probes

Peidong Shen, Wenyi Wang, Aung-Kyaw Chi, Yu Fan, Ronald W Davis, Curt Scharfe, Peidong Shen, Wenyi Wang, Aung-Kyaw Chi, Yu Fan, Ronald W Davis, Curt Scharfe

Abstract

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.

Figures

Figure 1
Figure 1
Probe construction, target capture and reciprocal paired-end sequencing.(a) Each cLPP contains a common linker flanked by post-capture amplification sites (red and green) and two target-specific capturing arms (blue and orange). Probe ends are trimmed (BsaI and MlyI) and 5'-phosphorylated to produce functional cLPPs. (b) Multiplex probe-target hybridization followed by gap-filling and ligation triggers probe circularization and target capture. (c) Capture libraries are multiplex-amplified using hybrid primers that anneal to the probes' amplification sites and add Illumina sequencing adaptors (P5 or P7) and sample-specific barcodes. This is done in two separate PCRs during which the adaptors swap positions at the ends of templates. Both PCRs are pooled for reciprocal PE sequencing of both DNA strands.
Figure 2
Figure 2
Coverage distribution across target regions. (a) Cumulative mean percent base coverage across 5,619 targets captured using cLPPs and ssLPPs, respectively, and shown separately for sequence read 1 and read 2. All bases have a minimum of 10× coverage. (b) Log ratio of coverage of read 1 and 2. Each boxplot corresponds to coverage distribution of a group of amplicons within a defined size range with number of amplicons, percent bases covered (≥10×) and average GC content shown for each group. All groups present a statistically significant distribution different from each other and each maintains a mean significantly different from 0.

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