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Sequence capture using PCR-generated probes: a cost-effective method of targeted high-throughput sequencing for nonmodel organisms.

Peñalba JV, Smith LL, Tonione MA, Sass C, Hykin SM, Skipwith PL, McGuire JA, Bowie RC, Moritz C

Recent advances in high-throughput sequencing library preparation and subgenomic enrichment methods have opened new avenues for population genetics and phylogenetics of nonmodel organisms. To multiplex large numbers of indexed samples while sequencing predominantly orthologous, targeted regions of the genome, we propose modifications to an existing, in-solution capture that utilizes PCR products as target probes to enrich library pools for the genomic subset of interest. The sequence capture using PCR-generated probes (SCPP) protocol requires no specialized equipment, is highly flexible and significantly reduces experimental costs for projects where a modest scale of genetic data is optimal (25-100 genomic loci). Our alterations enable application of this method across a wider phylogenetic range of taxa and result in higher capture efficiencies and coverage at each locus. Efficient and consistent capture over multiple SCPP experiments and at various phylogenetic distances is demonstrated, extending the utility of this method to both phylogeographic and phylogenomic studies.

DNA shearing

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March, 2014


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