The steps to synthesize a targeted capture probe set in non-reference taxa using four alternative approaches. In PCR capture, target sequences are identified and amplified with PCR. Amplicons are then biotinylated to create long single-stranded probes to capture these sequences across a diverse range of species. For de novo assembly capture, initial transcriptomic, RAD-seq, or WGS experiments are used to generate de novo assemblies from which a targeted capture probe set is designed. In transcriptome-based approaches, probes are designed to target regions within a single exon corresponding to a transcript or set of transcripts of a gene. For RAD-seq or WGS capture, probes may target population-informative markers from the assembly. Finally, using a reference genome assembly of a divergent species, genomic sequences of interest and their locations (e.g., often the chromosome and the start and stop position of the interval) are identified and a probe set is designed to tile across the targeted regions.
Mol Ecol. ;25(1):185-202.