The full details of transcriptome sequencing, probe design, library preparation, sequence captures, and bioinformatics pipelines are described in Portik et al.
More...The full details of transcriptome sequencing, probe design, library preparation, sequence captures, and bioinformatics pipelines are described in Portik et al. (2016b), but here we outline major steps of the transcriptome-based exon captures. We sequenced, assembled, and filtered the transcriptomes of four divergent hyperoliid species and selected 1,260 orthologous transcripts for probe design. We chose transcripts 500–850 bp in length that ranged from 5–15% average pairwise divergence. Five additional nuclear loci (POMC, RAG-1, TYR, FICD and KIAA2013) were also incorporated based on published sequence data (Portik & Blackburn 2016). The final marker set for probe design included 1,265 genes from four species and 5060 individual sequences, with a total of 995,700 bp of target sequence. These sequences were used to design a MYbaits-3 custom bait library (MYcroarray, now Arbor Biosciences) consisting of 120 mer baits and a 2X tiling scheme (every 60 bp), which resulted in 60,179 unique baits. Transcriptomes, target sequences, and probe designs are available on Dryad (Portik et al. 2016c).
Genomic DNA was extracted using a high-salt extraction method (Aljanabi & Martinez 1997) and individual genomic libraries were prepared following Meyer & Kircher (2010) with modifications described in Portik et al. (2016b). Samples were pooled for capture reactions based on phylogenetic relatedness, and the combined postcapture libraries were sequenced on three lanes of an Illumina HiSeq2500 with 100 bp paired-end reads. Raw sequence data were cleaned following Singhal (2013) and Bi et al. (2012), and the cleaned reads of each sample were de novo assembled, filtered, and mapped as described in Portik et al. (2016b). Less...