ddRAD-Seq of Gyrinophilus porphyriticus: tail tissue
Libraries were prepared following the double-digest restriction-associated DNA sequencing (ddRAD-seq) method (Peterson et al. 2012) with several modifications. Briefly, we used the restriction enzymes BspDI and SbfI to digest 300  1000 ng of genomic DNA per individual. We then performed fragment size selection using SPRI beads prior to adapter ligation. To enable detection of PCR duplicates, we introduced a random 8 bp sequence in the P2 adapter (Schweyen et al. 2014). The use of 12 unique barcode sequences in the P1 adapter allowed us to pool 12 individuals to form a single library. Fragments in the range of 360  440 bp were extracted from each library using a BluePippin size selection system (Sage Science). We used 39 uniquely indexed PCR primers, enabling us to pool all individuals into a single library for sequencing following PCR. The pooled library was sequenced on 3 lanes of Illumina HiSeq 2500 (125 bp paired-end) at Hudson Alpha Institute for Biotechnology, resulting in 616,385,491 forward and reverse reads.