target enrichment sequencing of 4485
DNA was extracted from samples using a salt extraction protocol (Sambrook and Russell, 2001), and 10,000ng per sample was sheared to approximately 200bp-500bp on a BioRuptor NGS (Diagenode) and dual-end size selected (0.8X-1.0X) with SPRI beads. Dual-indexed libraries were prepared from 375-2000ng of size selected DNA using KAPA LTP library prep kits (Glenn et al., 2017). These libraries were pooled (with samples from other projects) into batches of 16 samples at 250ng per sample (4,000ng total) and enriched in the presence of 30,000ng of c0t-1 repetitive sequence blocker (McCartney-Melstad et al., 2016) derived from T. carnifex (casualties from a removal action of an invasive population (Meilink et al., 2015)) by hybridizing blockers with libraries for 30 minutes and probes with libraries/blockers for 30 hours. Enriched libraries were subjected to 14 cycles of PCR with KAPA HiFi HotStart ReadyMix and pooled at an equimolar ratio for 150bp paired-end sequencing across multiple Illumina HiSeq 4000 lanes (receiving an aggregate of 18% of one lane, for a multiplexing equivalent of 128 samples per lane).