UCE target enrichment of Leptocoma-sperata-KU-20350: muscle/liver tissue
We sheared 500 ng of DNA of each sample (except for one sample extracted from the toepad of a museum study skin, which was not sheared)  to 400Đ600 bp in 50 _l volume using a Covaris S220 sonicator at 175 W peak incident power, 2% duty factor, and 200 cycles per burst for 45 seconds.  We performed end repair, A-tailing, adapter ligation, and amplification on sheared DNA using Kapa Biosystems Library Prep kits following the procedure of Faircloth et al. (2012) described in detail at http://ultraconserved.org.  We deviated from the standard protocol by using _ volume of reagents called for in the library prep kit, ligating universal iTru stubs instead of sample-specific adapters to allow for dual indexing, and adding a second AMPure XP bead clean up at 1.0x volume after stub ligation.  We incorporated dual-indexes to library fragments using a 17-cycle PCR with NEB Phusion High-Fidelity PCR Master Mix.  iTru stubs and adapters were developed and designed by T. Glenn et al (unpublished data). We quantified libraries using a Qubit 2.0 Fluorometer and pooled sets of eight  samples at equimolar ratios for enrichment. We performed sequence capture and post-enrichment amplification following standard protocols (Faircloth et al. 2012) using the Mycroarray MYbaits kit for Tetrapods UCE 5K version 1, which targets 5,060 UCE loci.  We hybridized biotinylated RNA probes with pooled libraries for 24 hrs., and which we enriched DNA targets using streptavidin-coated beads.  We amplified fragments with a 17-cycle PCR amplification step using NEB Phusion High-Fidelity PCR Master Mix.  After post-enrichment amplification, we quantified libraries using an Illumina Eco qPCR System and a commercial library quantification kit (Kapa Biosystems, Inc.), combined 96 libraries into a 10 ľM pool, and sequenced the pooled libraries in a high output, paired-end run of 100 cycles on an Illumina HiSeq 2500 System at the University of Kansas Genome Sequencing Core.