title: UCE target enrichment of Temnothorax_obturator_P111: whole organism
design: For all sequenced taxa I extracted DNA using Qiagen DNeasy Blood and Tissue kits (Qiagen Inc., Valencia, CA) and I fragmented up to 50 ng of input DNA to an average fragment distribution of 400-600 bp using a Bioruptor NGS sonicator (15 seconds of sonication, 100% amplitude, 90 seconds rest; Diagenode Inc., Denville, NJ). The amount of input DNA was calculated accurately by measuring the DNA concentration of extracts with a Qubit 2.0 fluorometer (Thermo Fisher Scientific Inc., Waltham, MA). Following sonication, I constructed sequencing libraries using Kapa Hyper Prep library preparation kits (Kapa Biosystems Inc., Wilmington, MA) and custom dual-indexing barcodes (Glenn et al. 2016). I followed the manufacturer’s protocol except that I performed all steps, except PCR, at one-quarter volume. I assessed success of library preparation following 12-14X PCR amplification by measuring DNA concentration and by visualizing libraries on an agarose gel. I purified post-PCR libraries by performing a 1.2X SPRI-bead clean-up using an AMPure XP substitute (Rohland & Reich 2012). For each UCE enrichment reaction, I pooled 10 libraries together at equimolar concentrations and adjusted pool concentrations to 147 ng/ul using a vacuum centrifuge. For each enrichment I used a total of 500 ng of DNA (3.4 ul each pool), and I enriched the DNA with an ant-specific bait library (Branstetter et al. 2017), diluted 1:4 (baits:water). I hybridized RNA bait libraries to sequencing libraries at 65 C for a period of 24 hours, and I enriched each pool following a standardized enrichment protocol (version 1.5; available from http://ultraconserved.org). I verified enrichment success with qPCR (CFX96 Touch, Bio-Rad Laboratories, Hercules, CA) by comparing amplification profiles of unenriched to enriched pools using PCR primers designed from several UCE loci. After verification, I used qPCR to measure the DNA concentration of each enriched pool, and I combined all pools together at equimolar ratios to produce a final pool-of-pools. I sent the final pool-of-pools to the High-Throughput Genomics Center at the University of Utah, where the samples were quality checked on an Agilent 2200 TapeStation (High Sensitivity D1K tape; Agilent Technologies, Santa Clara, CA), quantified with qPCR (Kapa Library Quant Kit), and sequenced on an Illumina HiSeq 2500 (2x125 v4 chemistry; Illumina Inc., San Diego, CA).
library_name: Temnothorax_obturator_P111