title: Targeted enrichment of Temnothorax turcicus: adult worker
design: For all sequenced taxa we extracted DNA using Qiagen DNeasy Blood and Tissue kits (Qiagen Inc., Valencia, CA) and we fragmented up to 50 ng of input DNA to an average fragment distribution of 400-600 bp using a Qsonica sonicator (15 seconds of sonication, 100% amplitude, 90 seconds rest). 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, we constructed sequencing libraries using Kapa Hyper Prep library preparation kits (Kapa Biosystems Inc., Wilmington, MA) and custom dual-indexing barcodes (Glenn et al. 2016).We followed the manufacturers protocol except that Iwe performed all steps, except PCR, at one-quarter volume. We assessed success of library preparation following 12-14X PCR amplification by measuring DNA concentration and by visualizing libraries on an agarose gel. We 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, we pooled 10 libraries together at equimolar concentrations and adjusted pool concentrations to 147 ng/ul using a vacuum centrifuge. For each enrichment we used a total of 500 ng of DNA (3.4 ul each pool), and we enriched the DNA with an ant-specific bait library (Branstetter et al. 2017), diluted 1:4 (baits:water). We hybridized RNA bait libraries to sequencing libraries at 65 C for a period of 24 hours, and we enriched each pool following a standardized enrichment protocol (version 1.5; available from http://ultraconserved.org). We 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, we used qPCR to measure the DNA concentration of each enriched pool, and we combined all pools together at equimolar ratios to produce a final pool-of-pools. We sent the final pool-of-pools to the High-Throughput Genomics Center at Novogene USA, where the samples were quality checked on an Agilent 2200 TapeStation (High Sensitivity D1K tape; Agilent Technologies, Santa Clara, CA), and sequenced on an Illumina HiSeq 2500 (2x125 v4 chemistry; Illumina Inc., San Diego, CA).