UCE target enrichment of Stenamma_snellingi_EX543_TCGCTGAG (CASENT0625875) [raw reads].
We employed a phylogenomic method that combines targeted enrichment of Ultra-Conserved Elements (UCEs) with multiplexed next-generation sequencing (Branstetter, Longino, Ward, & Faircloth, 2017; Faircloth, Branstetter, White, & Brady, 2015; Faircloth et al., 2012). The protocols are outlined in Faircloth et al. (2015) and described below. We extracted DNA using Qiagen DNeasy Blood and Tissue kits and checked DNA concentration using a Qubit 2.0 fluorometer (Life Technologies). We input up to 500 ng of DNA into library preparation and sheared the DNA to an average fragment distribution of 400-600 bp (verified on an agarose gel) using a Qsonica Q800R sonicator. Following sonication, we constructed sequencing libraries using either a standard Library Preparation Kit or the Hyper Prep Kit from Kapa Biosystems (now Roche). We performed all reactions at  volume except for the PCR step, which we ran at full volume (50 L). During library preparation we added either custom, single-indexing Truseq-style barcode adapters (Faircloth & Glenn, 2012), or a newer, dual-indexing set of adapter-primers called iTru (Glenn et al., 2019). We assessed success of library preparation by measuring DNA concentration with Qubit and by visualizing libraries on an agarose gel. For a subset of the samples, we removed adapter-dimers by performing a 0.7-0.8X SPRI-bead cleanup. For UCE enrichment we assembled enrichment pools containing 6-10 libraries at equimolar concentrations. We adjusted pool concentration to 147 ng/l using a vacuum centrifuge and we used a total of 500 ng of DNA (3.4 l of each pool) in each enrichment. We performed enrichments using a custom RNA bait library developed for Hymenoptera (Hym v1; Faircloth et al., 2015) and synthesized by MYcroarray (now Arbor Biosciences). The probe set includes 2,749 probes targeting 1,510 UCE loci. We enriched each pool of samples following the manufacturers protocol, except that for some pools, we left streptavidin beads bound to enriched DNA during PCR (on-bead PCR). The enrichment was performed at 65C for a period of 24 hours. We verified enrichment success with qPCR by comparing amplification profiles of unenriched to enriched pools for several UCE loci. After verification, we used qPCR to measure the DNA concentration of each pool and we then combined all pools into an equimolar final pool. At most we combined 110 samples together into the same sequencing lane (including samples from other projects). To remove fragments that were either too large or too small for sequencing, we size-selected pools from 300-800 bp using the Blue Pippin size selection instrument (Sage Science). We mailed size-selected pools to either the UCLA Neuroscience Genomics Core, Cornells Institute of Biotechnology, or the High Throughput Genomics Facility at the University of Utah, and we had the samples sequenced as single lanes on an Illumina HiSeq 2500 (2x150 Rapid Run, or 2x125 v4 Run).