UCE target enrichment of Polistes_poeyi_EX399: whole organism
For all newly sampled taxa, we extracted DNA using Qiagen DNeasy Blood and Tissue kits (Qiagen Inc., Valencia, CA) and we fragmented up to 500 ng of input DNA to an average fragment distribution of 400-600 bp using a Qsonica Q800R sonicator (Qsonica LLC, Newton, CT). Following sonication, we constructed sequencing libraries using Kapa library preparation kits (Kapa Biosystems Inc., Wilmington, MA) and custom sample barcodes. We assessed success of library preparation following PCR amplification by measuring DNA concentration and visualizing libraries on an agarose gel. We purified reactions following PCR using 0.8 to 1.0X AMPure substitute. For UCE enrichment we pooled 6Đ10 libraries together at equimolar concentrations and adjusted pool concentrations to 147 ng/_l. For each enrichment we used a total of 500 ng of DNA (3.4 _l each pool), and we performed enrichments using a custom RNA bait library developed for Hymenoptera and synthesized by MYcroarray (MYcroarray, Ann Arbor, MI). The probe set includes 2,749 probes, targeting 1,510 UCE loci. We hybridized RNA bait libraries to sequencing libraries at 65źC for a period of 24 hours, and we enriched each pool following a standardized protocol (version 1.5; protocol available from http://ultraconserved.org). We verified enrichment success with qPCR (ViiA 7, Applied Biosystems, Waltham MA) 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 pool, and we combined all pools together at equimolar ratios to produce a final pool-of-pools. To remove overly large and small fragments, we size-selected the final pools to a range of 300Đ800 bp using a Blue Pippin size selection instrument (Sage Science, Beverly, MA). We mailed size-selected pools to either the UCLA Neuroscience Genomics Core or the Cornell University Biotechnology Resource Center (http://www.biotech.cornell.edu/brc/genomics-facility), where the samples were quality checked on a Bioanalyzer (Agilent Technologies, Santa Clara, CA), quantified with qPCR, and sequenced on an Illumina HiSeq 2500 (2x150 Rapid Run; Ilumina Inc, San Diego, CA).