title: UCE trimmed reads of Parasyscia sp MG CASENT0732076
design: I extracted DNA using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). I quantified DNA for each extraction using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and sheared <5-50 ng of DNA to a target size of approximately 400-600 bp. The shearing was done by sonication on a Bioruptor instrument (Diagenode Inc., Philadelphia, PA, USA). The library preparation protocol that follows was slightly modified from Blaimer et al. (2015). I used a KAPA Hyper Prep Library Kit (Kapa Biosystems, Inc., Wilmington, MA, USA) with magnetic bead cleanup (Fisher et al. 2011) and a SPRI substitute (Rohland and Reich 2012) as described in Faircloth et al. (2014). I used TruSeq adapters (Faircloth and Glenn 2012) for ligation followed by PCR amplification of the library using a mix of HiFi HotStart polymerase reaction mix (Kapa Biosystems), Illumina TruSeq primers, and nuclease-free water. After rehydrating in 23 uL pH 8 Elution Buffer (EB hereafter) and purifying reactions using 1.1-1.2X speedbeads, I pooled nine to eleven libraries at equimolar ratios for final concentrations of 132-212 n/uL. I enriched each pool with 9,446 custom-designed probes (MYcroarray, Inc.) targeting 2,524 UCE loci in Hymenoptera (Branstetter et al. 2017). I followed library enrichment procedures for the MYcroarray MYBaits kit (Blumenstiel et al. 2010), except I used a 0.1X of the standard MYBaits concentration, and added 0.7 uL of 500 muM custom blocking oligos designed against the custom sequence tags. I ran the hybridization reaction for 24 h at 65 degrees C, subsequently bound all pools to streptavidin beads (MyOne C1; Life Technologies), and washed bound libraries according to a standard target enrichment protocol (Blumenstiel et al. 2010). I used the with-bead approach for PCR recovery of enriched libraries as described in Faircloth (2015). I combined 15 uL of streptavidin bead-bound, enriched library with 25 uL HiFi HotStart Taq (Kapa Biosystems), 5 uL of Illumina TruSeq primer mix (5 muM each) and 5 uL of nuclease-free water. Following PCR I purified the resulting reactions using 1.1-1.2X speedbeads, and rehydrated the enriched pools in 22 uL EB. Following enrichment I quantified 2 uL of each pool using a Qubit fluorometer (broad range kit). I verified if the enrichment was successful by amplifying four UCE loci targeted by the probe set. I set up a relative qPCR by amplifying two replicates of 1 ng of enriched DNA from each pool for the four loci and comparing those results to two replicates of 1 ng unenriched DNA from each pool. I performed qPCR using a SYBR FAST qPCR kit (Kapa Biosystems) on CFX Connect Real-Time PCR Detection System (Bio-Rad). Based on the size-adjusted concentrations estimated by qPCR, I pooled libraries at equimolar concentrations. The pooled libraries were then subjected to further quality control on a Bioanalyzer and sequenced using one full and one partial lane of a HiSeq 125 Cycle Paired-End Sequencing v4 run.