UCE target enrichment of Colobostruma_biconvexa_1576
To generate the UCE dataet we performed the following steps: DNA extraction, DNA shearing, library preparation, UCE enrichment, sample pooling, and sequencing. Most of the methods follow those presented in Faircloth et al. (2015), and a detailed outline is presented here. We extracted DNA using Qiagen DNeasy Blood and Tissue kits (Qiagen Inc., Valencia, California, U.S.A.). However, for several rare samples that had been extracted prior to our study, we performed whole-genome amplification to increase available DNA using Illustra Ready-To-Go GenomiPhy HY (GE Healthcare). For all samples, we measured DNA concentration with a Qubit 2.0 fluorometer (Life Technologies Inc., Carlsbad, CA) and we input up to 500 ng of DNA into shearing and library preparation. We sheared the DNA to an average fragment distribution of 400-600 base pairs (bp) (verified on an agarose gel) using a Qsonica Q800R sonicator (Qsonica LLC, Newton, CT). Following sonication, we constructed sequencing libraries using either the standard Library Preparation Kit or the newer Hyper Prep Kit from Kapa (Kapa Biosystems, Wilmington, MA). We performed all reactions at quarter volume except for the PCR step, which we assembled at full volume (50 _L). During library preparation we added custom, single-indexing Truseq-style barcode adapters to most samples. However, for a few libraries, we used a custom, dual-indexing set of adapter-primers. We assessed success of library preparation by measuring DNA concentration with Qubit and by visualizing the libraries on an agarose gel. For a subset of the samples, we removed adapter-dimers by performing a 0.7-0.8X bead cleaning using an AMPure substitute. After library preparation, we pooled 6-10 libraries together at equimolar concentrations for enrichment. Pool concentration was adjusted to 147 ng/_l using a vacuum centrifuge and 3.4 _l of pool was input into the enrichment process. UCE enrichment was performed using a custom RNA bait library developed for use in Hymenoptera by Faircloth et al. (2015) and synthesized by MYcroarray (MYcroarray, Ann Arbor, MI). The bait set includes 2,749 probes targeting 1,510 UCE loci. Enrichment followed a standardized, in-solution enrichment protocol (version 1.5; protocol available from http://ultraconserved.org). Enrichment incubation was performed at 65źC for 24 hours. Following enrichment, we measured the DNA concentration of pools using qPCR (Kapa qPCR reagents; ViiA7 instrument, Thermo Fisher Scientific Inc.) and we combined pools at equimolar ratios. No more than 110 individual samples were pooled together into the same sequencing pool. To remove fragments that were either too large or too small for sequencing, we size selected the pools to a range of 300-800 bp using a Blue Pippin size selection instrument (Sage Science, Beverly, MA). We sent size-selected pools to either the UCLA Neuroscience Genomics Core or CornellŐs Institute of Biotechnology for sequencing as single lanes on an Illumina HiSeq 2500 (2x150 rapid run; Illumina Inc., San Diego, CA).