UCE target enrichment of Progne-subis-LSUMNS-25508: tissue
We mechanically sheared DNA from each sample with an Episonic Multi-Functional Bioprocessor to an approximate average length of 500bp, as assessed by gel electrophoresis. We used a KAPA HyperPrep Kit (Roche Sequencing Systems) to prepare a barcoded library for Illumina sequencing from each sample by ligating dual indexed iTru adapters (Glenn et al. 2016) to the fragmented DNA. We followed the kit protocol, but used half reagent volumes for the end-repair and A-tailing, adapter ligation, and post-ligation cleanup steps. Libraries were amplified with nine cycles of PCR not including the initial denaturation, final extension, and holding cycles, and quantified with a Qubit 2.0 both before and after amplification. We combined groups of eight libraries into eight equimolar pools. We used hybrid capture to enrich each pool for 5,060 UCE loci. This process entailed denaturing the libraries and hybridizing a set of 5,472 biotinylated RNA probes (Tetrapods-UCE-5Kv1 sold by MYcroarray; Faircloth et al. 2012) to their target sequences on the single-stranded DNA. These probe-target hybrids were then captured on streptavidin coated magnetic beads, allowing the unhybridized, non-target DNA to be washed away. The target DNA was then released and amplified. We followed the protocol in the MYbaits manual v3.0.1 through bait-target hybridization, hybrid binding, and bead washing steps. After washing the beads to remove non-target DNA, we resuspended the enriched libraries along with the beads in 10mM Tris-Cl with 0.05% TWEEN and performed 15 cycles of limited-cycle PCR on the suspension with KAPA HiFi HotStart polymerase. We purified the PCR product with a 1.2X AMPure bead cleanup and verified the absence of adapter dimers by determining the size distribution of the enriched, amplified libraries with an Agilent Bioanalyzer. We combined the eight pools of enriched, amplified libraries with six pools of libraries prepared from passerine DNA for other projects. These libraries were sequenced together on one paired-end 150bp lane of Illumina HiSeq 3000 at the Oklahoma Medical Research Foundation Clinical Genomics Center (OMRF).