UCEs of white-eyes
We sheared 500 ng of DNA of each sample in 50 l volumes using a Covaris S220 sonicator at 175 W peak incident power, 2% duty factor, and 200 cycles per burst for 45 seconds.  We performed end repair, A-tailing, adapter ligation, and amplification on sheared DNA using Kapa Biosystems Library Prep kits following the procedure of Faircloth et al. (2012) and described in detail at http://ultraconserved.org.  We deviated from the above protocol in three ways: we used  volume of reagents called for in the library prep kit, we ligated universal iTru stubs (Glenn et al. 2016) instead of sample-specific adapters to allow for dual indexing, and we added a second AMPure XP bead clean up at 1.0x volume after stub ligation.  Dual-indexed iTru adapters (Glenn et al. 2016) were added to DNA fragments through a 17-cycle PCR using NEB Phusion High-Fidelity PCR Master Mix.  Libraries were quantified using a Qubit 2.0 Fluorometer and subsequently combined in pools of 8 equimolar samples for enrichment. We performed sequence capture and post-enrichment amplification following standard protocols (Faircloth et al. 2012) using the Mycroarray MYbaits kit for Tetrapods UCE 5K version 1, which targets 5,060 UCE loci.  Briefly, biotinylated RNA probes were hybridized with pooled libraries for 24 hrs.  Targeted DNA fragments were then recovered with the use of streptavidin-coated beads.  These fragments were then amplified in a 17-cycle PCR amplification step using NEB Phusion High-Fidelity PCR Master Mix.  After post-enrichment amplification, libraries were quantified using an Illumina Eco qPCR System, then sequenced in a high output, paired-end run of 100 cycles on an Illumina HiSeq 2500 System at the University of Kansas Genome Sequencing Core.
SUB7321659
zosterops_kulambangrae_76278