UCE target enrichment of Mestonia_acris_TAS063
Libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems), using up to 250 ng DNA (i.e., half reaction of manufacturers protocol) as starting material. After end-repair and A-tailing, 5 M universal adapter stubs (University of Georgia, EHS DNA Lab) were ligated onto libraries. Libraries were then amplified in a 50 l reaction, which consisted of 15 l adapter- ligated DNA, 1X HiFi HotStart ReadyMix, and 0.5 M of each Illumina TruSeq dual- indexed primer (i5 and i7) with modified 8-bp indexes. Amplification conditions were 98 C for 45 s, then 16 cycles of 98 C for 15 s, 60 C for 30 s, and 72 C for 60 s, followed by a final extension of 72 C for 60 s. Samples were quantified to ensure amplification success. Equimolar amounts of libraries were combined into 1000 ng pools consisting of eight samples each. Target enrichment was performed on pooled libraries using the MYbaits Arachnida 1.1K version 1 kit following the Target Enrichment of Il- lumina Libraries v. 1.5 protocol (http://ultraconserved.org/#protocols). Hybridization was conducted at 65 C for 24 hours, then libraries were bound to streptavidin beads and washed. Following hybridization, pools were amplified in a 50 l reaction consisting of 15 l of hybridized pools, 1X Kapa HiFi HotStart ReadyMix, 0.25 M of each of TruSeq forward and reverse primers, and 5 l dH20. Amplification conditions consisted of 98 C for 45 s, then 16 cycles of 98 C for 15 s, 60 C for 30 s, and 72 C for 60 s, followed by a final extension of 72 C for 5 minutes. Following an additional cleanup, libraries were quantified using a Qubit fluorometer. Molarity was determined with an Agilent 2100 Bioanalyzer and equimolar mixes were prepared for sequencing.