UCE target enrichment of Cicurina_sp_CIC1766
Library preparation followed Starrett et al. (2017), with some modifications. Libraries were prepared using the KAPA Hyper Prep Kit (Kapa Biosystems), using up to 250 ng DNA as starting material. Ampure XP beads (Beckman Coulter) were used for all cleanup steps. For samples containing <250 ng total DNA, all available DNA was used in library preparation. After end-repair and A-tailing, universal adapters were ligated onto libraries at varying concentration depending on amount of input DNA. Libraries were then amplified in a 50 _l reaction, with 15 _l adapter-ligated DNA, 1X HiFi HotStart ReadyMix, and 0.5 _M of each Illumina TruSeq dual-indexed primer (i5 and i7) with modied 8-bp indexes (Glenn et al. 2016). Amplification conditions were 98 C for 45 s, then 18 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 again to ensure amplification success. Equimolar amounts of libraries were combined into 1000 ng total pools consisting of eight samples each (125 ng per sample). Target enrichment was performed on pooled libraries using the MYbaits Arachnida 1.1K version 1 kit (Arbor Biosciences; Faircloth 2017) following the Target Enrichment of Illumina Libraries v. 1.5 protocol (http://ultraconserved.org/#protocols). Hybridization was conducted at 60 or 65 C for 24 hours. 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 or 18 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 and equimolar mixes were prepared for sequencing on an Illumina HiSeq 2500 (Brigham Young University DNA Sequencing Center) using 125 bp PE reads.