UCE target enrichment of Trachinotus_paitensis_SIO_07_91L
We prepared dual-indexed libraries for targeted enrichment using the HyperPrep Kit (KAPA Biosystems, Wilmington, MA) following the manufacturers protocols. We used a probe set targeting 1314 UCE loci informative for phylogenetic analyses of Carangiformes and other acanthomorph fishes across evolutionary time scales. We enriched libraries following protocols for the MYcroarray MYBaits kit v3.02 (Arbor Biosciences, Ann Arbor, MI) with three modifications: 1) we added 100 ng of MYBaits were added to each reaction, 2) we used 500ng custom blocking oligos that included 8 inosines to block the 8 nucleotide index sequences, and 3) we used chicken C0t-1 DNA (Applied Genetics Laboratories, Melbourne, FL) in place of the human C0t-1. We ran the hybridization reaction at 65C for 24 hours. Following hybridization, all pools were bound to streptavidin beads (MyOne C1; Life Technologies) and washed according to a standard target enrichment protocol (Blumenstiel et al. 2010). Samples were re-suspended with 30 L ddH20 while still bound to the streptavidin beads, and we proceeded immediately to PCR recovery of the enriched libraries. The PCR protocol used 15 L of streptavidin bead-bound, enriched library in water with 25 L HiFi HotStart Taq (Kapa Biosystems), 5 L of Illumina TruSeq primer mix (5 M each), and 5 L of ddH2O. We amplified each library using the following thermal profile: 98 C for 45s; 16 cycles of 98 C for 15s, 60 C for 30s, 72 C for 60s; and a final extension of 72 C for 5m. We performed a 1X cleanup of the enriched DNA using Speed Beads and re-hydrated the resulting enriched DNA in 33 L of ddH2O. To remove adapter-dimers <150 bp, we performed a clean-up step using the GeneRead Size selection kit (Qiagen, Inc.) on all enriched libraries, followed by quantification with a Qubit fluorometer. We estimated the size of the enriched pools by running each on an Agilent Bioanalyzer. Enriched libraries were sequenced using 150 base pair paired-end sequencing on an Illumina HiSeq 4000 (Oklahoma Medical Research Foundation Clinical Genomics Center).