2145
Rapture (RADseq+sequence capture) for Etheostoma cragini
We used the BestRad protocol following Ali et al. 2016 to generate RADseq libraries. Briefly, genomic DNA (100 - 200 ng) from each individual was aliquoted into a separate well on a 96-well plate and digested with a restriction enzyme (Sbfl-HF). Each well in the 96-well plate was then indexed with a biotinylated RAD adapter containing an individual-specific barcode and then pooled. Pooled DNA was sheared to 500 bp fragments using a Covaris sonicator (Covaris, Woburn, MA, USA). Shearing efficiency was evaluated with a fragment analyzer. Dynabeads M-280 streptavidin magnetic beads (Thermo Fisher Scientific, Waltham MA, USA) were used to physically isolate the RAD-tagged DNA fragments. The DNA was then eluted in TE buffer. Each pool was amplified with the addition of an Illumina index specific to each 96-well plate. After BestRAD library preparation, we combined four pools of 96 samples each and performed sequence capture using a custom bait set following the MyBaits protocol provided by Arbor Biosciences. Briefly, this involved performing a hybridization step at 65C for at least 16 hours, isolating bait-target hybrids using streptavidin-coated magnetic beads and washing to remove non-target DNA, and performing PCR amplification of captured DNA for sequencing. We submitted pooled libraries for sequencing at the MSU Genomics Core facility in five batches of 380 samples each using paired-end 2x150 bp reads on an Illumina HiSeq 4000, with a single lane of sequencing used for each batch.
2145
SUB7243821
2145