2.2 | Molecular analysis
DNA was extracted from the muscle plug from the 515 individual fish into 96-well plates with the QIAGEN DNeasy Blood and Tissue Kits as described by the manufacturer (QIAGEN, Inc.). In brief, small pieces of tissue (~20 mg) were excised from each muscle plug. The tissue pieces were digested in a proteinase solution for at least 3 hours at 55ºC. Protease digestions were performed in 96 well plates. After digestion, the samples were purified with either QIAxtractor or Corbett X-tractor robot producing eluted DNA which was stored at -20 ºC.
RADseq library preparation was done for all 515 samples plus eight samples that were replicates according to Ali et al. (2016) and refined by Andrews et al. (2018) using the Sbf1 restriction enzyme, which cuts at an eight-base recognition site. Custom eight-base biotinylated barcodes were ligated to the cut site allowing multiplexing of groups of 96 samples. The multiplexed samples were then sheared to 400 bp using Covaris M220 sonicator. This was followed by a Streptavidin bead assay to exclude sheared fragments that did not include the biotinylated barcodes. Illumina’s NEBNext ultra DNA library prep kit was then used to add Illumina adapters with indexes unique to each of the multiplexed groups of 96 samples to allow further pooling and Illumina sequencing compatibility. 150 bp paired end sequencing was done on two lanes at the Berkeley Genomics Center Laboratory (https://qb3.berkeley.edu/gsl/) using Illumina HiSeq 4000.