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.