DNA extractions and sequencing
DNA was extracted from a ~2mm clip of the pectoral fin of each fish using a Qiagen DNeasy Blood and Tissue Kit. The protocol was modified slightly to increase yield by washing the fins in dH­2O before lysis and by repeating the elution step twice using half the volume of buffer. The DNA samples were checked for fragmentation using gel electrophoresis, quality tested with an Implen N60 Nanophotometer, and concentration was measured using a Qubit 3.0 with three replicates per sample. Samples with low quality (A260/A280 < 1.8; A260/A230 < 2.0) or low quantity (concentration < 8ng/μL) were re-extracted. Any sample that failed three re-extractions was removed. This quality check was repeated after pooling DNA samples (see below).
Individual DNA samples were pooled together by population before library preparation (see Table S2 for quality scores of pools). The DNA pools were sent to Genome Québec (McGill University and Génome Québec Innovation Centre, Montréal, Canada) for library preparation and paired-end whole genome shotgun sequencing on their Illumina HiSeqX platform. The estimated coverage of each pool was set as double the number of individuals in the sample (2Nx), so that ideally each chromosome of each individual was sequenced once. A PCR step was performed, even though it is not advised for Pool-seq protocols (Schlötterer et al. 2014), because the mass of DNA in the pools did not meet Genome Québec’s minimum threshold for PCR-free sequencing.