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.