2.2 Genotyping and sequencing
Genomic DNA was extracted from c. 50 mg muscle tissue from each of the 140 individuals using the KingFisher cell and tissue DNA kit (ThermoScientific, MA, USA) according to the manufacturer´s instructions and eluted in 100 μl elution buffer. DNA quality was assessed by electrophoresis through a 1% agarose gel and subjectively assessing the proportion of high-molecular weight DNA relative to degraded DNA. The extraction procedure was the same for DNA used for genotyping using a 96 SNP array as for individual whole-genome sequencing (WGS). Double-stranded DNA was quantified using a Qubit fluorometer (ThermoScientific, MA, USA) and normalised to 30–50 ng/μl.
We genotyped all 140 fish using an EP1TM 96.96 Dynamic array IFCs genotyping platform (Fluidigm, San Francisco, CA) comprising 96 SNPs shown to be variable in Danish brown trout and evenly distributed on 40 linkage groups, selected from 3,782 SNPs identified in brown trout (Bekkevold et al., 2020; their Table S8). Using TBLASTN (E. values <0.0001 & bitscore 80; Altschul, Gish, Miller, Myers, & Lipman, 1990) we identified the location of these 96 SNPs on the brown trout reference genome (https://vgp.github.io/genomeark/Salmo_trutta/). We located 95 of the SNPs on chromosomes, 1 SNP was located on an unplaced scaffold. Two chromosomes contain 1 SNP each, while all others carry 2-3 SNPs. Using bedtool’s ‘intersectBed’ (v2.27.1; Quinlan & Hall, 2010), we investigated if SNPs were located within coding regions. We visualised SNPs with karyplotR in R (Gel & Serra, 2017; Supporting Information Figure S3). The results from the SNP analyses supported the existence of the two demes (see Results), and random samples of n =50 individuals per deme were used for Pool-seq. Also, we randomly selected n =2 individuals per deme for WGS.
DNA extraction for resequencing followed the same extraction protocol as above but with an additional RNase A treatment. DNA quality was assessed by visual inspection of DNA fragmentation on agarose gels and absorbance at 260/280. DNA with high molecular weight from each of 50 individuals per population was quantified using fluorometry (Qubit;
Thermo Scientific) and pooled at equal concentrations to achieve 3 μg pooled genomic DNA in a volume in the range of 65–120 μl; samples were pooled at equal concentrations per deme. Pool-seq and individual WGS samples were sent to the National Genomics Infrastructure (NGI) at the Science of Life Laboratory (SciLifeLab), Stockholm, Sweden. NGI conducted the construction of PCR-free paired-end libraries with an average insert size of 350 bp followed by Illumina HiSeq 2000 sequencing using read length 150 bp.