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.