Library preparation and resequencing
The adipose fins of the 70 individuals selected above that were
collected non-invasively and preserved in 99% ethanol were used for DNA
extraction. DNA extraction was performed using the DNeasy Blood and
Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s
protocol. DNA concentrations were measured using the Qubit dsDNA HS
Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). Agarose
electrophoresis was performed using KANTO ST (Kanto Chemical, Tokyo,
Japan) high gel strength (1% agarose with TAE) to confirm the quality
of genomic DNA.
Libraries were prepared from the DNA extracts following the Hackflex
protocol (Gaio et al., 2022). For
indexes, we used UD Index Adapter (Integrated DNA Technologies, Iowa,
USA). Concentrations of the DNA library were measured by a Qubit dsDNA
HS Assay Kit (Thermo Fisher Scientific) and the KAPA Library
Quantification Kit (Kapa Biosystems) for the StepOnePlus Real-Time PCR
System (Thermo Fisher Scientific).
Library size was measured using
an automated gel electrophoresis system (2200 TapeStation high
sensitivity D1000 [Agilent] or 4200 TapeStation high sensitivity
D1000 [Agilent]). Fragments in the DNA library were sequenced by
using a genome sequencer (HiSeq X; Illumina, California, USA) at 151 bp
paired ends.