Genomic DNA Isolation and SNP Calling in Groupers

A total of 24 tail fins of 12 brown-marbled groupers (E. fuscoguttatus) and 12 giant groupers (E. lanceolatus) were used for genomic DNA isolation. Total DNA was extracted using a TIANamp Marine Animals DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). The quality and quantity of the total DNA were determined using a NanoDrop 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). Paired-end sequencing libraries with an insert length of 350 bp were constructed using the Illumina Truseq DNA PCR free Library Preparation Kit (Illumina, San Diego, CA, USA). The obtained libraries were then sequenced using the Illumina HiSeq X Ten platform.
The raw data were filtered and trimmed using Trimmomatic [25 (link)]. Clean reads from each sample were aligned to the assembled genome using BWA. SNPs were called using BCFtools (version 1.10.2) [65 (link)] with mpileup, call and filter programs (https://github.com/samtools/bcftools). SNPs with a depth of <8, minor allele frequency (MAF) of <0.05, >20% missing positions, a minimum quality of <30 and a number of alleles of >2 were removed using VCFtools (version 0.1.16) [66 (link)]. The F_ST values were calculated using VCFtools with parameters including a window size of 100 Kb and an F_ST window step of 10 Kb.