Materials and Methods
Sampling
We collected 49 individuals of S. japonica from five populations
distributed throughout the coastal waters of China and Japan (Figure 1
and Table 1). All individuals were identified on the basis of
morphological features. A piece of muscle tissue was obtained from each
individual and preserved in 95% of ethanol or frozen for DNA
extraction.
Sequencing, read mapping, and SNP calling
Genome sequencing was performed using the Illumina HiSeq 2500 platform
by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).
Quality control and removal of potential adaptor sequences from raw
reads were performed to ensure the accuracy of bioinformatics analysis.
Paired-end reads from each individual were aligned to the reference
genome (unpublished data) using the Burrows–Wheeler Aligner (BWA) with
the option “mem-t 4-k 32 -M” (Li & Durbin, 2009). After mapping, the
resulting bam files were sorted using SAMtools (Li, Handsaker, Wysoker,
Fennell, & Ruan, 2009), and duplicated reads were removed.
Raw SNPs were identified on the basis of mpileup files generated by
SAMtools. The filtering threshold for raw SNPs was set to require a
quality score of ≥20. Furthermore, an SNP was discarded if the total
coverage of the SNP was less than one third or greater than five times
the overall coverage. If two SNPs were <5 bp apart, both SNPs
were removed.