Variants with minimum coverage of six reads with minimum base calling quality (Q) of 30 at the respective position, a minimum SNP-call quality (QUAL) of 160 (QUAL = −10 log10 (probability of wrong call) [ 56]) and with more than 67% of all quality reads calling the SNP were retained.
Based on a previous study [ 20], we called only variants that were present at sites with a minimum read depth of 40× and a minimum average base call quality of 20.
These SNPs were examined to remove any SNPs resulting from the ends of the reads and filtered requiring a minimum of three confirming reads per base call per allele.
SNVs were filtered for a minimum Phred quality score of 30, allowing 99.9% base call accuracy.
Only a few thresholds were incorporated within the commands for the initial calling of the variants, viz., minimum base quality of 20, minimum map quality of 20 and InDel alleles supported by at least two reads.
SNPs and indels were then called using the mpileup function in samtools, requiring a minimum base quality of 30.
For these programs, we used the following parameters: 20,000 = maximum number of reads for calling a SNP, 20 = minimum mapping quality, and 20 = minimum base quality to identify putative SNPs.
A minimum coverage of 8 reads mapping to variant sites, minimum base phred quality of 20 and a P-value of 0.05 were used for SNP calling.
This threshold is the minimum base quality at a position, for a read, required for that read to be included in the variant call for that position.
The first setting (high stringency) required variant calls on each strand with each base having a minimum coverage of 8X, a minimum base quality of 40, a strand minimum mapping quality of 40, and at least 20% of the reads to have the novel allele.
