The minimum read base quality value (--QRL) aggregateum alleleate allele quality value (--QAL) flags had to be set to a zero threshold because the assembly software used assigns low base quality scores to the consensus sequence at positions where there is a high degree of variability, such as at SNPs [ 40].
We used a minimum coverage of 20 reads, a maximum coverage of 8000, minimum phred quality of 20 and a minimum allele count of 4 for identifying the variants.
Each output line contains the read name, allele position on the reference, reference and observed allele, quality score of the allele base, average and minimum quality of both the entire read as well as the five bases on either side of the allele-calling base, and auxiliary data.
The resulting VCF files were filtered using VCFTools to include only SNPs with minimum quality score of 30, minimum depth of 8.0, and minimum allele frequency of 0.90 [ 24– 26].
A position to make a call, the following criteria have to be satisfied: (1) minimum read depth (2)minimum number of reads with variant allele (3) minimum variant allele frequency (4) minimum base quality (5) max p value.
The working dataset of high coverage contigs was screened for SNPs using the CLC Genomics Workbench v. 5.5 (minimum coverage 8×, minimum variant frequency 10%, minimum number of reads per allele = 2, minimum central quality 20).
Within the high coverage data set, 8,339 contigs contained SNPs that fell within our detection parameters (minimum coverage 8×, minimum variant frequency 10%, minimum reads per allele = 2, minimum central quality 20).
The working data set of high coverage contigs was screened for SNPs using the CLC Genomics Workbench v. 5.5 (minimum coverage 8×, minimum variant frequency 10%, minimum reads per allele = 2, minimum central quality 20).
Using a custom Perl script and the full data set, we identified 2000 SNPs (minor allele count = 4; minimum coverage = 8; minimum base quality = 20) on chromsome 3R, which are separated by at least 10,500 bp.
