The minimum base quality ('minQ') and the coverage depth ('minDepthMeth' for the methylation profiling) thresholds might be also important parameters to control de quality of methylation profiling and SNV calling.
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.
Genomic variation was annotated using SnpEff (ver. 3.3; minimum coverage = 5X, minimum base quality = 20).
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.
The maximum coverage was based on the observed maximum SNP coverage of 7961 reads with a minimum base quality of 20.
For SNPs other than the high confidence SNPs we required the following criteria: minor allele count >1, a minimum coverage of 4 and a minimum base quality of 20.
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.
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.
The novelty and redundancy of each assembly were determined by the proportion of base coverage by the other five assemblies with a high threshold (a minimum overlap length of 50 bp and minimum identity of 99%).
This FPKM of 0.5 corresponded to a minimum of 41 reads per kilobase of transcript, which equated to an average base coverage of approximately 8.2.
