Given the potential for false positives identified in genome sequencing, a stringent read depth of 50 and a call quality of 100 (calculated by complete genomics and based on a phred scale) were chosen as parameters for initial variant filtering.
Using the stringent read mapping criteria described above, read pairs were mapped to all eight alternative conformations.
SNP-o-matic is a fast, memory-efficient and stringent read mapping tool offering a variety of analytical output functions, with an emphasis on genotyping.
Merged and trimmed reads were then aligned to corresponding reference sequences that were bisulfite-converted in silico to allow stringent read alignment parameters in read mapping (C nucleotides remained in CpG dinucleotides for reads containing low methylation percentages).
We set a stringent read quality for each data set, retaining only those reads with a quality score above 20 in 80% of their nucleotides, and we also restricted the read length to 13 nucleotides (nt) after adapter trimming.
We also analyzed breed specificity for 280 putative deletions that resulted from applying a less stringent read mapping quality constraint, which was also applied in previous SV detection studies [ 19, 25].
For characterization of low quality ones, however, VarScan 2 was inferior to the other tools primarily due to its stringent read-depth cutoffs and our application of its high-confidence setting in this study.
An important advantage of the application of RNA-seq data in polyploid species is that it facilitates the distinction among homoeologues and recently-diverged paralogous genes by allowing the application of stringent read mapping thresholds.
With a stringent read filter of 30 reads per SNP and twofold AEI ratio, we detect 443 genes with likely AEI in any region such as Huntington's Disease-associated: ELMO1, NTRK2, WNK1 and associated with Schizophrenia: NTRK2, PIK3R1 and RTN4.
This stringent reads-mapping cutoff was used to give high confidence that the genes in this set are not sequencing artifacts or a result of assembly errors, and the 22,315 genes above the 1 CPM cutoff were used for all differential expression and functional analyses.
