Dear All,

I would like to ask you about an issue am facing. While doing exome sequencing I earlier did an estimation to what extent my reads were falling on the target regions, ie the target intervals that are used for target enrichment and I found for my samples the reads were around 75% spanning the exonic region. But now when am translating them into the variants and trying to find the somatic variants and annotating the somatic variants with all 3 annovar, oncotator and snpEff I find only 30% SNPs(novel ones which are not in dbSNP) are actually on the exons. The rest are spanning the intronic,intergenic,splice ,UTRs etc. Is this a likely scenario? How often do you see the SNPs mostly annotated in the non-exonic regions even when you use the exome sequencing provided your reads have high coverage in the exonic intervals used for target enrichment? I would like to know your advice in such cases.

Thanks
Does this include synonymous mutations? If you filtered out synonymous exonic mutations you'll find that is a large contributor as well. Most of the non-exonic regions you listed are all either targeted, or will be captured due to overlap (some areas extending into introns) and the majority of all mutations in a cell happen to occur in non exonic regions simply because exonic regions make up such a small percentage of the genome. Of course since you have filtered out off-target variants in this case, you will enrich for exonic variants.

Coupled with filtering out mutations that are also in dbSNP (which many valid somatic mutations may overlap or match identically mutations found in dbSNP by the way, plenty of known somatic variants that appear in COSMIC, TCGA, etc are also found in dbSNP) the number you are reporting (30%) doesn't surprise me that much.