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I'm working on rather standard whole-exome sequencing data and treat it the same as whole-genome sequencing data (aligning it to the full GRCh38 reference assembly and calling variants with no exome-related parameters).

I recently noticed that my variant calling results contain numerous variants mapped to a non-exomic region, incidentally, some of them were significantly associated with the phenotype I study.

My questions are as follows:

  1. Are the non-exome variants in my exome data invalid? or could it be that the exome kit captured more than it was designed to capture?
  2. Should I treat raw exome sequencing data any different than genome sequencing data, and if so, what parts of my pipeline should be modified to accommodate exome data?
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These are called off-targets due to unspecific binding of DNA to the capturing beads during library preparation. This is normal and expected since capturing is not a perfect process. It is correct to align against the whole genome but you should simply limit variant calling to exons (or basically the on-target regions) and ignore everything else.

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  • $\begingroup$ Thanks for the reply, makes perfect sense. Now, considering one of my significantly associated variant is from an off-target region, should I report it as a legitimate direction for further research? Consider that the variant has adequate coverage, passes strict filtering in the entire cohort and heavily implicated in my phenotype of interest. $\endgroup$
    – shepp
    Jun 14, 2020 at 22:16
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    $\begingroup$ I would also call variants a few bases out of the exons to cover splice site mutations. $\endgroup$
    – Wouter De Coster
    Jun 15, 2020 at 7:42

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