Intronic/intergentic variants using NGS WES

[ramujana]

Hello folks,

Currently I'm looking for cost effective approaches to cover intronic and intergenic SNPs variants using Illumina NGS workflow? Some targeted enrichment protocol address that issue (i.e. TSCancer) and include probes for selected SNPs (~240), however I wonder is it possible to capture intronic variants using whole exome analysis? The conventional genotyping arrays would be the answer, however personally I consider this methodology outdated and looking for applicable alternative with NGS (whole genome seq would be ideal, however it's still too costly). Are there any protocols to customise exome enrichment kits and add desired non-exonic variants content?

thank you in advance for help,

R.

[Brian Bushnell]

The goal of exome capture is to exclude non-exomic content, so if you are interested in that, you should use whole-genome sequencing. The point of exome enrichment kits is to A) not be customizable and B) not give you intronic content, which is considered off-target.

You might be able to capture more of the intronic content that abuts exons using longer-insert paired-end libraries. For anything more than an insert-size away from a target, there's nothing you can do unless you define your own custom capture kit.

You can, of course, call variants on very low coverage regions... like, say, 3x. The accuracy will be low for a given sample, but if you have a lot of samples, that could make up for it. I've proposed mixing half exome-capture and half whole-genome together into the same library and sequencing it, since I think intronic variants are very important (and this was in the context of families, in which variants could be verified using inheritance), but I was unable to convince anyone else that it was a good idea, as they believed that exonic variants were the "low-hanging fruit". Still, in your situation, you might consider this approach.

[ramujana]

Thanks Brian for insights.