We are running a project where we are Sanger sequencing gDNA for a specific gene (DPYD), and doing sequencing reactions for each of the exons. The reads we get include intronic bp on each side of the exons.
We have an automated system for looking at variations, and wondered how far out each side into the intronic region we need to look for anything helpful related to splicing issues/transcript variation. Would 5bp each side be enough? More is better?
Hi Cam
in fact each baes can have an impact in splicing events, but the most important ones are for acceptor and donor splice sites, ie the last and first 2 bases before and after each exons. if you get more, yes you can take this way, but never less.
fred
Thanks Fred! That sounds good yes. We ended up doing 10 intronic bp flanking each exon, as we had the data for it, and after reviewing gnomAD variants database, there were recorded splice region variants up to about +/- 8bp out.
https://gnomad.broadinstitute.org/gene/ENSG00000188641?dataset=gnomad_r2_1
But you're right, that makes sense the donor/acceptor sites are the most important to bother with.
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