I targetted a protein-coding exon with Cas9, amplified a ~ 150bp product in the region and sequenced with Illumina MiSeq. I now have > 1000X coverage of the region, mapped to my reference genome. Many of these reads have indels, thus potentially inducing a frameshift (if not a multiple of 3).
Is there any tool available to predict premature stop codons in my reads (or even non-sense mediated decay)?
I should add I do not want to create an assembly/consensus of my reads. That is because there is mosaicism in the mutations induced by Cas9 (eg. half of my reads have a 1bp deletion, the other half have a 9bp insertion). In the simplest form, I would want a tool that scans for stop codons each of my mutated read, and in the correct frame (i.e. the frame used by the cell to translate that specific mRNA).
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call variants (vcf) and annotate with VEP. stop_gained and NMD_transcript_variant might be of interest to you. other variations such as transcript ablation, frameshift mutations might be of interest to you. https://asia.ensembl.org/info/genome/variation/prediction/predicted_data.html
Thanks. It's very helpful; but I think the variant caller will not do the job I need. I would essentially need a variant call on each individual read, if that makes sense?
Just had a shot with a read now. I manually wrote the variant (it is just a 10bp deletion in the middle of the read). It does a pretty good job for part of it: looking up which transcript the variant will affect and calling a frameshift. I wish it would go transcribe/translate downstream in the new frame to see if any premature stop codon pops up though. Any clue why it does not do that? Am I missing something?
You generally expect that any out of frame frameshift is going to run into a stopcodon soon, but I'm not aware of any annotating software which formally assesses that.
Right, I see. It's a shame, it'd be nice data to have!
Cross post at https://bioinformatics.stackexchange.com/questions/7340/how-to-predict-stop-codons-in-illumina-reads
Yes, I wrote that. I did not get satisfying answers though. Is that an issue you think? Should I delete the stack post?
Not necessarily, but it's best to be open about that.