Entering edit mode
6.5 years ago
Sara
▴
240
I have aligned my RNAseq data and 3 mismatches were allowed and sorted the bam files (also have .bai files). I am trying to visualize the bam files on IGV. actually I am looking for the mutations (in our experiment we did mutation). but when I look at the samples on IGV there is no mutation even there is no SNP or sequencing error. this is not really normal. I think in the alignment, which was done once using tophat2 and once using STAR, at some point mutations are removed. have you ever had such problem? if so, how id you solve it?
If you have introduced the mutations (SDM I assume), extract regions of interest from bam, post alignment see if there is a variation. In general, viewers like IGV do not modify loaded files (bam or vcf etc).. If there is no variation, do an RT-PCR to check if there is a variation. Btw, did you recalibrate your alignments with variation db (such asdbSNP)? In addition, I am not sure if RNAseq is right tool to study variations (among NGS tools).
You really have to give some more details on where and why you expect mutations, so both about the experiment and your pipeline so far. Also provide a screenshot of a potentially (expected) mutated region.
What does that mean?
Are these supposed to be point/site directed mutations? Or are the longer deletions/insertions. It is possible that if they are the latter then tophat/STAR may not have aligned the reads right. You may need to consider using local realignment (e.g. with ABRA) to see these types of cases.