Question: how to visualize the RNAseq data on IGV
0
gravatar for Sara
2.5 years ago by
Sara90
Sara90 wrote:

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?

rna-seq • 1.7k views
ADD COMMENTlink written 2.5 years ago by Sara90
1

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).

ADD REPLYlink modified 2.5 years ago • written 2.5 years ago by cpad011212k

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.

ADD REPLYlink written 2.5 years ago by ATpoint31k

in our experiment we did mutation

What does that mean?

ADD REPLYlink written 2.5 years ago by WouterDeCoster43k

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.

ADD REPLYlink written 2.5 years ago by genomax80k
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