Question: RNA-Seq variant calling dependent on expression?
gravatar for Dave Tang
3.1 years ago by
Dave Tang180
Dave Tang180 wrote:

If a deleterious variant, such as a stop gain, leads to the degradation of the mRNA via nonsense-mediated decay, it would be impossible to call this variant using RNA-Seq data?

Related to this, the quality of variants called from lowly expressed genes would be lower than highly expressed genes? This seems logical but as I have never performed variant calling on RNA-Seq data, it may not be the case in practice.

I was just thinking about why we bother doing exome sequencing at all if we can call variants from RNA-Seq data, since from RNA-Seq, we also get expression and splicing information.


rna-seq variant calling • 1.3k views
ADD COMMENTlink modified 3.1 years ago by Devon Ryan90k • written 3.1 years ago by Dave Tang180
gravatar for Devon Ryan
3.1 years ago by
Devon Ryan90k
Freiburg, Germany
Devon Ryan90k wrote:

As you surmised, coverage (required for accurate variant calling) is highly variable in RNAseq. Anything that causes non-sense mediated decay or any cases where you have allele-specific expression will cause wrong results if you do variant calling on them. Avoid calling variants on RNAseq data unless you absolutely need to.

ADD COMMENTlink written 3.1 years ago by Devon Ryan90k

Yes as Devon rightly pointed out, calling variants is not an ideal scenario unless you have no genetic data for your samples, in that case you can do that but yes the problem of splicing and coverage is in fact a bottleneck for the variants that can be called from RNA-Seq. So you end up with false positive variants in fact. However the GATK pipeline with STAR 2-pass somewhat makes the call less spurious (still there is high false positive and false negative calls) as till the end you end up with large call sets, in that case if you have some specific variants that you might be interested you can see them through the final realigned/recalibrated bam that was subjected to haplotypcaller or the final vcf file in any browser to see if they truly exist or you might score them with some tools to assign the functional/structural identity to those variants to rank their impact but having said that the problem of low coverage will still persist.

ADD REPLYlink written 3.1 years ago by ivivek_ngs4.8k

Thanks Devon. I just needed some confirmation.

ADD REPLYlink written 3.1 years ago by Dave Tang180

Although it gets interesting if you can compare variant calls on exome sequencing and RNA-seq.

ADD REPLYlink written 3.1 years ago by WouterDeCoster39k

Here is a case report for comparing in tumor:

ADD REPLYlink written 2.3 years ago by shuomou0
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