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RNA-Seq requirements for both gene expression quantification and variant calling

I would like to use RNA-Seq on fresh tumor tissue for both gene expression quantification and variant calling.

Do I need stranded RNA information?

Do I need polyA enrichment based or rRNA depletion based rRNA removal?

How many data (sequencing reads or coverage) per sample do I need?

Thanx!

rna-seq variant calling gene expression
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updated by Devon Ryan Devon Ryan • written by Ozzie • Ozzie

Being curious, would like to know the basis of your sample size.

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written by cpad0112 cpad0112

1 answer

  1. No, but it's not much more expensive and you get a bit nicer data.
  2. You can do either, but poly-A enrichment would probably make the most sense.
  3. As much as you can afford if you want to do variant calling. Honestly, I'd do 15-20 million reads for RNAseq and then pair that with exome sequencing. Otherwise you're going to have to really up your depth to get decent coverage of lowly-expressed genes and will still be left with the problem of allele-specific expression.
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written by Devon Ryan Devon Ryan

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