What to pay attention when analyzing RNA-Seq samples from FFPE ?

Question

[Nicolas Rosewick]

Hello,

I will analyze soon RNA-Seq samples build using a TruSeq RNA Exome (illumina) from FFPE. I've already a standard RNA-Seq pipleline that works well on fresh tissue ( RiboZero kits). Pipeline is a classic STAR-DESeq2 + additional post-hoc analysis (splicing, fusion, pathways, etc..).

In this context, should I be carefull on some steps using capture-based RNA-Seq samples from FFPE samples ?

thank you

[GenoMax]

A local group uses STAR in TranscriptomeSAM quantmode followed by salmon quantification. They do a lot of FFPE samples so this method must work well.

[Kevin Blighe]

I would anticipate very low alignment %

Answer 1

[Bastien Hervé]

I would be interested to know if there are some criticals bias too !

A start in this Intro : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976456/

[Nicolas Rosewick]

Thank you for the information.

Answer 2

[Friederike]

FFPE samples often suffer from low quality RNA inputs, i.e. lots of degraded RNA, which often leads to pronounced 3' bias and sometimes dramatically reduced library complexity (i.e., only few genes were captured).

You should make sure to check the gene body coverages, exon coverages and library complexity (e.g. using QoRTs) because they may differ quite significantly from the fresh samples and may influence the types of comparison you can do.

Some nice assessments of the impact of degradation were done by Schuierer et al., 2017 and Li et al., 2014.

[Nicolas Rosewick]

Thank you for the information