Question: What to pay attention when analyzing RNA-Seq samples from FFPE ?
1
gravatar for Nicolas Rosewick
8 months ago by
Belgium, Brussels
Nicolas Rosewick8.6k wrote:

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

rna-seq ffpe • 327 views
ADD COMMENTlink modified 8 months ago by Friederike5.2k • written 8 months ago by Nicolas Rosewick8.6k

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.

ADD REPLYlink written 8 months ago by genomax77k

I would anticipate very low alignment %

ADD REPLYlink written 8 months ago by Kevin Blighe53k
3
gravatar for Bastien Hervé
8 months ago by
Bastien Hervé4.5k
Limoges, CBRS, France
Bastien Hervé4.5k wrote:

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/

ADD COMMENTlink written 8 months ago by Bastien Hervé4.5k

Thank you for the information.

ADD REPLYlink written 8 months ago by Nicolas Rosewick8.6k
3
gravatar for Friederike
8 months ago by
Friederike5.2k
United States
Friederike5.2k wrote:

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.

ADD COMMENTlink written 8 months ago by Friederike5.2k

Thank you for the information

ADD REPLYlink written 8 months ago by Nicolas Rosewick8.6k
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