RNA-Seq for FFPE samples

Question

[wbliu]

Hi there,

Has anyone worked on RNA-Seq data from FFPE samples? The library prep was rRNA depletion and Illumina HiSeq was used. It turned out the exonic mapping rates are only 10-30%, and intronic and intergenic rates are very high (20-40% each). My question is, are the exonic rates even acceptable? Any comments will be appreciated. Thanks!

Answer 1

[Charles Warden]

While it is difficult to officially say a sample can't be used, those exonic alignment rates seem low for an ribosome-depleted library.

[wbliu]

Thanks for your comment, Charles. It seems that a range for exonic mapping rate near 20% is 'normal' for FFPE samples prepared with rRNA depletion. I found this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4039489/figure/pone-0098187-g003/.
One surprise to me is that the RNA seq data from FF and FFPE (as long as 20 years in storage!) are very well correlated.

Plan to exclude those with extremely low rates e.g. <5%, for differential expression analysis. Any other comments will be appreciated.

[Charles Warden]

While I would typically expect the exonic rate to be lower for a ribosome-depleted library versus a polyA library, I guess I have seen some rRNA-depleted samples with an exonic rate of ~30% (if you count the exonic rate within aligned reads).

While I wanted to be cautious about giving you advice about performing additional experiments (particularity if it was possible that you might have problems with any kit), Figure 2 in Schuierer et al. 2017 pretty much matches my expectation (although your exonic percentage is lower than they present, even for their highly degraded sample):

I'm not exactly sure how the heat degradation compares to being archived for 20 or so years (and if low input concentration is an additional issue). However, I hope this can be somewhat helpful. Best of luck!