I was just wondering how feasible it is to look for mutations in RNA-seq data from FFPE mutations. I know that the FFPE process may cause base changes to the RNA so I was wondering if it would be at all meaningful to look for stuff like variants in the data? If so what does the pipeline seem to look like? Unfortunately I don't think I would be able to DNA sequencing for these samples so I wanted to know if it was all possible with RNA
I should be possible but I imagine without a lot of sequencing depth that it would be extremely difficult. My experience comes from standard RNA-seq and I imagine if FFPE adds additional mutations then this would be excessively more difficult.
These guys say its possible in DNA... so I'm sure it is possible in highly expressed genes. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520241/
My sequencing depth is high as I only focused the RNA-seq on a panel of genes (targeted sequencing). So based on that I guess it might be alot more possible. I have never analysed DNA sequencing but would the pipeline be similar for RNA-seq data?
Have you been able to identify the FFPE samples in the TCGA? - how did you do that? FFPE DNA suffers fragmentation, cross-linking, and bases can 'decay' into other bases. So, it is frought with issues.
If anything, it would be a useful PLoS ONE or Scientific Reports manuscript to compare the TCGA FFPE versus fresh tissue data.
I haven't done that comparison yet but I believe there have been a few studies to compare paired FFPE and fresh frozen samples but there have been some mixed results.
https://pubmed.ncbi.nlm.nih.gov/26202458/ https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170632#pone.0170632.ref015
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