Before sending some RNA seq data from formalin-fixed samples to the graveyard, I wanted to try one last time to assess if it is publishable. My reluctance to let them go stems from the significant amount of labor that was expended by a member of my lab in the production of samples. Also, I foolishly already did some analysis of the data and found some interesting stuff before even doing the QC, so I am also kind of invested at this point...
To obtain some objective foundation as for why this data isn't trash, I searched the web and found this nice paper which basically concludes: having FFPE samples isn't the end of the world and the results are somewhat comparable to the freshly-frozen samples as long as the following quality metrics are OK: Percent spliced index [PSI], Gene body coverage, GC content, paired-end inner distances, median transcript integrity number [TIN] and distribution of mismatches across reads...
So as long as these metrics turn out to be ok, I can save my face.
So I started with computing the splicing index and I would have wanted to do this with Spladder, as the study suggests.
A prerequisite to that part was to produce bam files from Fastq files, for which was my question - should I go for this double-pass mode in STAR that apparently allows a better quantification of novel splicing:
The 1-pass mapping mode generates all required data essential for many downstream analyses such as differential gene expression analysis. However, if your goal is to robustly and accurately identify novel splice junctions for differential splicing analysis and variant discovery, it is highly recommended to use the STAR in 2-pass mode.
In the two-pass mode, the genome indices are re-generated from splice junctions obtained from a 1-pass mode with the usual parameters and then run the mapping step (2-pass mapping).
For the assessment of the RNA degradation we are not looking for novel splicing, we rather just look for alternative splicing between R1 and R2, right? Would that be actually beneficial for my purposes, I wonder??
