Hi All, I conducted paired-end total RNA sequencing with triplicates for both wild type and drug-treated conditions. Each wild type BAM file contains ~200 million mapped reads (processed with STAR using two-pass mode, read length 65 nt), while the treatment BAM files have ~400 million mapped reads. I analyzed splicing events, focusing on exon skipping, using rMATS-turbo (comparison-1).
To assess reproducibility, I randomly down sampled the reads in each BAM file to 90%, 80%, and 50% and repeated the splicing analysis (comparison-2). I compared the skipped exons identified in comparison-1 and comparison-2 but still observed many skipped exons.
Then i asked to rMATS developers how the splicing quantification was done between the sample group. The answer was it considers the junction counts but no normalization was done before comparison.
So, I'm asking is there any way to normalize the counts and compare it for the splicing quantification.
Also, i'm only considering the junction counts.
Thanks
Very nice. I agree with this.