exons skipping and intron retention definition

[sagishaked]

Hello,

I am using rMATS with a single sample that has multiple replicates, and I want to analyze the ratios within the sample—for example, the ratio of skipping exons to constitutive exons, and the ratio of retained introns to spliced introns.

I noticed that the output files list all skipping exons (e.g., in the skipping exon file), but I would like to understand the PSI threshold you use to classify an exon as skipping or constitutive.
Similarly, what is the PIR threshold used for introns?

Thank you! :)

[EricKutschera]

rMATS doesn't have a PSI or PIR threshold. It detects an event if it has some evidence (from the GTF or the alignments) for each exon and junction needed to define the event type at that location: https://github.com/Xinglab/rmats-turbo/tree/v4.3.0?tab=readme-ov-file#output

Since you only have 1 sample the events won't be filtered based on supporting read counts: #348 (comment)

This post describes how rMATS detects skipped exon events: #135 (comment)

Here's a related post that describes how rMATS detects retained intron events: #17 (comment)

[sagishaked]

Thanks for your detailed answer,
I have a follow-up question to your answer that,
are we able to know from the rMATS output the the numbers and ratios between alternative/constitutive exons and also spliced/retained introns?

[EricKutschera]

I don't think the rMATS output can be used for counts of alternative/constitutive exons or spliced/retained introns. rMATS can find some exons which are skipped and some introns which are retained, but it isn't designed for determining constitutive exons or introns which are never retained. rMATS is looking for regions that match the particular splicing event types: SE, A5SS, A3SS, MXE, RI. rMATS needs certain information from the --gtf to detect those events as described in the previous posts. Additionally, an exon might be involved in some more complicated splicing event which doesn't fit any of the rMATS event types (ex: consecutive skipping)