Time to KIS-AS: Keep It Simple for Alternative Splicing

In my previous post I stated that exciting things are coming to all you MAJIQ users for splicing detection/quantification/visualization. But you did not suspect it will involve KIS-AS, right?

OK, I guess some explanation is in order here about why we need to Keeping it Simple for Alternative Splicing (KIS-AS)….

Well, anyone who works on alternative splicing will tell you it gets complicated very fast: too many junctions obscure what is actually simple. And the more samples you add, the more things become hairy, like in this example:

These many junctions are likely NOT the result of technical errors (e.g. in mapping): in this case we see they are marked as dashed grey/red which means they are in the transcriptome annotation but never observed in your data/specific sample. In other cases junctions may be observed at very low levels (either de-novo or known junctions) i.e even if they are indeed real they represent some form of biological “noise” which is known to occur in splicing. The deeper you sequence, the more junctions you will find, but rarely/lowly expressed junctions are likely not that relevant for your work…. Consequently, such lowly included junction “obscure the view” and make it hard for us to see the forest from the trees. But here is the dilemma: On the one hand, you do not want to ignore complex events, possibly with de-novo junctions, which may be extremely important for the process you are studying. On the other hand, you can’t see the forest from the trees. So, what do you do? Well, it’s time to KIS-AS, by using our new MAJIQ/VIOLA simplifier!

MAJIQ’s simplifier is a part of the builder pipeline (the part that builds all the splice graphs etc.), and it can be triggered using the argument –simplify . This argument enables the simplification process that can be tweaked using a set of specific parameters.

• –simplify [psi_threshold] : Simplify enables the simplification step, the ratio used to simplify is specified by psi_threshold, 0.01 by default.
• –simplify-denovo [readnum] : Simplify all denovo junctions which total number of raw reads is lower than readnum. Default value is 0
• –simplify-annotated [readnum] : Simplify all annotated junctions which total number of raw reads is lower than readnum. Default value is 0
• –simplify-ir [readnum] : Simplify all intron retentions which total number of reads is lower than readnum. Default value is 0

To make things simpler (😉) only the –simplify argument is required to activate the simplification, in which case the rest of the arguments would be set to their default values. Note though that the above flags give users a lot of power/flexibility. For example users can sacrifice little in PSI accuracy by removing junctions who’s inclusion level (PSI) is minor. Junctions that do not pass the simplifier threshold(s) are deemed (putatively) irrelevant.

But if you use MAJIQ’s simplifier, what happens to the LSVs? Good question!

After the simplification steps, all the LSVs are defined and quantified without considering the irrelevant junctions. The output is “lighter” (e.g. file size) but note that any future quantification of the removed junctions would require re-running the builder step with different settings to include those in the quantification. “But if I need to do that again it will take long!” – Well, not really. First MAJIQ 2.0 is now significantly faster (see here), but we are also working on more goodies to make all these processes much faster….

OK, so now the next question: What happens if you run with the simplifier but then your PI’s favorite junction becomes irrelevant? (**GASP**) Or maybe you just want to see what you might have missed? Fret not, the VOILA visualization provides two views: 1) a simplified view of only the relevant junctions that were actually quantified by MAJIQ or 2) a non-simplified view showing all the irrelevant and relevant junctions in your data.

The effects of the simplification are visible using a new button than can be activated or deactivated to show the splicegraph structure w/wo the irrelevant junctions. They say an image is worth a thousand words, so let’s try this to show the effect of having the new simplifier built-in:

We hope you like the new MAJIQ/VOILA simplifier. It’s already available on the recent MAJIQ 2.1 release. You can send us feedback/suggestions on the user support group (here). And as promised, more cool features are coming soon so stay tuned!

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