[miso-users] Question about definition of assigned counts

[Yarden Katz]

The assigned counts parameter should not be used for anything; it's there only for internal debugging purposes.  It is the assignment of reads at the last iteration of the Monte Carlo simulation which is not an interpretable quantity for any downstream analyses.  If you want to get an expected number of reads assigned to each isoform, you can use the Psi values and the length of the isoforms for this.

To answer your question about RPKM: if you're using an exon-centric annotation (e.g. skipped exon trios), it would not be a good idea to use this to calculate RPKMs.  The reason is that there's no guarantee that the flanking exons are constitutive relative to the whole gene.  You can have an alternative exon with an upstream constitutive exon, and a downstream alternative exon (the latter could be "the skipped exon" in another event in the annotation.)  Even if the two flanking exons of the alternative exon of interest were constitutive, this would be a poor estimate of RPKM since it only relies on two exons (using other constitutive internal exons and long constitutive regions of the 3' UTR would give a much better estimate.)  In short, estimating RPKM based on constitutive exons is much easier than estimating isoform abundance, so it's best not to convert an easier problem into a much harder one. 

Yarden

> On Jul 7, 2016, at 5:03 PM, Wang, Ziheng <Ziheng_Wang at hms.harvard.edu> wrote:
> 
> Dear MISO users,
> 
> I am just wondering what the assigned counts parameter in the summary file is consisted of. Are constitutive reads from the exon regions included in this number? If I sum all the assigned counts up, can I theoretically calculate an RPKM for the gene?
> 
> Thank you,
> 
> Ziheng Wang
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