[miso-users] question about inclusion ratio and PSI

[yu_peng]

Hi, colleague:

I have several questions:
1. In your 2008 Nature paper,the 'inclusion ratio' is defined as the ratio of the number of 'inclusion¡¯ reads to inclusion plus ¡®exclusion¡¯ reads.
Are the reads used here those assigned by MISO using Bayesian method? 

2. When calculating PSI, the density of reads were used (reads per position), i.e. one need to divide the above (assigned) reads with some length.
However, I have not found the definition of the effective length in the paper. In the supplementary file, it was noted that 28nt were kept from both
sides of a junction (cause your original read is 32bp, thus can assure at least 4bp overlapping the next exon). So in my understanding, the effective
length of a junction is 28+28 (this could be changed to real read length minus 4 ), while for event including a middle exon, the length should be 28+28+length of included exon. Am I right? 
And for A5SS or A3SS events, as there are reads spanning the boundary of two events, how do you deal with them?

3. Currently I am analyzing a set of pair-end RNA-sequencing data. The insertion size is about 150nt but the read length is 101, so two reads overlap.
When using MISO with pair-end mode, it showed low read coverage for many events. Is MISO vulnerable for such condition?

4. And the last question. In my result of TandemUTR analysis, it is seen that the assigned read are greatly different between events, but the PSIs are 
similar (see below), is this normal?

event_name      miso_posterior_mean     ci_low  ci_high isoforms        counts  assigned_counts chrom   strand  mRNA_starts     mRNA_ends
2d  0.28,0.26,0.27,0.20     0.24,0.22,0.25,0.18     0.31,0.31,0.33,0.22     '2d.1.0','2d.2.0','2d.3.0','2d.4.0'     (0,0,0,0):1163,(0,0,0,1):6,(0,0,1,0
):1,(1,0,0,0):203       0:203,1:0,2:1,3:6       chr3    +       126545676,126543284,126518921,126303617 126546040,126543378,126521721,126304424
 
Thanks.

Peng  
 



***************************************
Peng Yu, Ph.D.
Institute of Molecular Medicine
Room 303, Yingjie Center
Peking University
Beijing 100871, China
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