[miso-users] Problem with discarded reads from strand-specific library

[Yarden Katz]

Hi Andreas,

The fr-secondstrand ought to have the following behavior: the forward read should match the target (genomic) strand.
Does this behavior match your library prep protocol?  If it does and that's not what you're getting from MISO, please send me an example
BAM file with a subset of reads (e.g. only the reads in the event below, "1:97661831:97662018:- at 1:97654600:97654689:- at 1:97643902:97646361:-") and your GFF, and I'll take a look.

Best, --Yarden


On Aug 7, 2013, at 2:53 PM, Andreas S. Richter wrote:

> Dear MISO users,
> 
> I recently started using MISO with input data from strand-specific libraries. I configured MISO accordingly by setting "strand = fr-secondstrand".
> 
> When inspecting the batch-logs generated by run_events_analysis.py, I noticed the following curious behaviour:
> * For the vast majority of events on the - strand, nearly all reads are discarded due to strand violation (example below)
> * For events on the + strand, only few reads are discarded by this filter.
> 
> I visually inspected the reads of the BAM file together with the annotation in IGV and both looked correct regarding strandedness.
> 
> Can someone please explain me this behaviour? I am using version 2 of mm10 alternative events and MISO version 0.4.9.
> 
> Thanks a lot in advance.
> 
> Best,
> Andreas
> 
> 
> Example output of run_events_analysis.py:
> Computing Psi for 1 genes...
>   - 1:97661831:97662018:- at 1:97654600:97654689:- at 1:97643902:97646361:-
>   ...
> Filtered out 61 read pairs that were on same strand.
> Filtered out 60 reads that had no paired mate.
>   - Total read pairs: 2458
> No. reads discarded due to strand violation: 2458
> Only 0 reads in gene, skipping (needed >= 20 reads)
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