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

[Andreas S. Richter]

Hi Yarden,

thanks a lot for your help.

Yes, our library prep protocol generated the forward read in sense orientation.
I will send you the example files in a separate email.

Best,
Andreas

----- Original Message -----
> From: "Yarden Katz" <yarden at mit.edu>
> To: "Andreas S. Richter" <arichter at ie-freiburg.mpg.de>
> Cc: miso-users at mit.edu
> Sent: Wednesday, 7 August, 2013 9:11:27 PM
> Subject: Re: [miso-users] Problem with discarded reads from strand-specific library
> 
> 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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> 
>