[miso-users] exon-centric analysis

[mali salmon]

OK, I have managed to run MISO after indexing each gff file and then
running MISO for each event type separately.
Now I would like to filter the results. Using the command suggested in the
manual, all the splicing events were filtered out
"python filter_events.py --filter input_bf --num-inc 1 --num-exc 1
--num-sum-inc-exc 10 --delta-psi 0.20 --bayes-factor 10 --output-dir
filtered"
Is this too stringent for exon-centric analysis?
Is it a matter of coverage? Would you suggest to sequence technical
replicates in order to increase the coverage?
And one last issue (for now :-) how does MISO accounts for the difference
in library size?
Looking forward for your reply
Thanks
Mali



On Mon, Apr 23, 2012 at 8:45 AM, mali salmon <shalmom1 at gmail.com> wrote:

> Thanks Yarden for your reply
> One more question. I have downloaded mm9 exonic-events gff file from your
> site. The uncompressed folder contains few gff files + ensGene.map and
> locuslink.map folders.
> Can you please explain the content of these two folders? which one is the
> indexed folder? Shall I index each gff file separately?
> I have tried to run
> "run_events_analysis.py --compute-genes-psi mm9 input.bam --output-dir
> output --read-len 36" but this command yield nothing, so I suppose the
> problem is with the indexing (I haven't run index_gff.py, assuming the
> folder downloaded from your site is "ready to use")
> Thanks
> Mali
>
>
>
> On Mon, Apr 23, 2012 at 1:28 AM, Yarden Katz <yarden at mit.edu> wrote:
>
>> Hi Mali,
>>
>> see replies below:
>>
>> On Apr 22, 2012, at 2:33 AM, mali salmon wrote:
>>
>> > Hello MISO users
>> > Two questions:
>> > 1. I have BAM files generated using Tophat that was given a RefSeq GFF
>> as input. Now I would like to do an exon-centric analysis using the ensembl
>> gff file provided in MISO.
>> > Is it a problem that the mapping was done with refseq annotations and
>> not Ensembl?
>>
>> You can use any annotation with MISO -- the only requirement is that the
>> chromosome naming schemes in your BAM file match the chromosome naming
>> scheme in your GFF file.  See note on this here:
>> http://genes.mit.edu/burgelab/miso/docs/#human-mouse-gene-models-for-isoform-centric-analyses
>>
>> > 2. Before I'm writing it myself,  is there a script provided for
>> generating exon-centric gff based on isoform-centric gff files?
>> > Thanks
>> > Mali
>> >
>>
>> There is not generic a script that takes isoforms and converts them into
>> events.  There are many ways to go about generating distinct types of
>> events (SE, AFE, ALE, ...) that require decisions to be made about what
>> counts as SE versus A3SS, etc.
>>
>> You can either write a script that makes these categorizations based on
>> rules that you find reasonable.  If you're using Drosophila, mouse or human
>> genomes, you can instead use the event annotations that we provide.  The
>> mapping from isoforms to events is complex and so there are many reasonable
>> ways of doing it -- the events we provide are just one annotation.
>>
>> Hope this helps.
>>
>> Best, --Yarden
>>
>>
>>
>>
>
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