[BioC] Detecting differential usage of introns from RNA-seq data.

[Alejandro Reyes]

Dear Fong Chun Chan,

Yes the IDs from the introns are causing some problems in the functions.

It is because DEXSeq uses feature names from the exonCountSet object the 
concatenation of the gene ID and the exon ID separated by a ":" 
character, so introducing additional ":" causes some parsing errors.  
Could you replace the ":" in your exon IDs for another character?
I think that should solve this problem!

Alejandro


Thanks for the reply. I've tried to use DEXSeq for intronic regions just 
like how I did it with exons and I am running in this the problem when I 
am trying to estimatelog2FoldChanges. This is the error:

exonCountSet <- estimatelog2FoldChanges( exonCountSet )
Error in do.call(`[[<-`, c(quote(coefIndices), as.list(lvlTbl[i, ]), 
coefNames[i])) :
   [[ ]] subscript out of bounds

My code to get to this point is (ignore the fact that the variables are 
called exon. I am just copying the code from my exonic DEXSeq run):
-----------------------------

print('Building ExonCountSet ...')
exonCountSet <- newExonCountSet( countData = sumExonsDf[selectedExons, 
samples], design = designMat, geneIDs = sumExonsDf[selectedExons, 
'geneID'], exonID = sumExonsDf[selectedExons, 3], exonIntervals = 
exonAnnotDf[selectedExons, c('chr', 'start', 'end', 'strand')] )
print('... Done')

print('Estimating size factors ...')
exonCountSet <- estimateSizeFactors(exonCountSet)
print('... Done')

print('Estimating dispersions ...')
if ( opt$trt ){
         print('Using the TRT functions ...')
         exonCountSet <- estimateDispersionsTRT( exonCountSet, nCores = 
opt$nCores )
} else{
         exonCountSet <- estimateDispersions(exonCountSet, nCores = 
opt$nCores)
}
print('... Done')

print('Fitting dispersions ...')
exonCountSet <- fitDispersionFunction( exonCountSet )
print('... Done')

print('Testing for differential exon usage ...')
if ( opt$trt ){
         print('Using the TRT functions ...')
         exonCountSet <- testForDEUTRT( exonCountSet, nCores = opt$nCores )
} else{
         exonCountSet <- testForDEU( exonCountSet, nCores = opt$nCores )
}
print('... Done')

exonCountSet <- estimatelog2FoldChanges( exonCountSet )
----------------------

Any idea what is happening here? Does it have something to do with 
exonIDs actually? Because I don't actually have proper intronIDs so what 
I am actually doing is using the genomic coordinates as the intronID. So 
an intronID will look like:

chr1:861181-861301

I wondering if this is causing a problem. Any help would be greatly 
appreciated. I am using DEXSeq 1.5.6 the version that I got from the svn 
repository so that I have access to the TRT functions.

Fong


>
>
> On Wed, Feb 6, 2013 at 1:06 AM, Alejandro Reyes 
> <alejandro.reyes at embl.de <mailto:alejandro.reyes at embl.de>> wrote:
>
>     Dear Fong Chun Chan,
>
>     Recently I have tried this, and works nicely you just need to
>     count the reads falling in the introns and them as "exonic bins"
>     in DEXSeq.
>
>     However, I think is recommendable to have strand specific data for
>     this, because sometimes intronic regions contain antisense
>     transcripts that if they are differentially expressed between your
>     conditions, they can look like intron retention differences in
>     your transcripts. Also, when adding the introns as "exonic parts"
>     in DEXSeq, the models become big and difficult to compute, so it
>     is necessary to use DEXSeq in the TRT context
>     (estimateDispersionsTRT and testForDEUTRT).
>
>     Best wishes,
>     Alejandro Reyes
>
>
>
>         Hi,
>
>         I am interested in trying to detect the intron retention in
>         RNA-Seq I was
>         wondering if anyone how ever tried to apply DEXSeq to looking for
>         differential intron usage between two groups of samples. It
>         seems like an
>         ideal software that could detect for this given that the
>         features are just
>         introns now instead of exons. Or does anyone else recommend
>         any other
>         Bioconductor package that looks for differential intron retention?
>
>         Thanks,
>
>         Fong
>
>                 [[alternative HTML version deleted]]
>
>         _______________________________________________
>         Bioconductor mailing list
>         Bioconductor at r-project.org <mailto:Bioconductor at r-project.org>
>         https://stat.ethz.ch/mailman/listinfo/bioconductor
>         Search the archives:
>         http://news.gmane.org/gmane.science.biology.informatics.conductor
>
>
>