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Question: count table generation using summarizeOverlaps
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gravatar for Gyan Prakash Mishra
16 months ago by
INDIA
Gyan Prakash Mishra0 wrote:

Hi,

I am using DESeq2 to analyze RNA-seq data

In this first step to generate count table I used following code.

library( "GenomicFeatures" )
hse  <- makeTxDbFromGFF( "Mus_musculus.GRCm38.84.chr.gtf", format="gtf" )
exonsByGene  <-  exonsBy( hse, by="gene")
fls <- list.files( "Bamfiles", pattern="bam$", full=TRUE )

library( "Rsamtools" )
bamLst  <-  BamFileList ( fls, yieldSize=100000 )

library( "GenomicAlignments" )
se <- summarizeOverlaps( exonsByGene, bamLst,mode="Union",singleEnd=FALSE,ignore.strand=TRUE,fragments=TRUE )

But I am getting total count as "0" for all the bam files. from this code

colSums( assay(se) )

I am not able to find the reason for so. I did alignment using tophat.

 

 

ADD COMMENTlink modified 16 months ago • written 16 months ago by Gyan Prakash Mishra0

Are all(seqlevels(bamLst) %in% seqlevels(hse)) ? Is the gtf file from the same build as the alignments?

ADD REPLYlink written 16 months ago by Martin Morgan ♦♦ 20k

Thanks Martin

I got my answer, gtf is not from same build.

ADD REPLYlink written 16 months ago by Gyan Prakash Mishra0
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