Question: Problem bam files 0 counts
0
gravatar for Pin.Bioinf
12 months ago by
Pin.Bioinf230
Malaga
Pin.Bioinf230 wrote:

Hello, I am trying to get the counts of my experiment from the .bam files by using summarized experiment function in R.

My reads are single ends, not strand specific. Here is some R code:

gtffile <- file.path(dir2,"Homo_sapiens.GRCh38.91.gtf")
(txdb <- makeTxDbFromGFF(gtffile, format="gtf"))
(genes <- exonsBy(txdb, by="gene"))
library("GenomicAlignments")
se <- summarizeOverlaps(features=genes, reads=bamfiles,
                        mode="Union",
                        singleEnd=FALSE,
                        ignore.strand=FALSE,
                        fragments=FALSE )

I am getting 0 counts as the output for every sample and every gene. Anyone knows if it could be the .gtf file or if my .bam files are not correct? Ive done it many times. Any alternatives?

counts rna-seq mapping • 357 views
ADD COMMENTlink modified 12 months ago • written 12 months ago by Pin.Bioinf230
1

Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time. Formatting bar

ADD REPLYlink written 12 months ago by RamRS20k

Didn't you get any error? What is the output of samtools idxstats file.bam ?

ADD REPLYlink written 12 months ago by venu5.8k

I get :

samtools idxstats: fail to load index for "24_control.bam"
ADD REPLYlink modified 12 months ago by RamRS20k • written 12 months ago by Pin.Bioinf230

Please use the formatting bar!

ADD REPLYlink written 12 months ago by RamRS20k

Then you should index 24_control.bam by doing samtools index 24_control.bam.

Top thing to check when zero counts manifest is to ensure that chromosome names match in your alignments and annotation (i.e. chr1 =\= 1).

ADD REPLYlink modified 12 months ago • written 12 months ago by genomax62k

Thank you a lot, i was helping another person who had stumbled upon this problem, and i realized she was using different genome anotations for mapping and for quantification.

ADD REPLYlink written 12 months ago by Pin.Bioinf230
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