Problem bam files 0 counts
0
0
Entering edit mode
4.5 years ago
Pin.Bioinf ▴ 300

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")
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?

RNA-Seq mapping counts • 1.1k views
1
Entering edit mode

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

0
Entering edit mode

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

0
Entering edit mode

I get :

samtools idxstats: fail to load index for "24_control.bam"

0
Entering edit mode

0
Entering edit mode

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).

0
Entering edit mode

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.