Problem bam files 0 counts
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3.2 years ago
Pin.Bioinf ▴ 290

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?

RNA-Seq mapping counts • 827 views
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Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time. Formatting bar

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

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I get :

samtools idxstats: fail to load index for "24_control.bam"
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Please use the formatting bar!

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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).

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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.

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