Question: featureCounts gives extreme low counts on highly expressed genes
gravatar for xxtomnyxx
15 days ago by
xxtomnyxx0 wrote:

I mapped paired-end sequencing with RNA-STAR and got the BAM file. I then use featureCounts to count the reads with a gene annotation file (an GTF file generated from UCSC table browser). But I found that some genes have extreme low counts. I checked the BAM file with IGV and the BAM file shows lots of reads in these low count genes.

Two genes for example:

GAPDG Gapdh locus

and T T locus

Whereas their counts are: enter image description here

Initially I though I input the wrong strand parameter so I tried run featureCounts with Unstranded, Forward and Reverse strand and check their results, but that doesn't solve the problem. (Btw my sequencing file should be "Reversed.")

I also checked the GTF file used as Gene annotation input file. NM_008084.3 and NM_009309.2 data in the GTF file

Their "exon" position is definitely correct in the mm10 genome! (I sure every of my data were processed with mm10)

Interestingly, if I run featureCounts with builtin Gene annotation file, the output file seems to have correct counts in these genes. But the builtin Gene annotation is not able to provide transcription isoform information due to that it only output the counts with the geneID.

Does anyone know how to fix it?

rna-seq featurecounts • 55 views
ADD COMMENTlink written 15 days ago by xxtomnyxx0

Hum..... I think I know what's wrong.... I should set the "Allow read to contribute to multiple features" parameter in "Advanced options" to "Yes." So I guess the problem is caused by that the reads belong to GAPDH and T (and other genes with same problem) were already counted on other RefSeq feature in the supported GTF file. Thus, if counts of each transcription isoform is desired, the parameter of "Allow read to contribute to multiple features" should be set to "Yes."

ADD REPLYlink written 15 days ago by xxtomnyxx0
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