Problems with the tool featureCounts: Fatal error: Exit code 255 ()

Hello Galaxy Community,

Im doing a RNASeq Analysis and having problems with the tool featureCounts. I will give a short overview of what Ive done so far and what error I get at the end.

  1. I checked the quality of my rnaseq files with FASTQC tool
  2. Since I Work with Pseudomonas I downloaded the genome reference (fasta) and annotated genome (gtf) from Pseudomonas Genome Database
    2b. I unpacked the files since they are in .x.gz format
  3. I used the tool hisat2 for the alignment of reads to the mentioned genome reference (fasta)
  4. I tried to link the aligned counts (hisat2 files) to genes with the annotated genome (gtf) and the tool featureCounts
    The job was cancelled and I got following error code: "Fatal error: Exit code 255 (). ERROR: no features were loaded in format GTF. The annotation format can be specified by the ‘-F’ option, and the required feature type can be specified by the ‘-t’ option…
    The porgram has to terminate. "
    The error code mentions something with format GTF. I double checked and the annotated reference genome was in the correct format. Maybe I did something along the line that I was not supposed to do.
    Im relatively new to galaxy and I would very much appreciate your help.


Hi Marcel,

I met the same problem as you. Did you figure it out?


Hi @Marcel and @q_thistle,
the problem is that by default featureCounts set exon as feature filter (Advanced options/GFF feature type filter), but this feature doesn’t appear in the Pseudomonas annotation file (third column). You need to specify CDS as feature type in featureCounts.


Hi there,

I faced the same issue, changing the feature type in the advanced settings to CDS did not solve the problem and I got the same error.
I am working with the chicken genome, downloaded from NCBI, do I have to index the annotation file as well ?

Best, Mila

Hi @Mila

If adjusting the tool setting is not enough, the problem could be with one or more of the input formats. These three FAQs tend to help.