Help in How to generate a GFF file

I am trying to do a differential gene expression analysis of the bovine oviduct. When using the featureCounts tool, I need to upload a GFF/GTF file in the “Gene annotation file”. The reference genome I selected in the Hisat2 tool was bosTau9, so in the “featureCounts” tool I choose the option “GFF/GTF file in your history” and upload a GFF file from the web page (, but it seems that the gene annotation file I retrieve (Index of /pub/release-109/gff3/bos_taurus) do not corresponds to the same reference genome (bosTau9) I used for the alignment with the Hisat2 tool because a received an error message: Fatal error: Exit code 255 ().
I hope someone can help me.
Many thanks.

Hi @vpabloa

The bosTau9 genome available and indexed in Galaxy is based on the UCSC version of the assembly. It uses slightly different chromosome names than the Ensembl version. Tools expect exact matches, and can fail or produce odd results when there is some mismatch.

Try using the GTF annotation from UCSC. There are a few choices, and each corresponds with a Gene and Gene Prediction track (see their browser for a description). The file you want is probably this one. Copy/paste the link into the Upload tool using all defaults and it will be ready to use.

FAQ Working with GFF GFT GTF2 GFF3 reference annotation

Thank you @jennaj, I uploaded the file and will continue the job.

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