I am following the Reference-based RNA-Seq data analysis (Reference-based RNA-Seq data analysis) and I have encountered a problem. I am trying to get gene body coverage. But this requires a BED12 format. The reference genome is from website “Zymoseptoria_tritici - Ensembl Genomes 57” in the format GFF3.
I try this tool: gffread Filters and/or converts GFF3/GTF2 records. and I have encountered a problem with this message "The server could not complete this request. Please verify your parameter settings, retry submission and contact the Galaxy Team if this error persists. "
Here is an example of a successful run: Galaxy
UCSC will also load BED12 directly if your genome is supported (tool → UCSC main).
Is the input formatted correctly?
Headers in GTF are out of specification, and will cause problems even outside of Galaxy, so remove all of them.
Many data providers still do include them to provide provenance information, but in practical use the original specification doesn’t include any, so tool authors write tools in a way that doesn’t expect or filter them out. Some Galaxy wrappers around those tools can handle headers properly, but it was not possible in all cases to make that kind of special adjustment. So, prep the data yourself, especially if you get an error (any error, even if it seems not related, it is the first troubleshooting step). Odd output – do the same.
And, GFF3 will always have at least one header (defines the file version and is required to be the first line) and multiple other headers are OK (define source, and other custom attributes). Internal comments or blank lines (if present) should be removed.
An internet search will find these exact file specifications if you want to learn more. This forum also has much discussion on the topic in various contexts.
For Galaxy help, also see Working with GFF GFT GTF2 GFF3 reference annotation
Hope that helps!