is the chromosome naming in the GFF3 data. Is it really for just one chromosome? You will need annotation for all chromosomes instead.
is the chromosome naming in the fasta, with
as the part of the fasta that is not description content
These don’t match up, so the tool cannot actually use the annotation.
How to use a custom reference genome. Be sure to follow the fasta formatting guidelines. Custom genome + custom build: How to use a genome that is not natively indexed at the server you are working at - #2 by jennaj
Then, you need reference annotation that is based on that exact genome assembly, including the same chromosome identifiers. The headers in your current annotation indicate that it is just annotation for one contig, not all in the assembly. Plus the identifier that is included is a mismatch.
What to do:
- Clean up the format of your genome fasta assembly.
- Find the reference annotation for that full genome assembly, and load it to Galaxy.
- Double check that both use the same chromosome identifiers, and that none are missing from either file. Then double check that your BAM data uses the same format, or consider remapping against your updated fasta.
- Once your BAM is ready, and you have “matching” annotation available, that is when you can adjust downstream tool form settings to match the annotation’s attributes (9th column).
I know this seems complicated, but you only need to get the assembly and annotation into Galaxy once, then formatted once. Consider putting the files in a dedicated history used just for storing the paired reference datasets. Copy those into histories for mapping and other downstream steps.
So far, you have all of the information you need except for the full matching reference annotation.