5 weeks ago by
I can't see your graphics - these need to be hosted by some public site then linked into questions here. I use https://imgur.com/ but any public link will work.
That said, I agree the problem is likely with the GTF (one of the most common reasons for failures). If you mapped against the hg38 genome indexed at Galaxy Main https://usegalaxy.org -- or UCSC was your genome source when indexing on your own server, try using the UCSC version of the annotation from iGenomes: https://support.illumina.com/sequencing/sequencing_software/igenome.html. Download the tar archive, unpack it locally, then upload just the genes.gtf to Galaxy (use FTP).
Give that a try first. If the job still fails:
- Double check you actually mapped against hg38 with Tophat ("rerun" or "job details" will have the original inputs recorded)
- Make sure there are hits in the resulting BAM datasets
- Try using HISAT2 instead since both Tophat and Tophat2 are considered deprecated
- Review your steps versus those in this tutorial (uses FeatureCounts, but Htseq-count can be used instead): https://galaxyproject.org/tutorials/nt_rnaseq/
If you cannot find the problem after doing the above, and you can reproduce this at Galaxy Main, a bug report can be sent in from one of the error datasets. Please leave the inputs/outputs undeleted and include the link to this Galaxy Biostars post in the comments. How-to: https://galaxyproject.org/issues/
Thanks! Jen, Galaxy team