Hi, I ran into this error running cuffdiff. I had a hard time debugging this user error, so I though it would be nice to share the solution. This was in a local instance, but I don't see why it wouldn't happen in Galaxy Main under the same circumstances. Tool execution generated the following error message: Error running cuffdiff. Error: cannot open reference GTF file CONDITION,CONTROL for reading The tool produced the following additional output: cuffdiff v1.3.0 () cuffdiff --no-update-check -q -p 4 -c 1000 --FDR 0.050000 -N -b --labels CONDITION,CONTROL /local/db/genomes/illumina/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf /local/opt/galaxy_central/database/files/001/dataset_1339.dat,/local/opt/galaxy_central/database/files/001/dataset_1391.dat /local/opt/galaxy_central/database/files/001/dataset_1452.dat,/local/opt/galaxy_central/database/files/001/dataset_1478.dat The problem ended being the use of "Perform Bias Correction"(-b) and a GTF file with no "Database/Build" associated. Looking at cuffdiff wrapper I found, if a FASTA reference is not selected from the history, the FASTA reference of the GTF file associated build is used. If there is not build association, your cuffdiff run will fail with this not so helpful error. My feeling is, cuffdiff should check for a non-dashed string after '-b' and complain if is absents, but this doesn't happen currently. Kind regards, Carlos