Ok, thanks for trying that. Your inputs are correct now.
The problem is likely with the tool itself. I didn’t notice this before but it was sourced from the test toolshed which is a sandbox/testing tool repository. And the tool wrapper plus the dependencies it uses haven’t been updated since 2015. My guess is that the tool is still hosted on the AU server due to legacy reasons – and it may work in some special cases/older workflows – but I’m not too surprised it isn’t working now.
Featurecounts instead. Those tools are current, work, and produce individual count files, which would be required by
DESeq2. You could combine those counts into a matrix, or use the individual count files, with
Limma. The format of a matrix is on those latter tool’s forms down in the help section. Tools in the group
Text Manipulation (example:
Multi-Join) could be used to merge the individual count files together into a matrix, but that isn’t required, and if you are not sure how to do that or have problems – skip that and input the individual count files instead.
Examples of RNA-seq DE analysis are covered in the GTN tutorials under the topic “Transcriptomics”. The three tutorials in the group “End-to-End Analysis” are the best place to start for an overview of current methods.
So – the bad news was that this tool won’t work (again, sorry for not noticing where it was sourced originally!) – but the GOOD news is that the changes/checks you made with your inputs would have been needed anyway when using the updated tools/methods.
Please give that a try!