23 days ago by
Leave the replicates as individual inputs or grouped into a dataset collection. Do not merge the data. The tool expects replication and if not used that way, the tool will fail.
This Galaxy tutorial shows how to set up two conditions with replicates: https://galaxyproject.org/learn/
Also please see the updated DeSeq2 help (it is more comprehensive now). Find this lower down on the tool form. There are also links to the Bioconductor help pages, which in turn link to their support forum where experimental design using their tools are discussed.
Featurecounts works a bit better than HTseq-count for this tool as it already places a header on each input count file. This is expected content, so if you really want to use HTseq, you'll need to add those headers, in the correct format. Run Featurecounts on one of your BAMs to see what the header format/content should be. Or, switch to using Featurecounts -- the final results will be the same (many have compared using the two and these discussions are online).
Thanks! Jen, Galaxy team