If you have replicates now, check that the number of lines for each count dataset are the same. There should be at least four unique count datasets input to the tool.
Each line represents a gene (plus optionally, one header line). Should the number of lines be different between count files, then that indicates a problem with the reference annotation or perhaps how you are counting (this tool expects counts by “gene”).
The FAQ above covers nearly all the issues that can come up with this tool and other similar tools. Your original posted error had two problems: 1) no replicates and 2) a different number of lines (genes) for each of the two inputs.
Check the new error message: If the line counts are different, you’ll need to review the upstream analysis to fix the annotation/counting problems. The “End to End” tutorials have an example. Galaxy Training!
Ps: Please help to keep the conversation on a single topic by avoiding cross-posting to other topics. I saw that you posted on the topic below about this same issue. That post is a good reference, specifically the sentence below, but let’s keep troubleshooting your issue in this topic. If you need or want to reference other topics, capture the link and post it back here.