HUMAnN 3.0: Error: Unexpected end of input files

Hmm, my first guess was the number of threads - we have seen errors in alignment with very large numbers of threads getting out of step with file I/O, leading to errors like this. In your demo run though it looks like you’re only using one thread though.

My next guess is an incompatibility with the newest version of DIAMOND (2.0.15). HUMAnN 3.0 was notably built/tested against DIAMOND 0.9.36, the end of the pre-2.0 lineage. I have seen it used successfully on DIAMOND 2.0+ but it’s possible something has changed recently that resulted in this error. I would try rolling back to an earlier DIAMOND and see if that fixes the problem.