Welcome @spongebob !
What a puzzling problem! The fact that classify-consensus-vsearch is also failing is a pretty good indicator that something has gone seriously wrong.
A few things could be at fault, but it sounds like it's probably an issue with the query sequences themselves. Could you walk through the following to troubleshoot? Stop once you find the problem.
- This is a better tutorial to work from: Fungal ITS analysis tutorial
- that tutorial (and the older one you linked to) both use mock community data as a toy dataset... want to try classifying the mock community data with your classifier? If you get the same issue, we know it's a classifier issue. If it works, then we know that your data are at fault!
- try NCBI BLASTing a few to see what you hit (e.g., if you hit non-fungal sequences that's a clear answer. Most ITS primers also hit plants and other euks, so maybe it's just non-target? That should be unclassified or classified at kingdom level since such seqs are not in the UNITE db)
If those steps don't shake out the issue, could you please share all of the QZVs that you listed above? Then I can work backwards through them to see if I spot any clues... you can share via DM if you don't want these posted publicly here.
Let me know what you find!