Thank you for testing that.
If vsearch gives the same classifications, then read orientation is not the issue.
It also looks like you used full-length 16S as a reference for vsearch? (if you did, good — then trimming is not the issue either). If so, the pre-trained classifiers will not help either.
At this point I think the most likely possibilities are:
- You have a large proportion of non-target DNA (e.g., host DNA)
- There is something wrong with your query sequences (e.g., adapters or other non-biological DNA in the sequences).
Check out this post, particularly the part about checking some of the unassigned sequences with NCBI blast… that will help figure out if you have some non-target DNA.
Let us know what you find!