HI @Daniela_Vargas,
Can you re-run your original feature-classifier classify-sklearn
command with the --p-read-orientation same
option? The default for this is --p-read-orientation auto
. For sklearn to work properly the reads must be in the same orientation as the reference database / classifier. Sometimes bad sequences can trick the orientation detector to map the reads as the reverse compliment, thus returning poor taxonomic assignment. It also explains why using different taxonomic results for your full data set vs your two same samples.
You can also sanity check this by using feature-classifier classify-consensus-vsearch
, which is not dependent on orientation. But if you obtain reasonable taxonomy, then that suggests that sklearn got confused about the orientation.... or that your reads are in fact in mixed orientation (you can search the forum for more details on this.