Issues assigning taxonomy when using V4V5 region

Thank you for the helpful information. I will be sure to follow up with those methods. The version of QIIME2 currently running on our HPC is quite out of date and will need to be updated before I proceed.

However, while I was waiting for a software update, I recalled I had previously analyzed this data using the older SILVA 132 reference data but with a newer version of QIIME2 than the one I am currently running. This analysis as done in an identical manner where the amplicon region was extracted using the primer sequence. These extracted reads were then used as the input to train the classifier. This was very successful as you can see in the bar plot.

For due diligence I took those extracted reads and taxonomy file from the SILVA 132 release that worked for me previously, and used then to train the feature classsifier using the older version of QIIME2 I am currently running. To my surprise it did not work at all. I got the same issue where everything was only classified to domain level. Very strange.

I am not sure what could be causing this. It's the same samples, and same region that was extracted using the same primer region on the same reference database (SILVA 132). The only difference is the version version of QIIME2, and slightly different denoising parameters.