Not sure if this was incorrectly characterized, or why this would appear after removing chloroplast sequences...

[SoilRotifer (Mike Robeson)]

@bkramer

I successfully got the pre-made Silva 138 classifier to run and tested it against my samples.

Great!

@bkramer

is that a hit for Eukaryotes shows up, specifically for Chlorophytes, or a species of the Desmodesmus genus

This should help:

silva database question

Remember the SILVA SSU contains not only Bacterial, Acrachaeal, and Eukaryal 16S rRNA gene sequences, but also contains the Eukaryal 18S rRNA gene sequences too (which are longer). That is, the 18S rRNA gene is a cystolic homologue of the 16S rRNA gene. -Mike

That is, Bacteria, Archaea, and Eukaryota are present. Eukaryotes, can be detected either by the mitochondrial / chloroplast 16S rRNA gene and/or the 18S rRNA gene.

So, even though you've removed plastid sequences (i.e. mitochondria and chloroplast), any 18S off-targets may remain. It is quite common for Bacterial & Archaeal specific primers to amplify eukaryote off-target sequences. Simply add "Eukaryota" to your exclusion list if you are only interested in Bacteria and Archaea like so:

Phylogenetic tree effect on downstream analysis

Hi @Yin_Hui_Cheok, Often I like to perform a variety of sequence and taxonomy filtering prior to inferring a phylogeny for use in downstream analyses. There are many ways to do this, but here are a few suggestions: Remove unwanted / poorly annotated taxonomic groups. The following example removes any sequence variants that do not have at least a phylum-level classification, removes eukaryotes, chloroplast, and mitochondria sequences, as well as any unclassified sequences. Feel free to modify …

Also, keep in mind that the QIIME formatted SILVA files provided by QIIME 2020.2 and earlier, differed in their preparation. Additionally, taxonomic and sequence curation can change between SILVA versions. You can read the original README file contained within the Silva_132_release.zip located here. However, you can use RESCRIPt to download and format fresh copy of SILVA 132 that is inline with similar processing of later :qiime2: releases. You can run the following to obtain the data and process how you'd like with RESCRIPt:

qiime rescript get-silva-data \
    --p-version 132 \
    --p-target SSURef_NR99 \
    --p-include-species-labels \
    --output-dir silva-132

-Mike