Training classifier for taxonomic analysis

[Linda_Abenaim]

Hi everyone!

I tried to train the classifier using the Greengenes 13_8 99% OTU dataset for the 16S rRNA gene V3-V4 regions. I followed the tutorial "Training feature classifiers with q2-feature-classifier".
For the "extract-reads" command I did:
qiime feature-classifier extract-reads
--i-sequences 99_otus.qza
--p-f-primer CCTACGGGNGGCWGCAG
--p-r-primer GACTACHVGGGTATTCTAATCC
--p-min-length 400
--p-max-length 500
--o-reads ref-seqs.qza

After which I trained the Naive Bayes classifier.

Unfortunately, already in the "taxonomy.qzv" file (which I attach) many features stop at the domain.

I then applied the taxa barplot command to my filtered table and didn't get great results. Furthermore, there are no statistically significant differences regarding abundance at taxa level 6.

For simplicity I attach all the files.
bsf-l6-ancom-treatment.qzv (420.0 KB)
taxa-bar-plots-bsf.qzv (356.2 KB)
taxonomy.qzv (1.3 MB)

I'll explain my work to you: I'm trying to analyse the intestinal microbiome of an insect species raised on a control diet and a treated diet to understand if there are any differences of bacterial community.

Do you have suggestions for improving the analysis? The analysis of alpha and beta diversity was all significant.

Thank you in advance

[colinbrislawn (Colin J Brislawn)]

Hello Linda,

@Linda_Abenaim

I'll explain my work to you: I'm trying to analyse the intestinal microbiome of an insect species raised on a control diet and a treated diet to understand if there are any differences of bacterial community.

This sounds like a great project!

Have you tried using Greengenes2 with non-v4-16s, as shown in the main tutorial?

EDIT: This does not make ASVs from your data like other pipelines. Instead, it directly compares your reads against the GG2 database entries, which already have a taxonomy.

Simply counting database hits is also known as 'closed-ref-clustering'.
This has very different tradeoffs than denoising -> taxonomy inference.

Introducing Greengenes2 2022.10

Introduction We are excited to announce a new release of the Greengenes database, full redesigned from the ground up, backed by whole genomes, with a focus on harmonizing 16S rRNA and shotgun metagenomic datasets. We're calling the new database Greengenes2 due to substantial changes in the design. However, like the original Greengenes, it relies on a de novo phylogeny, and expresses a taxonomy that is derived from the phylogeny. The focus of this forum post is on how to use the new Greengenes2…

While GG2 should work well with a Naive Bayes classifier, using the GG2 plugin
qiime greengenes2 non-v4-16s
is a great place to start and provides a point of comparison.

Keep us posted!

[Linda_Abenaim]

Thank you so much, Colin! I will try with your suggestion I will keep you posted