Taxonomic classification of shotgun metagenomic data of rhizospheric soil (bacteria + fungi)

Question

[sumah.1si21bt017]

I am currently working on shotgun metagenomic data from rhizospheric soil, and my focus is on both bacterial and fungal taxonomic classification. After going through the literature, I came across three commonly used approaches/tools: Kraken2 + Bracken (with PlusPF database), MetaPhlAn4, Kaiju

I wanted to check if I am proceeding in the right direction with these choices, or if there are other recommended methods specifically for classifying both bacteria and fungi in shotgun datasets.

Additionally, before proceeding with classification, do I need to separate prokaryotic and eukaryotic sequences (e.g., bacteria vs fungi), or can the classification tools handle this directly?

Any guidance or suggestions would be greatly appreciated. Thanks in advance!

Answer 1

[Mensur Dlakic]

GTDB-toolkit can classify only prokaryotes, but it does it really well. It will most likely classify all eukaryotes as Asgard-archaea.

https://github.com/Ecogenomics/GTDBTk

Tiara can classify the sequences between prokaryotes, eukaryotes and organellar genomes. That should be the first step after the assembly and binning.

https://github.com/ibe-uw/tiara

[sumah.1si21bt017]

That’s really good, sir, thank you for making me aware of the importance of separating prokaryotic and eukaryotic contigs first. I will definitely try using Tiara for that. Thank you for your advice!