how reliable are my taxonomic classifications if I did not identify species?


Also I had a query.
There are 25000 OTUs identified by Qiime2 in my samples, but after I got annotations only for 9000 at species level, in that too, species names are unassigned. How reliable these results are?

Sounds much more reliable than getting species identifications for everything! I recommend checking out the literature to see some extensive benchmarks on how read length, marker gene target, and subdomain targets impact taxonomic resolution and accuracy. See these articles for starters:


but I am worried about the genera or species, which are most abundant and still unassigned.

If the sequence is abundant but has no species label, that is typical for most short read sequences of 16S rRNA gene variable regions. Unassigned genus is still not uncommon for, e.g., V4.

If the sequence is abundant and is not even assigned at phylum level, it is probably non-target DNA… this is common, e.g., to amplify plant chloroplast DNA or other host DNA from some sample types.

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I have 16S rRNA PE data for V1-V3 region
What should I do at this stage?

Same issue. Species can be unreliable due to lack of species-level resolution in this region.

See my post above, but assuming you have family and genus level classifications, I’d say just move on. You could try out q2-clawback to help guide species-level classifications:

I have reduced my analysis till genus level…but here also I have found a lot of OTUs whose genera are unavailable.

These are one among most prevalent and most abundant genera.

This is really a common issue with 16S rRNA gene sequences, I must say.

I recommend reading the forum archives more to see how others have dealt with this issue (usually, by doing nothing, just accepting that short 16S reads have limited taxonomic resolution). The FAQs are a great start for various taxonomy-related topics:

Good luck!