# Issue with Taxonomic Classification Greengene2 for 16S V4 region - Unassigned data

**URL:** https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749
**Category:** User Support
**Tags:** taxonomy, greengenes2
**Created:** [March 14, 2025, 3:55pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749 "2025-03-14T15:55:46Z")
**Posts on this page:** 7
**Page:** 1

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### Author: ![emilia](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/e/db5fbb/32.png) [@emilia](https://forum.qiime2.org/u/emilia)
#### Post date: [March 14, 2025, 3:55pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749/1 "2025-03-14T15:55:46Z")

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Hello,

I am processing 16S rRNA V3-V4 data using QIIME2 2024.10 (miniconda qiime2-amplicon-2024.10). For the classification step, I am using GreenGene2 v2024.9 (`2024.09.backbone.v4.nb.qza`) with the `qiime feature-classifier classify-sklearn` command. However, the resulting taxonomy table/plot shows that most of the data is classified as "Unassigned." In some samples, up to 83% of the data was unassigned. For this I am using ASVs after Deblur.

However, when I tested the same reads with `2024.09.backbone.full-length.nb.qza`, the data was successfully classified.

This same pattern occurred with other datasets denoised with DADA2. Additionally, these two datasets are not related.

The command I used:

- With V4 classifier:  
qiime feature-classifier classify-sklearn   
--i-classifier 2024.09.backbone.v4.nb.sklearn-1.4.2.qza   
--i-reads rep-seqs-deblur.qza   
--p-n-jobs 6   
--o-classification taxonomy\_gg24\_v4.qza

- With full lenght classifier:  
qiime feature-classifier classify-sklearn   
--i-classifier 2024.09.backbone.full-length.nb.sklearn-1.4.2.qza   
--i-reads rep-seqs-deblur.qza   
--p-n-jobs 6   
--o-classification taxonomy\_gg24\_full.qza

Can someone help me understand what might be causing this? Can I use the data classified with the full-length classifier?

print of taxonomy.qzv after V4 classifier

 ![taxonomy_gg24_v4](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/3X/9/9/9957106f8bd6fe3985f40cf1d9eb0443904cb06d.jpeg)

print of taxonomy.qzv after full lenght classifier

 ![taxonomy_gg24_full_lenght](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/3X/d/c/dca366c9c0f7d7f84dcff849b7140d365c2653e8.jpeg)

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### Author: ![timanix](https://forum.qiime2.org/user_avatar/forum.qiime2.org/timanix/32/17879_2.png) [@timanix](https://forum.qiime2.org/u/timanix)
#### Post date: [March 14, 2025, 4:00pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749/2 "2025-03-14T16:00:08Z")

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Hello! Welcome to the forum!  
My guess is that V4 classifier was trained only on the V4 region of the 16S rRNA gene, while you are working with V3-V4 region, which is considerably longer and also exhibits higher variability.  
Your results obtained with full length classifier are much more trustworthy since they completely cover region you targeted, so you can use it.

Best,

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### Author: ![emilia](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/e/db5fbb/32.png) [@emilia](https://forum.qiime2.org/u/emilia)
#### Post date: [March 14, 2025, 5:27pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749/3 "2025-03-14T17:27:30Z")

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Hi!  
Thank you for the warm welcome and for your explanation! That makes a lot of sense—I hadn’t considered that before.  
I really appreciate your insight!

Best,

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### Author: ![wasade](https://forum.qiime2.org/user_avatar/forum.qiime2.org/wasade/32/2317_2.png) [@wasade](https://forum.qiime2.org/u/wasade)
#### Post date: [March 14, 2025, 10:52pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749/4 "2025-03-14T22:52:34Z")

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Hi @Emilia,

What were the specific primers were used?

Best,  
Daniel

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### Author: ![emilia](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/e/db5fbb/32.png) [@emilia](https://forum.qiime2.org/u/emilia)
#### Post date: [March 17, 2025, 3:34pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749/5 "2025-03-17T15:34:00Z")

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Hi @wasade,

Following [Illumina's protocol](https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf), we used primers to target the V3-V4 region of the 16S rRNA gene:

- Forward Primer: `5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'`
- Reverse Primer: `5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'`

Best,

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<div class="post-metadata">

### Author: ![wasade](https://forum.qiime2.org/user_avatar/forum.qiime2.org/wasade/32/2317_2.png) [@wasade](https://forum.qiime2.org/u/wasade)
#### Post date: [March 17, 2025, 4:31pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749/6 "2025-03-17T16:31:37Z")

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Thanks! Ya, I would not expect the V4 specific classifier to have representation for these data.

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### Author: ![system](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/3X/2/1/21af5fe23cb6f4579467c66a9ed94e55274ca7bd.svg) [@system](https://forum.qiime2.org/u/system)
#### Post date: [April 17, 2025, 10:31pm UTC](https://forum.qiime2.org/t/issue-with-taxonomic-classification-greengene2-for-16s-v4-region-unassigned-data/32749/7 "2025-04-17T22:31:52Z")

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