# Taxonomic analysis issue
**URL:** https://forum.qiime2.org/t/taxonomic-analysis-issue/11640
**Category:** Technical Support
**Created:** [September 23, 2019, 8:29pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640 "2019-09-23T20:29:00Z")
**Posts on this page:** 8
**Page:** 1
### Author:  [@microme](https://forum.qiime2.org/u/microme)
#### Post date: [September 23, 2019, 8:29pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/1 "2019-09-23T20:29:00Z")
From my reps-seqs file generated after DADA2 , I can see that I have host DNA in addition to the bacterial DNA. The region that I have amplified is V4-V6 region.
[rep-seqsPE.qzv](https://forum.qiime2.org/uploads/short-url/eqc2ocHA6PmYpEVlcmNXPCYPXM2.qzv) (269.2 KB)
I still proceeded with the subsequent steps of taxonomic analysis using SILVA 132, where I had trained it acc. to my primer region. In the end, I generate a taxonomy table having almost all of them as either unassigned or as D\_0\_\_Bacteria. I did this because I assumed that I could filter out the unwanted sequences using the qiime taxa filter-seqs & qiime taxa filter-table.
When I use the command (below) to filter out the unwanted taxa both in my reps-seqs and taxanomy table :
Command 1
qiime taxa filter-seqs
--i-sequences /test/rep-seqsPE.qza
--i-taxonomy /test/testsilva\_97\_v4-v6\_taxonomy.qza
--p-exclude archaea,eukaryota,mitochondria,chloroplast,Unassigned,
--o-filtered-sequences /test/rep-seqs-bac.qza
For command 1 I get: Plugin error from taxa:
All features were filtered, resulting in an empty collection of feature sequences.
Command 2
qiime taxa filter-table
--i-table /test/tablePE.qza
--i-taxonomy /test/testsilva\_97\_v4-v6\_taxonomy.qza
--p-exclude archaea,eukaryota,mitochondria,chloroplast,Unassigned
--o-filtered-table /test/table-bac.qza
However, for the command 2 I generate a table that looks like:
Command for visualizing : qiime metadata tabulate
--m-input-file /data/p281301/test/table-bac.qza
--o-visualization /data/p281301/test/table-bac.qzv
The taxonomy table I generate, consists of unassigned and bacteria. But in the reps-seqs file, when I was directed to the ncbi database, I did spot few bacterial genera (and species):this does not reflect in the taxonomy table. For example, ncbi database shows E.coli (lets say) as a result, so correspondingly I should get the taxa until genus level and not just the domain d\_0\_bacteria. Where am I going wrong? From my reps-seqs file generated after DADA2 , I can see that I have host DNA in addition to the bacterial DNA. The region that I have amplified is V4-V6 region.
[rep-seqsPE.qzv](https://forum.qiime2.org/uploads/short-url/eqc2ocHA6PmYpEVlcmNXPCYPXM2.qzv) (269.2 KB)
I still proceeded with the subsequent steps of taxonomic analysis using SILVA 132, where I had trained it acc. to my primer region. In the end, I generate a taxonomy table having almost all of them as either unassigned or as D\_0\_\_Bacteria. I did this because I assumed that I could filter out the unwanted sequences using the qiime taxa filter-seqs & qiime taxa filter-table.
When I use the command (below) to filter out the unwanted taxa both in my reps-seqs and taxanomy table :
Command 1
qiime taxa filter-seqs
--i-sequences /test/rep-seqsPE.qza
--i-taxonomy /test/testsilva\_97\_v4-v6\_taxonomy.qza
--p-exclude archaea,eukaryota,mitochondria,chloroplast,Unassigned,
--o-filtered-sequences /test/rep-seqs-bac.qza
For command 1 I get: Plugin error from taxa:
All features were filtered, resulting in an empty collection of feature sequences.
Command 2
qiime taxa filter-table
--i-table /test/tablePE.qza
--i-taxonomy /test/testsilva\_97\_v4-v6\_taxonomy.qza
--p-exclude archaea,eukaryota,mitochondria,chloroplast,Unassigned
--o-filtered-table /test/table-bac.qza
However, for the command 2 I generate a table that looks like:
Command for visualizing : qiime metadata tabulate
--m-input-file /data/p281301/test/table-bac.qza
--o-visualization /data/p281301/test/table-bac.qzv
The taxonomy table I generate, consists of unassigned and bacteria. But in the reps-seqs file, when I was directed to the ncbi database, I did spot few bacterial genera (and species):this does not reflect in the taxonomy table. Where am I going wrong?
i have already looked at [here,](https://forum.qiime2.org/t/removing-features-assigned-as-bacteria-0-bacteria-but-no-other-taxanomic-classifications/3261) [here,](https://forum.qiime2.org/t/too-many-unassigned-or-only-at-kingdom-level-features/2934) [here,](https://forum.qiime2.org/t/only-one-taxonimic-level/6562/10) [here.](https://forum.qiime2.org/t/error-while-using-metadata-based-filtering/3476/8).
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### Author:  [@microme](https://forum.qiime2.org/u/microme)
#### Post date: [September 23, 2019, 8:33pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/2 "2019-09-23T20:33:18Z")
Also to let you know, I have looked this up [here,](https://forum.qiime2.org/t/removing-features-assigned-as-bacteria-0-bacteria-but-no-other-taxanomic-classifications/3261) [here,](https://forum.qiime2.org/t/too-many-unassigned-or-only-at-kingdom-level-features/2934) [here,](https://forum.qiime2.org/t/only-one-taxonimic-level/6562/10) [here.](https://forum.qiime2.org/t/error-while-using-metadata-based-filtering/3476/8)
---
### Author:  [@ben](https://forum.qiime2.org/u/ben)
#### Post date: [September 23, 2019, 9:16pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/3 "2019-09-23T21:16:50Z")
Your first link is expired. Would like to take a look at it. Ben
edit: when I had this problem, the dada2 portion did not have enough overlap and ended up without partial reads.
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [September 23, 2019, 9:35pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/4 "2019-09-23T21:35:57Z")
Hey @ben, the first link is fine - [here is a view link to it](https://view.qiime2.org/visualization/?type=html&src=https%3A%2F%2Fforum.qiime2.org%2Fuploads%2Fshort-url%2Feqc2ocHA6PmYpEVlcmNXPCYPXM2.qzv). You might've run into a situation where you downloaded and pre-cached the viz. Thanks!
PS - you can also just download the QZV and open directly in [q2view.](https://view.qiime2.org)
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### Author:  [@microme](https://forum.qiime2.org/u/microme)
#### Post date: [September 23, 2019, 10:19pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/5 "2019-09-23T22:19:49Z")
Sorry, @ben was right, it was not working and then I edited the post by uploading the qzv file. But did not specify that I had correctwd, sorry for the confusion
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### Author:  [@microme](https://forum.qiime2.org/u/microme)
#### Post date: [September 26, 2019, 3:00pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/6 "2019-09-26T15:00:25Z")
I performed taxonomic analysis on the single end reads which is for the V4 region. Earlier when I did the taxonomic analysis ([here](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640)) using paired end reads for the same samples, I found almost all of the features had corresponded to either d\_0\_bacteria or unassigned. I assumed it might be because I did not successfully merge them.
Hence on repeating the same steps with the single end reads, for few features I did get family and genus level classification (but still very low). The remaining are still unassigned (which is from the host) while for several features I still see d\_0\_bacteria: what does this mean? Can anything be done to improve this classification? The now improved taxonomy file for single end reads is below.
[taxonomySE.qzv](https://forum.qiime2.org/uploads/short-url/cQgecmAxkW1cUFr2B4aA9PhmK55.qzv) (1.3 MB)
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### Author:  [@Nicholas\_Bokulich](https://forum.qiime2.org/u/Nicholas_Bokulich)
#### Post date: [September 26, 2019, 3:49pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/7 "2019-09-26T15:49:20Z")
> [@microme](#):
>
> using paired end reads for the same samples, I found almost all of the features had corresponded to either d\_0\_bacteria or unassigned.
chances are you were using an inappropriate reference dataset — you can search the forum for other examples, but 99% of the time this happens when, e.g., someone has V1-3 reads and are using a V4 classifier. Or even V4-6 and using a V4 classifier. The classifier needs to be trained on reads ≥ the target region (so e.g., full-length 16S is fine for classification of V4).
> [@microme](#):
>
> for few features I did get family and genus level classification (but still very low). The remaining are still unassigned (which is from the host) while for several features I still see d\_0\_bacteria: what does this mean?
One of two things:
1. the reads are too short
2. the reads are not really bacterial but are more host; spot check a few with NCBI BLAST (exclude uncultured/unclassified/unknown) to confirm.
3. same issue as #1; the classifier does not fully overlap your target region so you are getting bad classifications.
4. your reference database has poor coverage of the target taxa?
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### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [October 27, 2019, 9:49pm UTC](https://forum.qiime2.org/t/taxonomic-analysis-issue/11640/8 "2019-10-27T21:49:23Z")
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