# Large portion of Cyanobacteria
**URL:** https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232
**Category:** User Support
**Tags:** taxonomy
**Created:** [June 2, 2022, 12:12pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232 "2022-06-02T12:12:13Z")
**Posts on this page:** 20
**Page:** 1
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 2, 2022, 12:12pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/1 "2022-06-02T12:12:13Z")
I need help please. I assigned taxonomy using my own trained classifier for V3/V4 region.
The problem is when I removed the primers and got much of my reads kept as non-chimeric which is good, **I have dominance of Cyanobacteria in my taxonomy file**. **That dominance belonged to Proteobacteria in my previous taxonomy file generated** which makes more sense to me.
Here the the final taxonomy file i generated
[atra-taxa-bar-plots.qzv](https://forum.qiime2.org/uploads/short-url/lrNqkGDu36or67FjxwLGMXihlNV.qzv) (833.5 KB)
Here is the **prefinal taxonomy file i generated, although here much less reads were included as chimera was almost half of my sequences**
[taxa-bar-plots.qzv](https://forum.qiime2.org/uploads/short-url/qMk8GbYxb3PwgyjyBjx8U62oimS.qzv) (570.2 KB)
**What could I have done wrong to get that much Cyanobacteria which makes no sense to me?**
---
### Author:  [@timanix](https://forum.qiime2.org/u/timanix)
#### Post date: [June 2, 2022, 12:40pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/2 "2022-06-02T12:40:36Z")
Hi!
Please, always create a new topic if it is no more relevant to the original one. It helps moderators to solve the issue in a faster way and it will be more visible to other users with similar questions.
In your case, Cyanobacteria is dominant because of large amount of Chloroplast. I guess you are working with plant material. Here is a [tutorial](https://docs.qiime2.org/2022.2/tutorials/filtering/#taxonomy-based-filtering-of-tables-and-sequences) how to get rid of organelles DNA.
Probably after filtering dominance will return to the Proteobacteria. But not necessarily - your first run was biased by large portion of false-chimeric reads.
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 2, 2022, 4:05pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/3 "2022-06-02T16:05:45Z")
Actually, It is mainly sediment and gut samples not plant material.
---
### Author:  [@timanix](https://forum.qiime2.org/u/timanix)
#### Post date: [June 2, 2022, 4:23pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/4 "2022-06-02T16:23:36Z")
Still, you can remove organelles DNA from the dataset.
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 2, 2022, 4:56pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/5 "2022-06-02T16:56:33Z")
I have different Cyanobacteria subclassification in my taxonomy as follows and I am confused if I should exclude all
d\_\_Bacteria;p\_\_Cyanobacteria;c\_\_Cyanobacteriia; **o\_\_Chloroplast** ;f\_\_Chloroplast; **g\_\_Chloroplast** | 19.457%
d\_\_Bacteria;p\_\_Cyanobacteria;c\_\_Cyanobacteriia; **o\_\_Cyanobacteriales** ; **f\_\_Xenococcaceae** ;g\_\_Xenococcus\_PCC-7305
d\_\_Bacteria;p\_\_Cyanobacteria;c\_\_Cyanobacteriia; **o\_\_Cyanobacteriales** ;f\_\_Xenococcaceae;g\_\_Pleurocapsa\_PCC-7319
d\_\_Bacteria;p\_\_Cyanobacteria;c\_\_Cyanobacteriia; **o\_\_Synechococcales** ; **f\_\_Cyanobiaceae** ;g\_\_Synechococcus\_CC9902
d\_\_Bacteria;p\_\_Cyanobacteria;c\_\_Cyanobacteriia;o\_\_Cyanobacteriales;f\_\_Xenococcaceae;g\_\_Chroococcidiopsis\_PCC-6712
d\_\_Bacteria;p\_\_Cyanobacteria;c\_\_Cyanobacteriia;o\_\_Cyanobacteriales;f\_\_Cyanobacteriaceae;g\_\_Cyanobacterium\_CLg1
d\_\_Bacteria;p\_\_Cyanobacteria;c\_\_Cyanobacteriia;o\_\_Cyanobacteriales;f\_\_Phormidiaceae;g\_\_Trichodesmium\_IMS101
**So I should exclude all the orders of Cyanobacteria, or just chloroplast?**
**What confuses me is that their existence in sediment or seawater samples** could be normal but in **midgut samples with high abundace is not normal to me** , How they are there?
---
### Author:  [@timanix](https://forum.qiime2.org/u/timanix)
#### Post date: [June 2, 2022, 5:02pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/6 "2022-06-02T17:02:29Z")
No, you do not need to exclude the whole order. Just chloroplast and mitochondria, as it is shown in the tutorial.
I know that a lot of chloroplast can be found in human mouth samples just because subject ate some salad that day. Can be that they are coming from the food?
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 2, 2022, 6:30pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/7 "2022-06-02T18:30:54Z")
Do I have then to just use the new filtered-seq.qza file to create new taxa barplots? But I will be using then the same feature-table unfiltered, Is this okay, **or should i filter also the feature table.qza too??**
---
### Author:  [@timanix](https://forum.qiime2.org/u/timanix)
#### Post date: [June 2, 2022, 6:32pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/8 "2022-06-02T18:32:28Z")
Filter first your feature table and then filter rep-seq.qza file (optional) based on filtered feature table.
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 2, 2022, 7:00pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/9 "2022-06-02T19:00:54Z")
qiime taxa barplot
--i-table table.qza
--i-taxonomy **taxonomy.qza**
--m-metadata-file sample-metadata.tsv
--o-visualization taxa-bar-plots.qzv
So, finally when we generate taxa barplots after filtration of chloroplast, we will use the **filtered table.qza** **but still we will use the unfiltered taxonomy file, Is this correct?**** as we only filtering table.qza and seq.qza but not taxonomy.qza ?** correct?
**Then, I believe, I should run Phylogeny, alpha and beta diversity again using the new filtered table.qza and seq.qza files, correct?**
Thank you so much for your patience with me.
---
### Author:  [@timanix](https://forum.qiime2.org/u/timanix)
#### Post date: [June 2, 2022, 7:18pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/10 "2022-06-02T19:18:24Z")
> [@Sabrin](#):
>
> So, finally when we generate taxa barplots after filtration of chloroplast, we will use the **filtered table.qza** **but still we will use the unfiltered taxonomy file, Is this correct?** **as we only filtering table.qza and seq.qza but not taxonomy.qza ?** correct?
There is no need to filter taxonomy.qza file. It is not a problem when ASVs, present in taxonomy file, are missing in the feature table and representative sequences. The problem is when ASVs from feature table are missing in rep-seqs or taxonomy files.
> [@Sabrin](#):
>
> Then, I believe, I should run Phylogeny, alpha and beta diversity again using the new filtered table.qza and seq.qza files, correct?
That's correct!
> [@Sabrin](#):
>
> Thank you so much for your patience with me.
No worries, I am learning here as well by helping others.
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 14, 2022, 11:47am UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/11 "2022-06-14T11:47:47Z")
Hi again,
after filteration of my taxonomy file and removing cholroplast and mitochondria, I still have cyanobacteria dominating my midgut samples whhich makes no sense to have it dominating here
[atra-filtered-no-mitochondria-no-chloroplast-taxa-bar-plots.qzv](https://forum.qiime2.org/uploads/short-url/rTMU48iP50h5U32CAmIp6IHqqYK.qzv) (828.2 KB)
How I could please proceed with it without ruining my data? I have also cyanobacteria in seawater and sediment samples but in midgut samples is not logic
Where could that wired classification comes from?
---
### Author:  [@SoilRotifer](https://forum.qiime2.org/u/SoilRotifer)
#### Post date: [June 14, 2022, 12:27pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/12 "2022-06-14T12:27:19Z")
Hi @Sabrin,
> [@Sabrin](#):
>
> I still have cyanobacteria dominating my midgut samples
That is fine, as not all cyanobacteria are photosynthetic. Many are known to live in the gut too. See:
- [The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria | eLife](https://doi.org/10.7554/eLife.01102)
- [Melainabacteria - Wikipedia](https://en.wikipedia.org/wiki/Melainabacteria)
- [Frontiers | Long Term Diversity and Distribution of Non-photosynthetic Cyanobacteria in Peri-Alpine Lakes](https://doi.org/10.3389/fmicb.2018.03344)
- [https://doi.org/10.1093/gbe/evu073](https://doi.org/10.1093/gbe/evu073)
Also, if these are gut samples, it is entirely possible these could be dietary 'bycatch', that is, cyanobacteria living on surfaces that the host is eating. Possibly contamination too, but hard to say without knowing more about your study system.
-Mike
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 14, 2022, 12:52pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/13 "2022-06-14T12:52:32Z")
thank you so much for your quick response.
I am sampling from nature gut of sea cucumbers and **dividing the gut into three compartments, foregut, midgut, hingut**. Additionally sampling seawater and sediment samples.
It would make sense to me that these cyanobacteria comes from feeding on sediment if the foregut also was dominated by these cyanobacteria but it is not the case. So this means these cyanobacteria is enriched in the mid gut but if they are photosynthetic cyanobacteria then it makes no sense.
**How i could reach their metabolic activity? any suggestions!! I can not find them on literature in any gut study** ,
---
### Author:  [@SoilRotifer](https://forum.qiime2.org/u/SoilRotifer)
#### Post date: [June 14, 2022, 5:34pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/14 "2022-06-14T17:34:07Z")
> [@Sabrin](#):
>
> t would make sense to me that these cyanobacteria comes from feeding on sediment if the foregut also was dominated by these cyanobacteria but it is not the case.
Not necessarily true. The foregut may simply not be the right environment for them to "stay around", so they just keep moving through the gut until they find a region that they can do well and become enriched there (midgut). Or perhaps it was indeed diet, and you happened to sample at a point and time when the DNA from the Cyanobacteria had moved to the midgut. Of course, these are just a few thoughts 🤷♂️. Sounds like you may have an interesting research question blooming. 🙂
> [@Sabrin](#):
>
> if they are photosynthetic cyanobacteria then it makes no sense
Remember trying to identify very specific taxa at the genus and species level can be difficult with amplicon sequencing data. It could be there are no good representatives of non-photosynthetic cyanobacteria within the reference database. I've not looked very thoroughly myself. Though you can look [here](https://www.arb-silva.de/browser/). _Note, I did not find any Melainabacteria within SILVA_. So they may be present under another name or may simply not have been included yet. Thus, it is possible that the assigned taxonomy is simply a spurious result of the query sequence consistently mapping to the closest, but unrelated, taxon within the reference database. Also, keep in mind that taxonomy is always changing, and new sequence data is being generated.
You can always use the various tools within QIIME 2 and [RESCRIPt](https://github.com/bokulich-lab/RESCRIPt/) to fetch and append any 16S rRNA gene data of [Melainabacteria](https://www.ncbi.nlm.nih.gov/nuccore/?term=Melainabacteria+16S) from [GenBank](https://forum.qiime2.org/t/using-rescript-to-compile-sequence-databases-and-taxonomy-classifiers-from-ncbi-genbank/15947) into your existing SILVA or other reference database (Assuming the taxonomy labelling schema is constant between the files, _i.e._ GenBank and SILVA differ in some respects. But you can use `qiime rescript edit-taxonomy ...` to help). Then you can merge the new data into the existing reference database by using the `qiime feature-table merge-seqs ...` and `qiime feature-table merge-taxa ...` commands. Then you can train your new classifier.
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 16, 2022, 12:48pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/15 "2022-06-16T12:48:17Z")
@SoilRotifer thank you so much for your thoughts.
I have a question please regarding training my classifier, as i have fears it doesnot fit my sequences.
Here in this step in rescript,
qiime rescript filter-seqs-length-by-taxon
--i-sequences silva-138.1-ssu-nr99-seqs-cleaned.qza
--i-taxonomy silva-138.1-ssu-nr99-tax.qza
--p-labels Archaea Bacteria Eukaryota
--p-min-lens **900 1200** 1400
--o-filtered-seqs silva-138.1-ssu-nr99-seqs-filt.qza
--o-discarded-seqs silva-138.1-ssu-nr99-seqs-discard.qza
**Does --p-min-lens 900 1200 1400 means i am loosing any ref. sequences less than 1200 bp for 16S, could that be limiting my classifier to full length ref. 16S?** Would lowering that increase my chances and my ref. seq. by including shorter amplicon reads?
I am trying to understand why all my sequences are classified as bacteria and I have no unclassified results at all.
---
### Author:  [@SoilRotifer](https://forum.qiime2.org/u/SoilRotifer)
#### Post date: [June 16, 2022, 1:09pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/16 "2022-06-16T13:09:50Z")
> [@Sabrin](#):
>
> Does --p-min-lens **900 1200** 1400 means i am loosing any ref. sequences less than 1200 bp for 16S, could that be limiting my classifier to full length ref. 16S?
This is explained in the RESCRIPt documentation [here](https://forum.qiime2.org/t/processing-filtering-and-evaluating-the-silva-database-and-other-reference-sequence-data-with-rescript/15494#heading--fourth-header), as well as the help text, which you can access by:
`qiime rescript filter-seqs-length-by-taxon --help`
> [@Sabrin](#):
>
> Would lowering that increase my chances and my ref. seq. by including shorter amplicon reads?
Potentially. You can play around with the settings. If you do not need to differentially length-filter the sequences by taxonomy, you can simply use [qiime rescript filter-seqs-length ...](https://forum.qiime2.org/t/processing-filtering-and-evaluating-the-silva-database-and-other-reference-sequence-data-with-rescript/15494#heading--twelfth-header) instead. This way you can trim everything down to 900 bases, or whatever length you choose.
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 23, 2022, 1:07pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/17 "2022-06-23T13:07:32Z")
@SoilRotifer Hi again, As i have doubts still about my taxonomy, I am assigning taxonomy against the full classifier and not the specific V3V4 classifier, just to be sure..
Is these steps correct please , especially step 2? to assign directly against **silva-138-ssu-nr99-classifier.qza**
1. qiime feature- **classifier** fit- **classifier** -naive-bayes _--i-reference-reads_ **/user/asga9989/Taxonomy\_output/training-feature-classifiers/** **_silva-138-ssu-nr99-seqs-derep-uniq.qza_ _--i-reference-taxonomy_ /user/asga9989/Taxonomy\_output/training-feature-classifiers/ _silva-138-ssu-nr99-tax-derep-uniq.qza_ _--o-classifier_ /user/asga9989/Taxonomy\_output/training-feature-classifiers/** _ **silva-138-ssu-nr99-classifier.qza** _
2. qiime feature-classifier classify-sklearn --i-classifier /user/\*\*asga9989/Taxonomy\_output/training-feature-classifiers/\*\*_ **silva-138-ssu-nr99-classifier.qza** _ --i-reads /user/asga9989/atra-rep-seqs.qza --o-classification /user/asga9989/atra-vs-full-classifier-taxonomy.qza
3. qiime metadata tabulate --m-input-file /user/asga9989/atra-rep-seqs.qza --o-classification /user/asga9989/atra-vs-full-classifier-taxonomy.qza --o-visualization /user/asga9989/atra-vs-full-classifier-taxonomy.qzv
Is there is available full length classifier **silva-138-ssu-nr99-classifier.qza** that I might directly use? Just to check if my full classifier file is not corrupted!!
Best Regards,
Sabrin
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [June 23, 2022, 3:45pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/18 "2022-06-23T15:45:58Z")
@SoilRotifer I found _Melainabacteria within SILVA_ , here it is
But it is only four entries, which maybe still not enough?
---
### Author:  [@SoilRotifer](https://forum.qiime2.org/u/SoilRotifer)
#### Post date: [June 23, 2022, 4:28pm UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/19 "2022-06-23T16:28:02Z")
> [@Sabrin](#):
>
> Is there is available full length classifier **silva-138-ssu-nr99-classifier.qza** that I might directly use? Just to check if my full classifier file is not corrupted!!
You can find a few pre-made files on the [Data resources](https://docs.qiime2.org/2022.2/data-resources/) page.
> [@Sabrin](#):
>
> But it is only four entries, which maybe still not enough?
Likely not. Mainly because these labels only exist as organisms names, that have been used as the species labels. It is often quite difficult to obtain species-level classification with amplicon reads.
---
### Author:  [@Sabrin](https://forum.qiime2.org/u/Sabrin)
#### Post date: [July 5, 2022, 9:32am UTC](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232/20 "2022-07-05T09:32:38Z")
@SoilRotifer
I want now to add the Melainabacteria from NCBI to my classifier but so stuck how to start that.
How to only add Melainabacteria and not the whole 16S refs, or I should add the whole 16S?
> [@Using RESCRIPt to compile sequence databases and taxonomy classifiers from NCBI Genbank](https://forum.qiime2.org/t/using-rescript-to-compile-sequence-databases-and-taxonomy-classifiers-from-ncbi-genbank/15947):
>
> This tutorial will describe how to create custom reference databases from NCBI Genbank using [RESCRIPt](https://github.com/bokulich-lab/RESCRIPt). Read more about RESCRIPt here: [Processing, filtering, and evaluating the SILVA database (and other reference sequence data) with RESCRIPt - #7](https://forum.qiime2.org/t/processing-filtering-and-evaluating-reference-sequence-data-with-rescript/15494/7) If using NCBI Genbank data, please be aware of the [NCBI disclaimer and copyright notice](https://www.ncbi.nlm.nih.gov/home/about/policies/). Citation: If you use RESCRIPt or any RESCRIPt-processed data in your research, please cite the following pre-print: Michael S Robeson II, Devon R O'Rourke, Benj…
here it adds the whole 16S
qiime rescript get-ncbi-data
\*_--p-query '33175[BioProject] OR 33317[BioProject]' \*_
--o-sequences ncbi-refseqs-unfiltered.qza
--o-taxonomy ncbi-refseqs-taxonomy-unfiltered.qza
Thanks in Advance
[Next page](https://forum.qiime2.org/t/large-portion-of-cyanobacteria/23232.md?page=2)