Biom Format Questions

[mothur-westcott]

The user provided me with shared, constaxonomy and mothur created biom file. The file looked "weird" to him, more like a tree structure.

Response:

The make.biom command is designed to convert a shared file, http://www.mothur.org/wiki/Shared_file, into a biom file formatted file, http://biom-format.org/documentation/format_versions/biom-1.0.html#example-biom-files. When you provide a *.cons.taxonomy file, mothur will fill in the metadata information for each OTU with the consensus taxonomy.

12.24.15

[Vieira34]

Hi Sarah,

I'm not sure if here is the right place to post this question ... I get an error when I tried to use make.biom file command. I wanna try to make a biom file to use as input in picrust. I followed the instruction right util classify.otu command (I guess are right). I did the all pre-processing using both silva- and gg- database and the same error showed up.

Here is the error:

[ERROR]: could not find OTUId for k_Bacteria(100);k_Bacteria_unclassified(100)...

Looks like this group of sequence is not in the tax file or I did something wrong. Any idea?

Thank for your attention!

Fabricio.

[mothur-westcott]

What version of mothur are you using? Could you post the command you ran?

[Vieira34]

Hi Sarah, The last version of Mothur (v.1.39.0 - Linux). I followed the MiSeq SOP till shared- and constaxonomy- and then make.biom command: make.biom(shared=comp_list.shared, label=0.03, reftaxonomy=gg_13_8_99.gg.tax, constaxonomy=comp_list.0.03.cons.taxonomy, picrust=97_otu_map.txt) I tried using both database Silva v123 and gg for both aligment and classification and the same error showed up...: [ERROR]: could not find OTUId for k_Bacteria(100);k_Bacteria_unclassified(100)... Thank for your attention! Fabricio. 2017-03-03 12:18 GMT-05:00 Sarah Westcott <notifications@github.com>:

…

What version of mothur are you using? Could you post the command you ran? — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#187 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AY7v9DjeaovXWB6I6iTQZ2k8AAHoZWAMks5riEt6gaJpZM4G7Jgc> .

-- *Fabrício R. Vieira - Ph.D * *Visiting Research* *Penn State University* *Department of Plant Pathology and Environmental Microbiology*

[mothur-westcott]

We have a version 1.39.3 out with a few bug fixes. You can download it here, https://github.com/mothur/mothur/releases/tag/v.1.39.3. The upgrade shouldn't effect the issue you are having. The map file we have posted on the wiki is for gg_13_5_99.gg.tax. If you are using our map file there will be a mismatch with gg_13_8_99.gg.tax.

[DiliDaly]

Hi Sarah,
I'm also getting the same error when trying to make a biom file for use in PiCRUSt (mothur version 1.39.3, Mac).

I've tried with greengenes 13.8 and then 13.5, but get the same error. This is the command:

make.biom(shared=finalgg135.opti_mcc.0.03.subsample.shared, label=0.03, reftaxonomy=gg_13_5_99.gg.tax, constaxonomy=finalgg135.opti_mcc.0.03.cons.taxonomy, picrust=97_otu_map.txt)

: [ERROR]: could not find OTUId for k__Bacteria(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);. Its reference sequences are .

Within finalgg135.opti_mcc.0.03.cons.taxonomy I find that taxonomy listed 41 times.

Any tips?

Thanks,

Chris

[Vieira34]

Sarah,

Following the Chris tip I opened the cons.taxonomy and removed this OTUs ID (k__Bacteria(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);. Its reference sequences are .) and the biom command runs well. Any idea ?

Thanks,
Fabricio.

[DiliDaly]

Hi Fabricio - deleting those OTUs seemed to work for me too.

Chris

[mothur-westcott]

Thanks for reporting this. I have fixed the issue and the change will be part of our next release. The change is up on the 1.40.0 branch in case you want to pull it and build. Alternatively, If you send me your input files I can run the command for you.

[Vieira34]

Sarah,

Thanks for the effort. If you can run the command for me how can I send to you the input files ...? by email? (if yes, can you provide to me an email that I can use for send the files to you). The dataset that I'm using for this analysis is really small 4 samples (easy to run).

Thanks,
Fabricio.

[mothur-westcott]

You can send them to mothur.bugs@gmail.com :)

[jenlang]

Hello,

It looks like I'm having a similar problem but the origin seems to be in the taxonomy tree? I received files from a collaborator and I'm not very familiar with mothur (guilty of QIIME :) She said she used the standard pipeline to process data. I've tried both the 13_8 and 13_5 .tax files. My code is below. I have no idea how to address the error.

mothur > make.biom(shared=stability.an.0.03.subsample.shared.1000.txt, label=0.03, reftaxonomy=gg_13_8_99.gg.tax, constaxonomy=stability.an.cons.taxonomy.txt, picrust=97_otu_map.txt)

[ERROR]: Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; is not in taxonomy tree, please correct.

Thanks for the help!
Jen

[mothur-westcott]

Did you classify using Green Genes?

Their taxonomy looks like:

k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Comamonadaceae;

[jenlang]

No she used the https://www.mothur.org/wiki/MiSeq_SOP#OTUs, which uses Silva. Would it have to be redone using Green Genes? I thought the point of using the Green Genes files in the make.biom was to convert to Green Genes classification from other classification methods. Did I misunderstand? Thanks, Jennifer

…

On Thu, Apr 27, 2017 at 6:46 AM, Sarah Westcott ***@***.***> wrote: Did you classify using Green Genes? — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#187 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AJkiLHuuEK0gMzumaygxjdJq9mZa_OlOks5r0JxNgaJpZM4G7Jgc> .

-- Jennifer M. Lang, PhD Postdoctoral Researcher Division of Cardiology UCLA Department of Medicine MRL 3535 310-825-5778 (lab) 724-777-3522 (cell)

[mothur-westcott]

The make.biom command does not require a specific reference, unless you are using the picrust option. It does not convert taxonomies, but will include the consensus taxonomy information in the biom OTU metadata if provided. The picrust parameter is used to put this metadata in the form needed by picrust. For example:

Consensus taxonomy:
k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Comamonadaceae;g__;s__;

One of Green Genes Reference Sequences:
6940 k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Comamonadaceae;g__;s__;

Reference maps to Picrust OTUID:
6940 1137725

1137725 may be the reference OTUID for more than one OTU in the shared file. OTUs with the same reference OTUID are combined in the resulting biom file.

When mothur sees the classification of Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae; it does not match any reference sequences in the Green Genes reference, so the OTUIDs cannot be found.

[jenlang]

Oh ok, thanks for the explanation! So just to be explicit, if I want to use the picrust option the OTUs must have been classified with Green Genes? Thanks for the help! Jennifer

…

On Fri, Apr 28, 2017 at 6:34 AM, Sarah Westcott ***@***.***> wrote: The make.biom command does not require a specific reference, unless you are using the picrust option. It does not convert taxonomies, but will include the consensus taxonomy information in the biom OTU metadata if provided. The picrust parameter is used to put this metadata in the form needed by picrust. For example: Consensus taxonomy: k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__ Comamonadaceae;g__;s__; One of Green Genes Reference Sequences: 6940 k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__ Burkholderiales;f__Comamonadaceae;g__;s__; Reference maps to Picrust OTUID: 6940 1137725 1137725 may be the reference OTUID for more than one OTU in the shared file. OTUs with the same reference OTUID are combined in the resulting biom file. When mothur sees the classification of Bacteria;Proteobacteria; Betaproteobacteria;Burkholderiales;Comamonadaceae; it does not match any reference sequences in the Green Genes reference, so the OTUIDs cannot be found. — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#187 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AJkiLB_P5EqSOj2uwb94YK9nSmUHJS2Iks5r0esBgaJpZM4G7Jgc> .

-- Jennifer M. Lang, PhD Postdoctoral Researcher Division of Cardiology UCLA Department of Medicine MRL 3535 310-825-5778 (lab) 724-777-3522 (cell)

[mothur-westcott]

Yes, that's right.

[sandipansamaddar]

Hello,

I am using Mothur version 1.39.0 for processing my sequences.I would like to use PICRUST software for prediction of metabolic potential.On that note,I used the command in the make.biom command page but getting error every time.The command I used is : make.biom(shared=fileList.single.trim.contigs.good.unique.pick.pick.dgc.unique_list.shared, label=0.03, reftaxonomy=gg_13_8_99.gg.tax, constaxonomy=fileList.single.trim.contigs.good.unique.pick.pick.dgc.unique_list.0.03.cons.taxonomy, picrust=97.gg.otu_map)

The error I am getting is : [ERROR]: could not find OTUId for k__Bacteria(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);k__Bacteria_unclassified(100);. Its reference sequences are .

How can I fix the problem? I understand the problem has been discussed above but deleting the row from the cons.tax file shows me another error with another taxonomy.

If you would kindly help me how to proceed it will be very helpful.I am attaching the files used for your kind reference.

Looking forward.

Thanks,

Sandipan

Picrust.tar.gz

[mothur-westcott]

We fixed this bug in version 1.39.5, https://github.com/mothur/mothur/releases/tag/v1.39.5