[ERROR]: Bacteria;Firmicutes;Firmicutes_ is not in taxonomy tree, please correct.

[ayitbarek]

Hey rprops,

Thanks for the codes!

I keep getting the following error when I run make.biom: [ERROR]: Bacteria;Firmicutes;Firmicutes_ is not in taxonomy tree, please correct.

What might be the problem for getting the above error?

Thanks, Alex

[rprops]

Hi Alex,

I'm guessing the issue lies in the formatting of your taxonomy file. Could you provide me the first 10 lines of your taxonomy output by running the following one liner:

head -100 stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.an.unique_list.0.03.cons.taxonomy

Also did you classify your sequences in mothur, and if so with which version/database (e.g. SILVA vxxx)?

Thanks

Ruben

[ayitbarek]

Hi Ruben,

Here is the first 10 lines;

OTU Size Taxonomy Otu000001 105128 Bacteria(100);Firmicutes(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100); Otu000002 52120 Bacteria(100);Firmicutes(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100); Otu000003 51311 Bacteria(100);Bacteria_unclassified(100);Bacteria_unclassified(100);Bacteria_unclassified(100);Bacteria_unclassified(100);Bacteria_unclassified(100); Otu000004 44650 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);Ruminococcaceae(100);Ruminococcaceae_unclassified(100); Otu000005 40984 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);Clostridiales_unclassified(100);Clostridiales_unclassified(100); Otu000006 33963 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);Peptostreptococcaceae(100);Clostridium_XI(96); Otu000007 32443 Bacteria(100);Bacteria_unclassified(100);Bacteria_unclassified(100);Bacteria_unclassified(100);Bacteria_unclassified(100);Bacteria_unclassified(100); Otu000008 28016 Bacteria(100);Proteobacteria(100);Epsilonproteobacteria(100);Campylobacterales(100);Campylobacteraceae(100);Campylobacter(100); Otu000009 27421 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);Ruminococcaceae(100);Faecalibacterium(100); Otu000010 23700 Bacteria(100);Firmicutes(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100);Firmicutes_unclassified(100);

I used SILVA V4 that I got from the Miseq SOP site.

Thank you so much, Alex

[rprops]

Hi Alex,

I believe the issue is situated in the way mothur currently reports the taxonomy of unclassified levels. Previously these were just called "unclassified" while now the deepest known taxonomy is attached to that name, e.g. "Clostridiales_unclassified". Can you replace all of these new unclassified names to the old "unclassified" status: "Clostridiales_unclassified" -> "unclassified" You can quickly do that in R or unix. Let me know if you need help with that.

I believe this should fix the issue you're having with make.biom.

Ruben

[ayitbarek]

Hi Ruben,

I tried it by changing "*_unclassified" to "unclassified". Here are the first 10 lines; OTU Size Taxonomy Otu000001 105128 Bacteria(100);Firmicutes(100);unclassified(100);unclassified(100);unclassified(100);unclassified(100) Otu000002 52120 Bacteria(100);Firmicutes(100);unclassified(100);unclassified(100);unclassified(100);unclassified(100) Otu000003 51311 Bacteria(100);unclassified(100);unclassified(100);unclassified(100);unclassified(100);unclassified(100) Otu000004 44650 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);Ruminococcaceae(100);unclassified(100) Otu000005 40984 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);unclassified(100);unclassified(100) Otu000006 33963 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);Peptostreptococcaceae(100);Clostridium_XI(96) Otu000007 32443 Bacteria(100);unclassified(100);unclassified(100);unclassified(100);unclassified(100);unclassified(100) Otu000008 28016 Bacteria(100);Proteobacteria(100);Epsilonproteobacteria(100);Campylobacterales(100);Campylobacteraceae(100);Campylobacter(100) Otu000009 27421 Bacteria(100);Firmicutes(100);Clostridia(100);Clostridiales(100);Ruminococcaceae(100);Faecalibacterium(100) Otu000010 23700 Bacteria(100);Firmicutes(100);unclassified(100);unclassified(100);unclassified(100);unclassified(100)

However, I still get the following error; [ERROR]: Bacteria;Firmicutes; is not in taxonomy tree, please correct.

Sorry to bother you, and I really appreciate your help.

Alex

[rprops]

Hi Alex,

It only just hit me that you need to classify your sequences with the Greengenes taxonomy database and not SILVA. So just rerun the classification with Greengenes and make.biom should work.

[ayitbarek]

Thanks Ruben! I will try that.