Classify.seqs output in phylotype analysis

Commands in mothur
1

I am somewhat confused regarding the output of the classify.otu command when performing phylotype analysis. I ran the following commands as suggested on the mothur SOP for Illumina data for phylotyping to genus level:

mothur > phylotype(taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.taxonomy)
mothur > make.shared(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.tx.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table, label=1)
mothur > classify.otu(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.tx.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.taxonomy, label=1)

However, my output file: MOTEALL.trim.contigs.good.unique.good.filter.unique.precluster.pick.gg.wang.pick.tx.1.cons.taxonomy includes multiple species within the same genus, for example:

Otu0458 73 k__Archaea(100);p__Crenarchaeota(100);c__Thaumarchaeota(100);o__Cenarchaeales(100);f__Cenarchaeaceae(100);g__Nitrosopumilus(100);s__pIVWA5(100);
Otu0151 689 k__Archaea(100);p__Crenarchaeota(100);c__Thaumarchaeota(100);o__Cenarchaeales(100);f__Cenarchaeaceae(100);g__Nitrosopumilus(100);unclassified(100);

Shouldn’t these OTUs have been combined into one genus-level Nitrosopumilus OTU? Or is there something that I am not understanding about how the algorithm works?

I apologize if this is a repeated question, the search function for the forum is not working.

2

I’m going a bit from memory here, but I think when you phylotype ‘label=1’ is the deepest tip. So in that case, you would expect two different OTUs because the species label is different. If you repeat the make.shared command with label=2 you should get them collapsed into a genus-level OTU.

3

So that makes sense, but I’m still very confused as to why the SOP says: “The cutoff numbering is a bit different for phylotype compared to cluster/cluster.split. Here you see 1 through 6 listed; these correspond to Genus through Kingdom levels, respectively.” Wouldn’t it make more sense for it to say “Species through Kingdom levels”?

4

dwaite, you were correct, and I was misreading what you were saying. My “deepest tip” as you were saying was actually down to the OTU/species level so by selecting 1 I was binning everything into species. Thank you!

5

Yea, that is a bit confusing. I guess it’s because the SILVA and RDP taxonomies only go to genus, so for them 1 is the genus tip. It’s only Greengenes that adds in the species label.