Classify OTU Labels

Theory behind mothur
1

I keep coming across an issue at the classify OTU step.

The label is set at 0.03 to determine a 97% similarity between OTUS.
After I run the command (classify.OTU) mothur output says “Your file does not include the label 0.03. I will find the next smallest label 0.16”
This set alls of my data to 84% similarity. I’m not sure what I can do in the earlier steps to fix this problem. Does anyone have any idea what would cause this to happen?

Further Information:
I am using rumen microbiome data that is sequenced from the v4 region of 16sRNA

2

which version are you using? what is your clustering command?

3

I am using version 1.39.1 and my cluster command is as follows:
cluster.split(fasta=rumenAB.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=rumenAB.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.pick.count_table, taxonomy=rumenAB.trim.contigs.good.unique.good.filter.unique.precluster.pick.rdp.wang.pick.taxonomy, splitmethod=classify, taxlevel=4, cutoff=0.15)

I’m still extremely new to mothur so I appreciate any advice or ideas as to what I may be doing wrong. Thanks!

4

Ah the new clustering method has changed a lot of the clustering command. try this instead

dist.seqs(fasta=rumenAB.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta,  countends=F, cutoff= 0.03)
cluster(column=current, count=rumenAB.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.pick.count_table, method=opti)
5

I tried this and it worked perfectly!

Thank you for your help!