Your file does not include the label 0.03. I will use unique.

Commands in mothur
1

I performed cluster command on 16S Illumina MiSeq reads treated through MiSeq SOP protocol. I did cluster with a cutoff=0.03 but at the end it said “changed cutoff to 0.00947584”, Then When I run the classify.otu with my unique_list.list file appear a new message “Your file does not include the label 0.03. I will use unique.” what does it means? Is it a problem or not, are all my unique sequences considered as one unique OTU?

2

Hi,

Please see this:

https://mothur.org/wiki/Frequently_asked_questions#Why_does_the_cutoff_change_when_I_cluster_with_average_neighbor.3F


Also, with the latest release of mothur the default is now the opticlust algorithm, not average neighbor. Using opticlust will make this problem go away and result in improved OTU clusters.

Pat