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?
Please see this:
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.