In the MiSeq SOP step classifying.seqs, if I use Greengenes as the reference database, is the process of classify.seqs:
- Was greengenes used as reference when forming the OTUs i.e. closed ref otu picking or
- Were de novo otus clustered and then annotated against the greengenes database?
Kind of Option 2.
OTUs are constructed de novo in mothur through the (dist.seqs -> cluster -> make.shared) steps. The typical mothur pipeline is to classify all the individual sequences and then assign OTUs a consensus taxonomy of their constituent sequences. If you want to classify the representative OTU sequences themselves, you’d need to do something like the following:
There’s no direct ability to do closed-reference OTU picking in mothur.