I haven’t written this up as an SOP yet, but here’s my ITS2 batch file. I wrote this before opticlust came out so it’s using average. Also, I don’t chop the reads to the same length-so I should have ensured that the gap extension penalty was 0 but can’t remember if that’s what average in mothur does or not (Pat or Sarah??). Picking an OTU cutoff is pretty questionable for the rapidly evolving ITS, so I’d do both 3 and 5% and see if you are seeing the same patterns in your communities.
#RDP classifier using silva as the reference (similar results as RDP reference but some are classified to species)
classify.seqs(fasta=current, count=current, reference=UNITEv6_sh_dynamic.fasta, taxonomy=UNITEv6_sh_dynamic.tax, cutoff=60)
Once again displaying my newbie status with regard to ITS, but where did you get the UNITEv6 reference database, and how did you fix it so that MOTHUR could use it?