MiSeq SOP failing to generate 97% OTUs

What is the currently recommended method for making 97% OTUs for Illumina reads? I thought it was covered by the MiSeq SOP, but I’m only getting OTUs at 84%.

I was able to run the stability.batch file on the test data without getting any errors, but I did get some warnings.

I expected to find the OTU assignments here:


but there is only one line with label 0.16. The command:

classify.otu(list=current, count=current, taxonomy=current, label=0.03)

gives a warning because there is no label 0.03: “Your file does not include the label 0.03. I will use the next smallest distance. 0.16”

Also, there are discrepancies between the stability.batch file and the text in the wiki page, e.g. the chimera filtering in the batch file is done by chimera.uchime but in the wiki it is chimera.vsearch.

Log file is here:



The change of default method from average neighbor to opticlust means that you now need to set your cutoff to the cluster level you want. The way that opticlust works it doesn’t do all the levels up to your cutoff, it just clusters to the level you selected (I still don’t understand why exactly)

mothur > dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.fasta, cutoff=0.03)
mothur > cluster(column=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.dist, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table)

Thanks, but I don’t understand your response. The text of your message is greek to me, and I don’t see the relevance of the code snippet – the dist.seqs() and cluster() commands do not appear in the stability.batch file from the MiSeq_SOP download. This is for a benchmarking paper, so I want to be sure that I’m doing comparative validation against other methods using the authors’ published / documented / recommended procedures.

I’ve never looked at Pat’s stability.batch file. I wrote my own by going through the SOP. The code that I posed is what you need to replace the line for dist.seqs and cluster in your batch. If you’re wanting to compare to one of his older papers (pre opticlust which came out this year), add “method=average” to the cluster command in the batch

OptiClust showed up in v1.39.5. So if you have an older version of mothur, it will be running average neighbor by default. Here is an expanation of what is happening there:



Pat – I was using v.1.39.5, as you can see from the log file (link in original message above).