vsearch + otu.hierarchy= your file did not include the label ....

Hello,
new to the forum so forgive me if this is not the propper way to query.

I have a large dataset (1 million unique seqs, 1k samples).

I need to use otu.hierarchy for my research (various thresholds),
however it was not possible to generate the distance file to cluster
(well, I wasn´t allowed actually by the computer cluster facility
people as it seemed it was going to take forever and generate a ca.
20T file). After reading your peerJ paper I turned to (previously
following the Miseq SOP) vsearch:

'cluster(fasta=name.unique.good.filter.unique.precluster.pick.pick.fasta,
count=name.unique.good.filter.unique.precluster.uchime.pick.pick.count_table,method=dgc,
cutoff=0.25)
then
‘otu.hierarchy(list=name.unique.good.filter.unique.precluster.pick.pick.dgc.unique_list.list,
label=0.03-0.25)’
Your file does not include the label
0.03. I will use the next smallest distance.
Your file does not include the label
0.25. I will use the next smallest distance.

So it seems that my list file only contained clusters at 0.255.
I then re-run the same cluster command without cutoff, it said 'You did not set a cutoff, using 0.03.'and the apparently only produced OTUs at 0.03

So, is vsearch going to generate OTUs at a single threshold (hence the
cutoff has another meaning as in dist)? or else what am I missing?

If this was the case (I mean vsearch working at individual
thresholds), is there a way I could achieve performing the
otu.hierarchy approach at various labels (appart from running vsearch
at all levels desired and the writting a dedicated script to link OTUs
at different levels)? is it possible to tune otu.hierarchy to link
OTUs at several levels at the same time?

thanks,
Daniel
(and thanks for mothur too!)

Have a look at the subsection “Finer points / Missing Distances” in the cluster wiki. (http://www.mothur.org/wiki/Cluster)
I think that might be your answer.

The vsearch methods just cluster for a specific distance. You would need to run the clustering for the distances you want to input to otu.hierarchy.

mothur > cluster.split(…, cutoff=0.03)
mothur > cluster.split(…, cutoff=0.25)

manually copy OTU 0.25 clusters line into 0.03 file.

mothur > otu.hierarchy(list=name.unique.good.filter.unique.precluster.pick.pick.dgc.unique_list.list,
label=0.03-0.25)