Design file for beta diversity

Hey dear mothur community,
I hope somebody can help me realize where the error is. Hope enough detail is provided.
I am using the last version to analyze V4 18S MiSeq samples, based on MiSeq SOP. I have 15 samples, group in 5 sets of three samples each. I want to compare OW/SI and ACC/BS/WASW, but after merging my design file is not recognized correctly and mothur crashes.
After getting my occurrence table (create.database) I subsampled the shared file and merged groups according to my design file to run AMOVA and Unifrac.
Here is how my commands look like:
make.shared(list=18Sfcm.pick.pick.filter.opti_mcc.unique_list.0.02.pick.list, count=18S5fcm.pick.pick.pick.count_table, label=0.02)
sub.sample(shared=18Sfcm.pick.pick.filter.opti_mcc.unique_list.0.02.pick.shared, size=89233)
dist.shared(shared=18Sfcm.pick.pick.filter.opti_mcc.unique_list.0.02.pick.0.02.subsample.merge.shared, calc=braycurtis, output=lt)
clearcut(, verbose=t)

When I try to run:
unifrac.unweighted(, random=F, groups=ACC-BS-WASW,

It send an error claiming that my design file does not include ‘ACC’, etc.

Design file:
group categorie
12_0207.15 SI
12_0409.15 SI
12_0509.15 SI
12_0202.15 OW
12_0505.15 OW
12_0701.15 OW
13_0502.15 ACC
13_0701.15 ACC
13_0902.15 ACC
13_0201.15 WASW
13_1107.15 WASW
13_1909.15 WASW
13_0808.15 BS
13_0909.15 BS
13_1314.15 BS

Please, if anybody can give me guidance on what can be going wrong I will greatly appreciate it.

Again, thanks,


Can you post the contents of

Thanks Pat,

My distance file is:

BS 0.492949
OW 0.349359 0.478579
SI 0.518209 0.394409 0.358466
WASW 0.276381 0.416386 0.389086 0.486165


I think you’re using the wrong distance matrix. Instead of having ACC, BS, OW, etc in the first column, it should have 12_0207.15, 12_0409.15, 12_0509.15, etc.


Thanks again Pat!
Using the not merged shared file cleared the confusion with the groups and I have been able to do the analysis.