Questions about AMOVA analysis

Hi everyone, i performed a 16s rRNA analysis for studying the intestinal microbiome of mice and assess the effect of chronic and metabolic stress in mices. Also i’ taking in consideration the sex.
I have 17 samples:

  • Control Group: 3 males(CM) and 3 females (CH)
    *Group expossed to chronic stress: 3 males (EM) and 3 females (EH)
    *Group for Metabolic stress: 3 males (DM) and 2 females (DH)

And, for assessing effect of the treatments, a teacher suggest me for an ANOVA analysis.

I followed the MiSeq SOP. So i got my distance matrix from the follow code :
mothur > dist.shared(shared=final.opti_mcc.shared)

My design file are in the format .tsv.

It was correct to use this files?

this is what i obtained comparing treatments (control x Stress x Metabolic stress) :

Ctl-Dta-Est Among Within Total
SS 0.892772 5.3959 6.28867
df 2 14 16
MS 0.446386 0.385421

Fs: 1.15818
p-value: 0.003*

Ctl-Dta Among Within Total
SS 0.463626 3.49937 3.96299
df 1 9 10
MS 0.463626 0.388819

Fs: 1.1924
p-value: 0.022

Ctl-Est Among Within Total
SS 0.460655 3.87668 4.33733
df 1 10 11
MS 0.460655 0.387668

Fs: 1.18827
p-value: 0.025

Dta-Est Among Within Total
SS 0.413579 3.41576 3.82934
df 1 9 10
MS 0.413579 0.379528

Fs: 1.08972
p-value: 0.096

What does that abbreviations means… SS, df , MS and Fs ? These results are enough to prove a hypothesis?

I’ve seen from another methods (for example, Rstudio) that i need a Fstat file, or a genind file with my samples information. It is posible to obtain these files from Mothur?


Hi there,

Your design is considerably more complicated than the single classification ANOVA we can do with amova in mothur. I’d strongly encourage you to check out adonis2 in the vegan R package. You can use a distance matrix as input to adonis2. You can see a video I made about using adonis (basically the same interface as adonis2) here:

In our case, SS and MS are the sum squares and mean sum squares, df is the degrees of freedom, and Fs is the F statistic.


Thanks you very much for your help!

I made a mistake writing … sorry :sweat_smile: i mean AMOVA. What i made in Mothur was the AMOVA analysis

But now, i’ve performer your tutorial and have mi results …that’s PerMANOVA, right?
also perform a pairwise analysis with “pairwise.adonis2”

what is the difference between this two methods?
Again, thank you for your help :smiley:

It’s the same basic method but adonis2 allows you to use much more sophisticated experimental designs.


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