Alpha and beta diversity analysis problem metadata sample

Hello everyone!

I have a problem to be able to generate my diversity analysis both alpha and beta since I am told that none of the data in my metadate sample file meets the criteria to be able to throw an attached image of the screenshot of the error and my metadata

Hi @Nasute,

Based on your metadata file you only seem to have 4 samples in total, all of which are unique under all the metadata categories. In a statistical sense that means you only have an n=1 in each group and thus non of the statistical tests would make any sense as they cannot calculate means/medians/deviations etc.
You should still be able to construct some PCoA plots but you can’t do any real testing with 1 sample per group.
Perhaps the question you are trying to answer based on your 4 samples need to be revisited?


Hello @Mehrbod_EstakiRegular

Thank you very much for answering I explain: each one of those samples corresponds to four different sites sampled, in each of them it collects insects to which later the DNA extracted from the microbiota and they were marked with primers of RNA16s -Amplicons sequence-, then The question I seek to answer with alpha and beta diversity analysis is to find; In which of the four sites is a greater diversity of bacteria concentrated ?, that is, building a curve of rarefaction per site.

thanks a lot

Got it!
For your rarefaction curves you want to use the qiime diversity alpha-rarefaction command which shouldn’t have any restrictions on n/groups. The alpha group significance command requires more than one sample per site.
As for beta diversity, you’ll want to use one of either beta diversity
or beta diversity (phylogenetic) methods from here then visualize those using the emperor tool.
That should get you what you need, keep in mind however that as mentioned above with 1 sample per site you are not going to be able to calculate any type of meaningful statistics like p-values, effect size, etc.
Let us know if that helps!


Excellent thank you very much! for now I’m just looking for a qualitative description of my data (no significance value)

Thank you very much for the help


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