Alpha diversity command won't run. Error in me metadata file

Hello I am trying to run the alpha diversity command. Here is my code:
qiime diversity alpha-group-significance
--i-alpha-diversity ./core-metrics-results/faith_pd_vector.qza
--m-metadata-file Metadata_colon-distal.tsv
--o-visualization ./core-metrics-results/faiths_pd_statistics.qzv

The error message says that "Metadata does not contain any columns that satisfy this visualizer's requirements. There must be at least one metadata column that contains categorical data, isn't empty, doesn't consist of unique values, and doesn't consist of exactly one value".

My metadata file passes the keemei filter. It is a very simple table, I am practicing with an old dataset of 13 animals, I am analyzing the distal colon microbiome. This is what it looks like:

I have tried adding other columns to the metadata, either numerical (animals 1-13), or categorical (sex: all male) but I recognize it is of no value since they are all the same samples, same treatment.

What I don't understand is that alpha diverstity is supposed to tell you how many different types of ASVs or OTUs there are in a community. So why can't I calculate the alpha diversity in these samples? I have 13 of them, all the same treatment. I want to know how diverse the microbiome is in the distal colon of each of these animals. Later on, when I compute diversity for proximal colon, I will compare these two colon sections, but for now, I just want this metric for this colon segment. I don't understand the message saying that a column cannot "consist of one value".

Sorry this question may be basic but I am new to microbiome analysis. The other related posts (for example kyeong_yun who only had 2 samples) I also don't understand why they could not establish shannon´s diversity index or faith's with 2 samples. We should be able to calculate the diversity for one sample, if wanted.

Thanks for your advice!

PS: I am using the current verson of qiime 2022.2


By running this command, you are actually trying to perform comparisson of alpha diversity.

To compare alpha diversity of 2 or more different groups, you need to have different groups in your metadata file, not only 1 group (treatment).

Right, it should. It is already calculated and you already have it in

file and others. You can export it as tsv tables if needed to check alpha diversity distribution.

In short: you already have it in core-metrics folder (*_vector.qza).


Thanks, this clarifies!
I do have the core-metrics folder!! So that means I should have the combined data from all sections of colon before attemting the alpha diversity command.
Thanks for your response!

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