Can I perform ANCOM with 2 samples?

Hi everyone!

Im using qiime2-2020.8 on conda.

Im trying to perform ANCOM analysis to compare two samples, from high and low salinity. When I try to do so, I get the following error.

qiime composition ancom
–i-table 3.4-7.7/comp-k-3.4-7.7-18S-table.qza
–m-metadata-file …/Metadata.tsv
–m-metadata-column Salinity
–o-visualization 3.4-7.7/ancom-k.qzv

Plugin error from composition:

All values in grouping are unique. This method cannot operate on a grouping vector with only unique values (e.g., there are no ‘within’ variance because each group of samples contains only a single sample).

From this I understand that perhaps you can’t peform this analysis with only two samples.

Is my assumption correct?


Hi Jose,

You’re exactly right! As most (if not all) statistical tests, ANCOM has no statistical power with only one sample per group. If you want to visualize differences between groups, I would recommend simply plotting the taxa relative abundance per sample. This will not give you conviction about your results, but it sure is a good descriptive tool for this limited dataset.

Good luck,



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