A simple statistical test of taxonomic or functional homogeneity using replicated microbiome sequencing samples

- A statistical test of homogeneity of microbiome sequencing data is proposed.
- The test is applicable to both taxonomic and functional profiles.
- The test is easy to perform and has a rigorous statistical basis.
- We illustrate the test on human feces samples, proving rejecting homogeneity on the cm scale.
- An implementation is provided in the open source software MEGAN CE.

One important question in microbiome analysis is how to assess the homogeneity of the microbial composition in a given environment, with respect to a given analysis method. Do different microbial samples taken from the same environment follow the same taxonomic distribution of organisms, or the same distribution of functions? Here we provide a non-parametric statistical “triangulation test” to address this type of question. The test requires that multiple replicates are available for each of the biological samples, and it is based on three-way computational comparisons of samples. To illustrate the application of the test, we collected three biological samples taken from different locations in one piece of human stool, each represented by three replicates, and analyzed them using MEGAN. (Despite its name, the triangulation test does not require that the number of biological samples or replicates be three.) The triangulation test rejects the null hypothesis that the three biological samples exhibit the same distribution of taxa or function (error probability ≤0.05), indicating that the microbial composition of the investigated human stool is not homogenous on a macroscopic scale, suggesting that pooling material from multiple locations is a reasonable practice. We provide an implementation of the test in our open source program MEGAN Community Edition.