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A simple method to analyze microbiome beta-diversity computes mean beta-diversity distances from a test sample to standard reference samples. We used reference stool and nasal samples from the Human Microbiome Project and regressed an outcome on mean distances (2 degrees-of-freedom (df) test) or additionally on squares and cross-product of mean distances (5-df test). We compared the power of 2-df and 5-df tests with the microbiome regression-based kernel association test (MiRKAT). In simulations, MiRKAT had moderately greater power than the 2-df test for discriminating skin versus saliva and skin versus nasal samples, but differences were negligible for skin versus stool and stool versus nasal samples. The 2-df test had slightly greater power than MiRKAT for Dirichlet multinomial samples. In associating body mass index with beta-diversity in stool samples from the American Gut Project, the 5-df test yielded smaller P values than MiRKAT for most taxonomic levels and beta-diversity measures. Unlike procedures like MiRKAT that are based on the beta-diversity matrix, mean distances to reference samples can be analyzed with standard statistical tools and shared or meta-analyzed without sharing primary DNA data. Our data indicate that standard reference tests have power comparable to MiRKAT’s (and to permutational multivariate analysis of variance), but more simulations and applications are needed to confirm this.

Power of Microbiome Beta-Diversity Analyses Based on Standard Reference Samples