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Though statistical normalizations are often used in differential abundance or differential expression analysis to address sample-to-sample variation in sequencing depth, we offer a better alternative. These normalizations often make strong, implicit assumptions about the scale of biological systems (e.g., microbial load). Thus, analyses are susceptible to even slight errors in these assumptions, leading to elevated rates of false positives and false negatives. We introduce scale models as a generalization of normalizations so researchers can model potential errors in assumptions about scale. By incorporating scale models into the popular ALDEx2 software, we enhance the reproducibility of analyses while often drastically decreasing false positive and false negative rates. We design scale models that are guaranteed to reduce false positives compared to equivalent normalizations. At least in the context of ALDEx2, we recommend using scale models over normalizations in all practical situations.

The human gut teems with a diverse ecosystem of microbes, yet non-bacterial portions of that community are overlooked in studies of metabolic diseases firmly linked to gut bacteria. Type 2 diabetes mellitus (T2D) associates with compositional shifts in the gut bacterial microbiome and fungal mycobiome, but whether T2D and/or pharmaceutical treatments underpin the community change is unresolved. To differentiate these effects, we curated a gut mycobiome cohort to-date spanning 1,000 human samples across 5 countries and a murine experimental model. We use Bayesian multinomial logistic normal models to show that metformin and T2D both associate with shifts in the relative abundance of distinct gut fungi. T2D associates with shifts in the Saccharomycetes and Sordariomycetes fungal classes, while the genera Fusarium and Tetrapisipora most consistently associate with metformin treatment. We confirmed the impact of metformin on individual gut fungi by administering metformin to healthy mice. Thus, metformin and T2D account for subtle, but significant and distinct variation in the gut mycobiome across human populations. This work highlights for the first time that oral pharmaceuticals can confound associations of gut fungi with T2D and warrants the need to consider pharmaceutical interventions in investigations of linkages between metabolic diseases and gut microbial inhabitants., Competing Interests: Disclosure Statement: The authors report there are no competing interests to declare.