Insights into study design and statistical analyses in translational microbiome studies

Research questions in translational microbiome studies are substantially more complex than their counterparts in basic science. Robust study designs with appropriate statistical analysis frameworks are pivotal to the success of these translational studies. This review considers how study designs can account for heterogeneous phenotypes by adopting representative sampling schemes for recruiting the study population and making careful choices about the control population. Advantages and limitations of 16S profiling and whole-genome sequencing, the two primary techniques for measuring the microbiome, are discussed followed by an overview of bioinformatic processing of high-throughput sequencing data from these measurements. Practical insights into the downstream statistical analyses including data processing and integration, variable transformations, and data exploration are provided. The merits of regularization and ensemble modeling for analyzing microbiome data are discussed along with a recommendation for selecting modeling approaches based on data-driven simulations and objective evaluation. The review builds on several recent discussions of study design issues in microbiome research but with a stronger emphasis on the downstream and often-ignored aspects of statistical analyses that are crucial for bridging the gap between basic science and translation.