LAUSR

Background Since sequencing techniques have become less expensive, larger sample sizes are applicable for microbiota studies. The aim of this study is to show how, and to what extent, different diversity metrics and different compositions of the microbiota influence the needed sample size to observe dissimilar groups. Empirical 16S rRNA amplicon sequence data obtained from animal experiments, observational human data, and simulated data were used to perform retrospective power calculations. A wide variation of alpha diversity and beta diversity metrics were used to compare the different microbiota datasets and the effect on the sample size. Results Our data showed that beta diversity metrics are the most sensitive to observe differences as compared with alpha diversity metrics. The structure of the data influenced which alpha metrics are the most sensitive. Regarding beta diversity, the Bray–Curtis metric is in general the most sensitive to observe differences between groups, resulting in lower sample size and potential publication bias. Conclusion We recommend performing power calculations and to use multiple diversity metrics as an outcome measure. To improve microbiota studies, awareness needs to be raised on the sensitivity and bias for microbiota research outcomes created by the used metrics rather than biological differences. We have seen that different alpha and beta diversity metrics lead to different study power: because of this, one could be naturally tempted to try all possible metrics until one or more are found that give a statistically significant test result, i.e., p-value < α. This way of proceeding is one of the many forms of the so-called p-value hacking. To this end, in our opinion, the only way to protect ourselves from (the temptation of) p-hacking would be to publish a statistical plan before experiments are initiated, describing the outcomes of interest and the corresponding statistical analyses to be performed.