Rarefaction is currently the best approach to control for uneven sequencing effort in amplicon sequence analyses.

Sequencing 16S rRNA gene fragments has become a fundamental tool for understanding the diversity of microbial communities and the factors that affect their diversity. Due to technical challenges, it is common to observe wide variation in the number of sequences that are collected from different samples within the same study. However, the diversity metrics used by microbial ecologists are sensitive to differences in sequencing effort. Therefore, tools are needed to control for the uneven levels of sequencing. This simulation-based analysis shows that despite a longstanding controversy, rarefaction is the most robust approach to control for uneven sequencing effort. The controversy started because of confusion over the definition of rarefaction and violation of assumptions that are made by methods that have been borrowed from other fields. Microbial ecologists should use rarefaction.