Rarefaction is better than robust Aitchison PCA and other compositional data analysis methods at controlling for uneven sequencing effort.

Rarefaction outperforms robust Aitchison PCA and other compositional methods in controlling for uneven sequencing effort in microbial studies.

• Why it matters: Accurately accounting for variation in sequencing depth is crucial for valid microbial community analyses, yet existing methods like robust Aitchison PCA have been questioned for their effectiveness.
• What they did: The author re-evaluated benchmarking data, corrected coding errors, expanded datasets, and compared multiple normalization approaches, including rarefaction and compositional methods.
• The result: Findings show rarefaction remains the most effective method, enabling more reliable interpretations of microbial community structures despite the popularity of alternative compositional techniques.