Evaluation of normalization methods for predicting quantitative phenotypes in metagenomic data analysis

Significant advancements have been made in metagenomic research for the prediction of phenotypes based on microbiome data. While qualitative case-control predictions have received significant attention, less emphasis has been placed on predicting quantitative phenotypes. This emerging field holds great promise in revealing intricate connections between microbial communities and host health. However, the presence of heterogeneity in microbiome datasets poses a substantial challenge to the accuracy of predictions and undermines the reproducibility of models. To tackle this challenge, we investigated 22 normalization methods aimed at removing heterogeneity across multiple datasets, conducted a comprehensive review of them, and evaluated their effectiveness in predicting quantitative phenotypes using 3 simulations and 31 real datasets. The results indicate that none of these methods demonstrate significant superiority in predicting quantitative phenotypes or attain a noteworthy reduction in root mean squared error of the predictions. Given the frequent occurrence of batch effects and the satisfactory performance of batch correction methods in predicting datasets affected by these effects, we strongly recommend utilizing batch correction methods as the initial step in predicting quantitative phenotypes. In summary, the performance of normalization methods in predicting metagenomic data remains a dynamic and ongoing research area. Our study contributes to this field by undertaking a comprehensive evaluation of diverse methods and offering valuable insights into their effectiveness in predicting quantitative phenotypes. ### Competing Interest Statement The authors have declared no competing interest.