Group-wise normalization in differential abundance analysis of microbiome samples.

backgroundA key challenge in differential abundance analysis (DAA) of microbial sequencing data is that the counts for each sample are compositional, resulting in potentially biased comparisons of the absolute abundance across study groups. Normalization-based DAA methods rely on external normalization factors that account for compositionality by standardizing the counts onto a common numerical scale. However, existing normalization methods have struggled to maintain the false discovery rate in settings where the variance or compositional bias is large. This article proposes a novel framework for normalization that can reduce bias in DAA by re-conceptualizing normalization as a group-level task. We present two new normalization methods within the group-wise framework: group-wise relative log expression (G-RLE) and fold-truncated sum scaling (FTSS).

resultsG-RLE and FTSS achieve higher statistical power for identifying differentially abundant taxa than existing methods in model-based and synthetic data simulation settings. The two novel methods also maintain the false discovery rate in challenging scenarios where existing methods suffer. The best results are obtained from using FTSS normalization with the DAA method MetagenomeSeq.

conclusionCompared with other methods for normalizing compositional sequence count data prior to DAA, the proposed group-level normalization frameworks offer more robust statistical inference. With a solid mathematical foundation, validated performance in numerical studies, and publicly available software, these new methods can help improve rigor and reproducibility in microbiome research.