Integrative analysis across metagenomic taxonomic classifiers: A case study of the gut microbiome in aging and longevity in the Integrative Longevity Omics Study

There are various well-validated taxonomic classifiers for profiling shotgun metagenomics data, with two popular methods, MetaPhlAn (marker-gene-based) and Kraken (k-mer-based), at the forefront of many studies. Despite differences between classification approaches and calls for the development of consensus methods, most analyses of shotgun metagenomics data for microbiome studies use a single taxonomic classifier. In this study, we compare inferences from two broadly used classifiers, MetaPhlAn4 and Kraken2, applied to stool metagenomic samples from participants in the Integrative Longevity Omics study to measure associations of taxonomic diversity and relative abundance with age, replicating analyses in an independent cohort. We also introduce consensus and meta-analytic approaches to compare and integrate results from multiple classifiers. While many results are consistent across the two classifiers, we find classifier-specific inferences that would be lost when using one classifier alone. Both classifiers captured similar age-associated changes in diversity across cohorts, with variability in species alpha diversity driven by differences by classifier. When using a correlated meta-analysis approach (AdjMaxP) across classifiers, differential abundance analysis captures more age-associated taxa, including 17 taxa robustly age-associated across cohorts. This study emphasizes the value of employing multiple classifiers and recommends novel approaches that facilitate the integration of results from multiple methodologies.