Microbiome sequencing and methodological challenges: implications for personalized nutrition and medicine

Abstract

This study investigates the impact of sequencing methodologies on microbial composition and functional dynamics in the context of dietary patterns. By analyzing the same stool samples using both 16S rRNA and shotgun metagenomics, we uncovered significant discrepancies in taxonomic resolution, with shotgun metagenomics identifying a broader range of microorganisms, including 22 phyla and 1,489 species, compared to the 15 phyla and 509 species identified by 16S rRNA.Our results highlight the dependency of microbial abundances on sequencing methodology, raising concerns about the generalizability of associations reported in the literature. Notably, despite the herein selected dietary patterns, plant-based and Western, key genera and phyla displayed markedly different relative abundances, illustrating the complexity and variability inherent in microbiome research. Furthermore, we employed metatranscriptomics to differentiate between potential and active metabolic pathways, revealing that not all detected pathways at the DNA level are actively expressed, emphasizing the need for multi-omics approaches in future studies. The limited sample size in our analysis restricts comprehensive comparisons between dietary groups, underscoring the necessity for larger cohort studies. This work calls for standardized protocols in microbiome research and highlights the importance of addressing methodological sequencing biases, which can critically affect interpretations and applications aimed at the development of actionable strategies for personalized nutrition and future health interventions.