Moving from a taxonomic to a functional perspective in global microbiome analysis requires optimizing multiplexing ratios

ABSTRACT Next-generation sequencing has revolutionized microbiome research, yet the transition from taxonomic to functional profiling remains a major technical challenge. While marker gene sequencing provides a widely accessible ecological view, it often lacks the resolution for actionable insights. This perspective argues that shifting to whole metagenomic sequencing is essential for mapping functional potential, such as antimicrobial resistance, and metabolic pathways. However, we identify a critical bottleneck: excessive multiplexing. High multiplexing ratios reduce the number of unique molecules per sample, leading to high duplication rates and the stochastic dropout of low-abundance genes. We demonstrate that functional profiles are far more sensitive to these library complexity issues than taxonomic ones. We recommend prioritizing total sequencing depth and reducing multiplexing to ensure sufficient unique coverage. Additionally, adopting long-read or hybrid architectures is vital for providing the genomic context necessary for strain-level resolution. These optimizations are prerequisites for robust global microbiome synthesis and translational science.