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

Area of Science:

• Microbiome Research
• Metagenomics
• Genomic Sequencing

Background:

• Next-generation sequencing transformed microbiome studies, but functional profiling remains challenging.
• Marker gene sequencing offers ecological insights but lacks resolution for actionable data.
• Whole metagenomic sequencing is vital for understanding functional potential, including antimicrobial resistance and metabolic pathways.

Purpose of the Study:

• To highlight the technical challenges in transitioning from taxonomic to functional microbiome profiling.
• To identify library complexity and excessive multiplexing as critical bottlenecks in whole metagenomic sequencing.
• To propose solutions for improving the accuracy and resolution of functional microbiome analysis.

Main Methods:

• Comparative analysis of marker gene sequencing versus whole metagenomic sequencing.
• Evaluation of the impact of multiplexing ratios on library complexity and gene detection.
• Assessment of sequencing depth and read length on functional profile accuracy.

Main Results:

• Functional microbiome profiles are more sensitive to library complexity issues than taxonomic profiles.
• High multiplexing ratios lead to increased duplication rates and stochastic gene dropout, particularly for low-abundance genes.
• Inadequate unique molecular coverage significantly compromises the reliability of functional gene detection.

Conclusions:

• Prioritizing total sequencing depth and reducing multiplexing are essential for robust functional microbiome profiling.
• Adopting long-read or hybrid sequencing technologies is necessary for achieving strain-level resolution and genomic context.
• Optimizing sequencing strategies is a prerequisite for advancing global microbiome synthesis and translational applications.