Whole metagenome sequencing: not deep enough for complete microbial function recovery.

Area of Science:

• Microbiome research
• Metagenomics
• Bioinformatics

Background:

• Whole metagenome shotgun sequencing (WMS) is vital for microbial function profiling.
• Technical variability and batch effects in WMS can obscure true biological patterns.
• Systematic evaluation of these factors' impact on functional inference is needed.

Purpose of the Study:

• To systematically evaluate the effects of technical factors and batch effects on functional inference in WMS data.
• To quantify how sequencing depth and annotation strategies shape microbial function recovery.
• To develop a framework for optimizing WMS study design.

Main Methods:

• Analysis of oral-rinse WMS data from 671 Nigerian youths (aged 9-18).
• Sequencing performed on two Illumina platforms.
• Microbial functionality annotated using mi-faser/Fusion and HUMAnN 3/EC numbers pipelines.

Main Results:

• Annotation strategy impacts functional coverage breadth and specificity.
• Low-prevalence functions are lost at shallow sequencing depths, affecting study resolution.
• Increasing sequencing depth does not proportionally improve functional recall; even full depth recovered only an estimated 60% of the functional repertoire.

Conclusions:

• Findings provide practical guidelines for WMS study design and interpretation.
• Coordinating sequencing depth, annotation strategy, and batch control is essential for robust microbial function detection.
• A depth-to-function mapping framework aids in ensuring reproducible microbiome insights.