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 profiling microbial function.
• Technical variability, including batch effects, complicates large-scale WMS data analysis.
• Systematic evaluation of factors influencing functional inference in WMS is needed.

Purpose of the Study:

• To evaluate the impact of technical factors on functional inference in WMS data.
• To assess the influence of sequencing depth and annotation strategies on microbial function recovery.
• To develop a framework for optimizing WMS study design.

Main Methods:

• Analysis of oral-rinse WMS data from 671 Nigerian youths.
• Annotation of microbial function using mi-faser/Fusion and HUMAnN 3/EC numbers pipelines.
• Quantification of technical factor effects on functional recovery.

Main Results:

• Annotation strategy impacts the breadth and specificity of functional coverage.
• Low-prevalence functions are lost at shallow sequencing depths, affecting study resolution.
• Increasing sequencing depth does not proportionally improve functional recall; significant portions of the functional repertoire remain undetectable.

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.
• Reproducible microbiome insights depend on rigorous methodological planning.