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Modern Molecular Taxonomy01:29

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Whole Metagenome Sequencing: not Deep Enough for Complete Microbial Function Recovery.

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    Whole metagenome shotgun sequencing (WMS) technical variability can obscure microbial function. Optimizing sequencing depth and annotation strategy is crucial for accurate microbiome functional inference and reproducible results.

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    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.