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DNA Viral Size Fraction Metagenomics for Human Stool Samples
Published on: March 24, 2026
Wastewater Metavirome Diversity: Exploring Replicate Inconsistencies and Bioinformatic Tool Disparities
André F B Santos1, Mónica Nunes2, Andreia Filipa-Silva3
1Global Health and Tropical Medicine (GHTM), Associate Laboratory in Translation and Innovation Towards Global Health (LA-REAL), Instituto de Higiene e Medicina Tropical (IHMT), Universidade NOVA de Lisboa, 1349-008 Lisboa, Portugal.
Summary
Wastewater viral analysis using metagenomics showed significant variability due to sample processing and bioinformatics tools. CrAssphage showed potential as a public health surveillance proxy.
Area of Science:
- Environmental microbiology
- Bioinformatics
- Public health
Background:
- Wastewater surveillance is crucial for monitoring public health.
- Metagenomic analysis offers insights into viral communities in wastewater.
Purpose of the Study:
- To evaluate four bioinformatic tools for viral composition analysis in wastewater.
- To assess the impact of sample processing and sequencing on viral community profiling.
- To explore the utility of crAssphage as a public health surveillance marker.
Main Methods:
- Metagenomic sequencing of 32 wastewater samples from two treatment plants.
- Application of four distinct bioinformatic pipelines (Genome Detective, CZ.ID, INSaFLU-TELEVIR, Trimmomatic + Kraken2).
- Development of a custom Python pipeline for automated data processing and analysis.
Main Results:
- Wide variations in viral read counts (3 to 288,464) across 128 sample-tool combinations.
- Inconsistency in viral abundance and diversity between sample replicates and pools, indicating matrix heterogeneity.
- Significant differences in community profiling based on the chosen bioinformatic tool.
- A positive correlation observed between crAssphage abundance and human pathogens.
Conclusions:
- Bioinformatic tool selection and sequencing depth critically influence wastewater viral community analysis.
- CrAssphage shows promise as a reliable indicator for public health surveillance in wastewater.
- Standardized pipelines are essential for reproducible and accurate wastewater-based epidemiology.
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