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Constructed wetlands (CWs)Constructed wetlands (CWs) offer a sustainable, nature-based solution to wastewater treatmentWastewater treatment, supporting diverse and dynamic microbial communitiesMicrobial communitiesMicrobial communities that drive nutrient cycling, pollutant degradation, and pathogen removal. This chapter presents an end-to-end methodology for performing shotgun metagenomicShotgun metagenomics analyses on microbial populations from CWConstructed wetland (CW) influent and effluent. We detail approaches for site selection, sample collection, filtration, DNA extraction, and the incorporation of positive and negative controls to ensure reproducibility and data quality. Two modular bioinformatic workflows encompassing quality controlQuality control (QC), assembly, taxonomic/functional annotation, and metagenome-assembled genomeMetagenome-assembled genome (MAG) recovery are described alongside options for detecting antimicrobial resistanceAntimicrobial resistance (AMR) genes, pathogens, toxins, and plasmids. In addition, an example workflow for the calculation of alpha and beta diversity is provided. Guidelines for data standardization, replication, and compliance with community-driven reporting standards (MIMS, MIMAG) are also included. Incorporating this protocol will facilitate standardized, reproducible insights into CWConstructed wetland (CW) microbial dynamics, thereby informing ecological understanding and guiding practical interventions that enhance wastewater treatmentWastewater treatment efficacy and improve public health outcomes.

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Shotgun Metagenomic Analysis of Microbial Community Dynamics in Wastewater Treatment Through Constructed Wetlands

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  • 发表日期：

    2026年

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  • Georgios Miliotis,
  • Anna Tumeo

摘要

Constructed wetlands (CWs)Constructed wetlands (CWs) offer a sustainable, nature-based solution to wastewater treatmentWastewater treatment, supporting diverse and dynamic microbial communitiesMicrobial communitiesMicrobial communities that drive nutrient cycling, pollutant degradation, and pathogen removal. This chapter presents an end-to-end methodology for performing shotgun metagenomicShotgun metagenomics analyses on microbial populations from CWConstructed wetland (CW) influent and effluent. We detail approaches for site selection, sample collection, filtration, DNA extraction, and the incorporation of positive and negative controls to ensure reproducibility and data quality. Two modular bioinformatic workflows encompassing quality controlQuality control (QC), assembly, taxonomic/functional annotation, and metagenome-assembled genomeMetagenome-assembled genome (MAG) recovery are described alongside options for detecting antimicrobial resistanceAntimicrobial resistance (AMR) genes, pathogens, toxins, and plasmids. In addition, an example workflow for the calculation of alpha and beta diversity is provided. Guidelines for data standardization, replication, and compliance with community-driven reporting standards (MIMS, MIMAG) are also included. Incorporating this protocol will facilitate standardized, reproducible insights into CWConstructed wetland (CW) microbial dynamics, thereby informing ecological understanding and guiding practical interventions that enhance wastewater treatmentWastewater treatment efficacy and improve public health outcomes.