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

Constructed wetlands (CWs) offer a sustainable, nature-based solution to wastewater treatment, supporting diverse and dynamic microbial communities that drive nutrient cycling, pollutant degradation, and pathogen removal. This chapter presents an end-to-end methodology for performing shotgun metagenomic analyses on microbial populations from 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 control, assembly, taxonomic/functional annotation, and metagenome-assembled genome recovery are described alongside options for detecting antimicrobial resistance 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 CW microbial dynamics, thereby informing ecological understanding and guiding practical interventions that enhance wastewater treatment efficacy and improve public health outcomes.