How sequencing technology shapes our understanding of river water microbiomes and resistomes: a comparative study.

River ecosystems are vital for supporting biodiversity and supplying freshwater, but are increasingly impacted by microbial pollution, including the spread of antibiotic resistance genes (ARGs), which poses growing public health concerns. While high-throughput sequencing technologies have advanced our ability to study microbial communities and resistomes, their varying capabilities and biases require comparative analysis. In this study, we compared three sequencing approaches-Illumina 16S rRNA amplicon, Illumina shotgun metagenomics, and Oxford Nanopore-based long-read metagenomics-to profile microbial communities, ARGs, and virulence factors (VFs) in 48 river water samples. All methods identified Proteobacteria and Actinobacteria as dominant phyla, but substantial differences emerged at finer taxonomic levels. Long-read metagenomics and 16S data showed greater consistency at the genus level, while Illumina metagenomics differed, detecting more potential pathogens and fewer native freshwater taxa. For ARG and VF profiling, unassembled Illumina data yielded higher diversity and abundance, but assembled Illumina data showed comparable results to long-read metagenomics data in terms of dominant genes and host associations. Although Illumina provides high sensitivity, the use of short reads and associated assembly limitations can compromise functional accuracy. In contrast, long-read metagenomics facilitates gene-level resolution and direct host linkage, providing a more comprehensive understanding of environmental microbiomes. Our findings highlight the strengths and limitations of each method and support Oxford Nanopore technology (ONT)-based long-read metagenomic sequencing as a cost-effective and informative tool for high-resolution taxonomic and functional analysis of complex environmental samples.

Importance: Accurate characterization of microbial communities and their functional traits, such as antibiotic resistance, is essential for evaluating water quality and associated public health risks. However, the selection of sequencing methods can substantially influence the detection and interpretation of microbial community composition and functional potential in environmental samples. By directly comparing amplicon, short-read metagenomic, and long-read metagenomic sequencing across 48 freshwater samples collected across different sites and time points, this study builds upon earlier work that typically focused on only two methods or less complex communities. It provides a comparative evaluation of three widely used sequencing approaches, demonstrating how methodological differences affect the resolution and reliability of taxonomic and functional profiling in complex environmental microbiomes. By highlighting the strengths and limitations of each platform, these findings enhance our understanding of how sequencing strategy shapes environmental microbiome analyses and contributes to evidence-based method selection in environmental microbiology and antimicrobial resistance monitoring.