Long-read nanopore shotgun metagenomic DNA sequencing for river biodiversity, wildlife, pollution, and environmental health monitoring.

As the human population expands and global temperatures rise, species, populations, and biodiversity decline at unprecedented rates, while the frequency of infectious disease emergence increases. Therefore, it is more vital than ever to accurately understand the current state of natural habitats and their constituent species. We assess the feasibility of a single assay: long-read shotgun metagenomic sequencing of environmental DNA (eDNA), to monitor species from across the tree of life, from viruses to complex multicellular organisms, across a representative Irish river system (Avoca River, Co. Wicklow). We conducted aquatic eDNA sampling and long-read shotgun metagenomic sequencing from a mountain tributary through to the sea. This approach could detect and quantify organismal DNA present in environmental samples, from microbes (including DNA viruses) to mammals. Rather than the traditional siloing of microbial and multicellular studies of DNA recovered from environmental samples, simultaneously considering viruses, microbes, and eukaryotes (animals, plants, and fungi) can provide deeper insights. This single assay can simultaneously quantify differences in DNA abundance for a broad range of species and pathogens across sites and sample types, enabling wide-ranging biodiversity assessments. This included human, wildlife, plant, and microbial pathogens and parasites with health, agricultural, and economic importance. The environmental genomic data enabled animal phylogeny and transmissible cancer analysis (blue mussel,