Benchmarking Quantitative Metagenomics for Wastewater-Based Surveillance of AMR

Wastewater samples from a local VA WWTP were uniformly processed, extracted, and spiked with logarithmically decreasing concentrations of synthetic DNA standards (sequins) to evaluate the quantitative capacity of the Illumina platform. The samples were then used to explore the technical boundaries of wastewater-based surveillance of antibiotic resistance genes by establishing the limit of quantification and detection for the workflow. Each sample was sequenced at approximately 100 Gb to achieve near 100% metagenomic coverage. The dataset includes 10 influent, 10 activated sludge, and 10 secondary effluent samples each spiked with standards in a uniform manner to increase the statistical power.