Rapid absolute quantification of pathogens and ARGs by nanopore sequencing

Compositional nature of relative abundance data in the current standard microbiome studies limits microbial dynamics interpretations and cross-sample comparisons. Here, we demonstrate the first rapid (1-h sequencing) method coupling Nanopore metagenomic sequencing with cellular spike-in to facilitate the absolute quantification and removal assessment of pathogens and antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs). Nanopore sequencing-based quantification results for both simple mock community and complex real environmental samples showed a high consistency with those from the widely-used Illumina and culture-based approaches. Implementing such method, we quantified 46 predominant putative pathogenic species, and 361 ARGs in three WWTP sample sets. Though high log removals of dominant pathogens (2.23 logs) and ARGs (1.98 logs) were achieved, complete removal of all pathogens and ARGs were not achieved. Noticeably, Mycobacterium spp., Clostridium_P perfringens, and Borrelia hermsii exhibited low removal, and 13 ARGs even increased in absolute abundance after the treatment. Our proposed approach manifested its profound ability in providing absolute quantitation information guiding wastewater-based epidemiological surveillance and quantitative risk assessment facilitating microbial hazards management.