摘要
      当前标准微生物组研究中相对丰度数据的组成性质限制了微生物动力学解释和跨样本比较。在这里，我们展示了第一种将纳米孔宏基因组测序与细胞刺突蛋白偶联的快速（1小时测序）方法，以促进废水处理厂（WWTP）中病原体和抗生素抗性基因（ARGs）的绝对定量和去除评估。简单模拟社区和复杂真实环境样本的基于纳米孔测序的量化结果与广泛使用的Illumina和基于培养的方法的结果高度一致。通过实施这种方法，我们在三个污水处理厂样本集中量化了46种主要的假定致病物种和361种ARG。尽管优势病原体（2.23个对数）和ARGs（1.98个对数）的去除率很高，但并没有完全去除所有病原体和ARGs。值得注意的是，分枝杆菌、产气荚膜梭状芽孢杆菌和热疏螺旋体的去除率较低，治疗后13个ARG的绝对丰度甚至增加。我们提出的方法在提供绝对定量信息指导基于废水的流行病学监测和定量风险评估以促进微生物危害管理方面表现出了强大的能力。
Abstract
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.

https://www.sciencedirect.com/science/article/abs/pii/S0048969721072661