CAREER: Quantitative Viral Metagenomics for Water Quality Assessment

目前对微生物水质的监测和监管方法依赖于粪便指示细菌（FIB），由于病毒的代表性不足，无法充分保护公众健康。由于环境中可能存在病毒病原体的多样性（200），用传统方法直接测量病毒病原体往往是不完整的。此外，由于历史上对FIB的依赖，对废水中病毒病原体的存在和多样性的了解相对较少。PI计划开发定量病毒宏基因组学，对病毒DNA/RNA进行测序，用于病毒水质监测。拟议的技术发展不仅将大大促进水质监测，而且还将大大促进其他环境（例如食品安全）中的病毒病原体检测。病毒水质评价的提议也将为学生（高中到研究生院）、水质专业人员和普通大众提供教育发展机会。具体的技术目标包括开发一种快速的病毒分离和浓缩方法，以及一种改进的方法，从非活病毒中去除游离核酸，一个开源的生物信息学管道，从DNA序列数据中定量识别病毒病原体，以及一项关于污水中病毒病原体多样性的全国调查。这些发展可能会导致监测微生物水质的新方法以及病毒水质监测的方法，以阐明水环境中病毒病原体的存在和多样性。具体来说，目前的宏基因组学方法是非定量的（例如，在相对丰度的基础上），并且注释的准确性未经验证；病毒浓度法在实际应用中速度太慢，目前的游离DNA去除方法也不一致。最后，对污水影响系统中的病毒病原体多样性和动态知之甚少。病毒宏基因组学的应用将有助于为疾病生态学的研究提供信息。每项任务都代表了一个独立的进步，其应用范围超出了公共卫生、微生物学和生物信息学领域的水质。此外，生物信息学任务将适用于任何测序平台，并将作为开放获取的生物信息学工具发布，供全世界的研究人员使用。实现定量和独立于目标的病毒检测将有助于改善水质管理，减少对人类健康的总体影响，并为基础设施升级提供信息。具体的教育目标包括为高中生提供一个动手教学模块，以表征微生物水质；为高中生和本科生提供研究经验；扩大目前由项目负责人在卡内基科学中心建立的互动展览；以及通过水环境和再利用基金会举办的年度病毒水质网络研讨会。这一努力将导致发布一个开源的生物信息学病毒注释工具和一个用于高中课堂的教育模块，将技术和教育发展的影响扩展到当前项目范围之外。

PI Name: BibbyProposal Number: 1653356 Current monitoring and regulatory approaches for microbial water quality, relying on the Fecal Indicator Bacteria (FIB), insufficiently protect public health due to a poor representation of viruses. Direct measurement of viral pathogens by conventional means is often incomplete due to the diversity of viral pathogens (200) that may be in the environment. In addition, due to a historical reliance on FIB, there is relatively little knowledge regarding viral pathogen presence and diversity in wastewater. The PI plans to develop quantitative viral metagenomics, sequencing of viral DNA/RNA, for viral water quality monitoring. The proposed technical developments will significantly advance not only water quality monitoring but also viral pathogen detection in other settings (e.g., food safety). The proposed efforts in viral water quality assessment will also offer an educational development opportunity for students (high school through graduate school), water quality professionals, and the general public. Specific technical goals include developing a rapid viral isolation and concentration approach and an improved methodology to remove free nucleic acids from non-viable viruses, an open-source bioinformatics pipeline to quantitatively identify viral pathogens from DNA sequence data, and a national survey of viral pathogen diversity in sewage. These developments may lead to new methods for monitoring microbial water quality as well as a methodology for viral water quality monitoring to elucidate the presence and diversity of viral pathogens in the water environment. Specifically, current metagenomics methods are applied non-quantitatively (e.g., on a relative abundance basis) and annotation accuracy is unverified; viral concentration methods are too slow for practical application and current methods for free DNA removal are inconsistent. Finally, little is known about viral pathogen diversity and dynamics in wastewater impacted systems. Application of viral metagenomics will help to inform investigations into the ecology of disease. Each task represents a discrete advance with applications beyond water quality in public health, microbiology, and bioinformatics. Additionally, bioinformatics tasks will be applicable to any sequencing platform and will be released as an open-access bioinformatic tool for use by researchers worldwide. Enabling quantitative and target-independent virus detection will facilitate improved water quality management, reducing overall human health impacts and informing infrastructure upgrades. Specific educational goals include a hands-on educational module for high school students to characterize microbial water quality, research experiences for high school and undergraduate students, expanding a current interactive exhibit established by the project PI at the Carnegie Science Center, and annual viral water quality webinars to be conducted through the Water Environment and Reuse Foundation. This effort will result in the release of an open-source bioinformatics viral annotation tool and an educational module for the high school classroom, extending the impact of technical and educational developments beyond the immediate project scope.