PROJECT SUMMARY Overview. This U01 proposal is dedicated to advancing the Human Virome Project (HVP) by addressing several key objectives outlined in RFA-RM-23-018 NOFO. The project's focus is on the development and validation of innovative technologies that enhance rigor and reproducibility of virome discovery and characterization, particularly in human tissue samples. By addressing challenges such as low-biomass sample analysis, host and environmental DNA contamination, and the need for more effective viral quantification and enrichment techniques, this proposal aims to significantly advance the field of virome research. Goals and Objectives. The project is structured around three specific aims: Development of Viral-MEM: An innovative viral enrichment technology that operates independently of viral-like particles (VLP). Viral-MEM is designed to effectively process high-host load tissue samples by removing host nucleic acids while preserving and separating viruses and other microbes. This technology, building on our validated microbial enrichment method, is crucial for deep characterization of viral and bacterial fractions, improving limits of detection in sequencing, and aiding in the identification of novel viruses. Development of Viral StochQuant: A novel experimental and computational approach designed to increase the rigor and reproducibility of viral sequencing. This method uniquely combines sequencing measurements with absolute anchoring measurements to accurately track the absolute numbers of molecules throughout the sequencing process. It addresses the challenges of low target abundance and high background signal, and uses anchoring measurements and stochastic simulations for deriving limits of detection, measurement noise, differential abundance analyses, and contamination detection. Validation of Developed Technologies: Validation will address both biological and technical variabilities and be conducted in three distinct and challenging human tissue sample sets—daily sampled vaginal swabs, saliva samples paired with small-intestine biopsies, and paired biopsies from four locations in the human lower gastrointestinal (GI) tract. This approach will facilitate study of intricate phage-bacterial dynamics, connections between different human viromes, and the quantitative biogeography of the human virome along the GI tract. Impact. The successful implementation of this proposal will dramatically enhance the accuracy, cost- effectiveness, and scalability of virome analyses in human tissues. The technologies developed will enable a more comprehensive integration of virome data with broader human microbiome research and will offer new insights into virome dynamics and interactions. Importantly, these innovations will diversify the HVP's research capabilities, provide access to new sample types, and improve data quality, particularly for low-biomass samples. Overall, this project is designed to provide tools that significantly deepen our understanding of the human virome and its implications in health and disease.

PROJECT NARRATIVE This U01 proposal aims to advance the Human Virome Project by developing and validating innovative technologies that address critical challenges in virome analysis, especially in human tissue samples. Our focus is on the development of Viral-MEM and Viral StochQuant, which are designed to enrich viromes, preserve microbial context, and enhance the accuracy and reproducibility of viral sequencing. These advancements may significantly improve our understanding of the human virome and its interactions with the immune system and the rest of the microbiome, paving the way for new diagnostic and therapeutic approaches.