PROJECT SUMMARY The widespread adoption of inaccurate and biased methods has impeded effective analysis and stymied comprehensive understanding of the composition and dynamics of the human virome. In this project we aim to develop innovative and novel laboratory techniques and computational methods to overcome the major challenges in identifying and characterizing human viruses. Our research team has successfully developed and deployed robust viromics methods for soil microbial communities, leading to a revolution in understanding of soil viral ecology. Preliminary data strongly indicate that these methods are also effective on human stool samples. Here, we will first optimize laboratory methods for recovery and quantification of RNA and DNA viruses from human stool (Aim 1). This aim directly addresses multiple specific interests described in the NOFO by developing techniques for 1) viral isolation and viral detection, 2) viral quantification, 3) viral enrichment for downstream sequencing, 4) eliminating environmental background and contaminating sequences, and 5) proper human sample procurement and storage. We have specific plans to complete this aim by leveraging our research team’s extensive experience in viromics, software development, and the human microbiome. Next, we aim to comparatively evaluate stool viromics techniques by applying them to a longitudinal human cohort (Aim 2). Here we will assess DNA and RNA viromics and other omics techniques for their ability to capture viral dynamics and develop a computational approach to translate the data generated from different methods to enable meta- analyses across studies. This directly addresses the specific interest to develop in silico methods to compare viromes across studies. We will complete this aim by leveraging our research team’s extensive experience developing, publishing, and maintaining user-friendly and highly used software programs. Finally, we aim to extend our technique’s applicability to diverse types of human samples, including those with low biomass (Aim 3). Priority sample types will be chosen in consultation with the full HVP Consortium. This work addresses the key technological and methodological challenges that are currently hindering robust interrogation of the constituents and functionality of the human virome.