Biological validation of phage host-range identified by proximity guided metagenomics

Abstract Phages are viruses that infect bacteria and have the potential to be used as a clinical tool for treating infectious diseases and gut microbiome-related disorders. Understanding phage-host interactions can improve the ability to use phages as a treatment option, and potentially lead to the development of new therapies. However, current methods for studying these interactions are low-throughput and culture-based, limiting progress in phage therapy research. Proximity ligation sequencing, which uses in vivo crosslinking between phage and host genomes, is a high-throughput sequencing tool which can be used to read out these associations. However, no rigorous experimental validation has yet been performed to verify these interactions, nor to characterize the analytical performance of this approach. The objective of this project is to establish proximity ligation sequencing as a reliable scientific tool for studying phage-host associations. We propose to use a combination of gold-standard microbiological methods and advanced sequencing tools to rigorously validate and define the performance of proximity ligation sequencing for studying phage-host interactions. We will determine the sensitivity and specificity of proximity ligation sequencing using both defined microbial communities and natural fecal microbiome communities. In addition, we will estimate the positive predictive value of proximity ligation-identified phage-host associations in complex microbial communities using phage isolation techniques. These experiments will provide key statistical parameters of reliability, unlocking a high-throughput, culture-independent method of characterizing phage-host interactions. This approach would enable new avenues in phage-based applications, including the development of phage-based therapies for infectious disease, gut dysbiosis, and the targeted manipulation of microbiomes.