Project Summary/Abstract: The project objective is to develop an organism-agnostic deep sequencing-based diagnosis of ocular infectious disease from microliter-sized biopsies in less than 24 hours and for less than $1000. Current diagnostic approach relies on bacterial and fungal cultures that are time-consuming (~2-3 days) and often leads to the overuse of broad-spectrum antibiotics. In Phase I, we successfully developed and validated the EYE-Path™ system, demonstrating an integrated Scalable Metagenomics Alignment Research Tool (SMART) with an automated DNA extraction system for sample preparation. This system achieved a low limit of detection (104 cells/mL) and a turnaround time of <3 hours for bacterial infections. In Phase II, we plan to optimize the adaptive sequencing software for the SMART Adapt algorithm, validate the system in a multi-site clinical study (University of California, Los Angeles and University of Washington) in patients with corneal ulcer and suspected endophthalmitis, validate the system as a CLIA ready test and initiate a pre-submission meeting with the FDA to determine the appropriate regulatory strategy for eye-PATH to be considered as a CLIA waived in vitro-diagnostic device. The success completion of this project could significantly impact ocular infection diagnosis and treatment planning, with broader applications in point-of-care devices and non-ophthalmic diagnostics, helping to combat antimicrobial resistance.