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Purpose: Corneal ulcers pose a significant threat to vision, with the need for prompt and precise pathogen identification being critical to effective treatment. This study assesses the efficacy of using next-generation portable sequencing (Nanopore Technology) to detect and identify bacterial pathogens directly from tear samples, providing a non-invasive alternative to traditional corneal scraping and culture, which are limited by high false-negative rates., Design: Prospective observational study., Participants: Ten participants diagnosed with corneal ulcers., Methods: Tear samples were collected from the ocular surface using Schirmer strips. Corneal scrapings and cultures were performed as medically indicated. The 16S rRNA gene was amplified directly from the tear samples using polymerase chain reaction (PCR), and Nanopore sequencing was used for bacterial species identification and taxonomic classification. Comparative analysis was conducted to evaluate the concordance between Nanopore sequencing results and traditional culture methods., Main Outcome Measures: Comparison of bacterial species detected via Nanopore sequencing with those identified through traditional culture methods., Results: Bacterial DNA was identified in 8 of the 10 samples analyzed using the tear-based sequencing method. Notably, Nanopore sequencing accurately identified the causative bacteria in all 4 samples that exhibited bacterial growth on culture. Additionally, it detected bacterial pathogens in 2 of the 4 ulcers that did not show bacterial growth on culture. In 2 cases where cultures could not be obtained due to the small size of the ulcer, tear sequencing successfully identified bacterial species, highlighting potentially overlooked pathogens in corneal ulcers., Conclusions: PCR amplification of 16S RNA directly from tears followed by Nanopore sequencing is an effective, non-invasive method to identify bacterial pathogens in corneal ulcers, offering non-inferior results to traditional culture methods. This technique not only allows for the detection of traditionally hard-to-culture organisms, providing immediate diagnostic value to guide treatment, but also enhances our understanding of the microbiological landscape of corneal ulcers, thereby informing more effective treatment strategies.