PROJECT SUMMARY The exposed epithelial surfaces of the body, including the skin, gut, female reproductive tract, and airways, are colonized by microorganisms that contribute to homeostasis and disease. While the resident microbiota has been extensively characterized in many of these sites, the ocular surface microbiome is relatively understudied. The use of metagenomic sequencing approaches has facilitated moving beyond targeted culturing approaches to more fully characterize the breadth of organisms present in a specimen. However, the relative microbial biomass at the ocular surface is much lower than other mucosal surfaces, such as the gut. Metagenomic characterization of low microbial biomass specimens presents numerous challenges, as sources of contamination not only arise during the sampling procedure and from the environment itself, but even from laboratory processing methods. As a result, the lack of protocol standardization and omission of key controls for sources of contamination limits the interpretation and potential for comparison across studies. To address these challenges, we have assembled a large multidisciplinary team of experts in topics including (i) development of standardized protocols and clinical validation of diagnostic tests utilizing metagenomic sequencing for low biomass biospecimens, (ii) development of open source metagenome analysis tools, (iii) clinical assessment of ocular surface and external eye findings among a large, diverse patient population, and (iv) wet lab characterization of microbes under strict cleanliness guidelines. We previously described the use of metagenomic sequencing to detect the presence of pathogens in biopsies from the brain, paraffin embedded ocular tissue specimens, and cerebral spinal fluid (CSF). By comparing a range of specimen pre-treatment and processing approaches and sophisticated software tools, we were able to optimize the methods to maximize organism detection and minimize or remove contamination. We validated our metagenomic sequencing and analytical approaches to the rigor required for use as a diagnostic test. Here, we hypothesize that following similar approaches with low microbial biomass ocular specimens will facilitate the characterization of the healthy ocular surface microbiome. In Aim 1, we will compare specimen processing approaches and validate our analytical methods. In Aim 2, we will compare specimen collection approaches, including collection materials and procedures. We will then use our optimized specimen collection and processing approaches to collect ocular specimens longitudinally for characterization of organism persistence. In Aim 3, we will identify a subset of participants with persistent and/or unique organisms to collect additional specimens for validation of composition and characterization of viability. Upon study completion, we will have contributed to the characterization and understanding of the healthy ocular surface microbiome, and developed proto...