REVIEW article
Front. Ophthalmol.
Sec. New Technologies in Ophthalmology
This article is part of the Research TopicOcular Surface Disorders- An InsightView all 7 articles
Sequencing the Ocular Surface Microbiome: A Review of Methodological Practices and Considerations
Provisionally accepted
- 1Morgan State University, Baltimore, United States
- 2University of Maryland, College Park, United States
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Purpose: The human ocular surface microbiome (OSM) plays a vital role in ocular health, infection prevention, and immune modulation. However, use of sequencing technology for researching the OSM is challenged by low sample biomass, high sample variability, and methodological inconsistencies. This review systematically evaluates existing literature on OSM research, identifying methodological challenges and proposing standardization strategies to enhance data quality, comparability, and clinical relevance. Methods: A comprehensive analysis of peer-reviewed studies was conducted to assess methodologies used in sequencing-based OSM research, with focus on considerations in scope: sample size, selection, choice of eye, time frame, recruitment and enrollment criteria; sample collection and handling: sampling environment, topical anesthesia, sample collection tools and ocular region; sample preservation: temperature and use of buffers; and sample analysis: DNA extraction, quantification, and sequencing approach. Advantages and limitations of different approaches were identified, and best practices for standardization were explored. Results: This review identified substantial variations in sample collection and processing methodologies, many of which are known to impact OSM composition. However, the influence of certain approaches remains unclear. Additionally, large reporting gaps were observed, as many studies failed to describe critical methodological elements, including specific sample handling procedures and sequencing parameters. Conclusions: While sequencing technologies offer valuable insights, our findings highlight the need for further investigation of different methodological approaches to determine best practices and establish standardized methodological protocols, as well as the need for standardized reporting protocols in OSM research. These standards are essential for enhancing data reliability and translating findings into clinical applications.
Keywords: ocular surface microbiome, Microbiome sequencing, methodologicalstandardization, Minimum reporting guidelines, microbiome reproducibility, ophthalmicmicrobiome
Received: 06 Jul 2025; Accepted: 03 Nov 2025.
Copyright: © 2025 Mehravaran and Pop. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Shiva Mehravaran, shiva.mehr@gmail.com
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.