Current ocular microbiome investigations limit reproducibility and reliability: Critical review and opportunities

Abstract

Enthusiasm for research describing microbial communities using next‐generation sequencing (NGS) has outpaced efforts to standardize methodology. Without consistency in the way research is carried out in this field, the comparison of data between studies is near impossible and the utility of results remains limited. This holds true for bacterial microbiome research of the ocular surface, and other sites, in both humans and animals. In addition, the ocular surface remains under‐explored when compared to other mucosal sites. Low bacterial biomass samples from the ocular surface lead to further technical challenges. Taken together, two major problems were identified: (1) Normalization of the workflow in studies utilizing NGS to investigate the ocular surface bacteriome is necessary in order to propel the field forward and improve research impact through cross‐study comparisons. (2) Current microbiome profiling technology was developed for high bacterial biomass samples (such as feces or soil), posing a challenge for analyses of samples with low bacterial load such as the ocular surface. This article reviews the challenges and limitations currently facing ocular microbiome research and provides recommendations for minimum reporting standards for veterinary ophthalmologists and clinician scientists to limit inter‐study variation, improve reproducibility, and ultimately render results from these studies more impactful. The move toward normalization of methodology will expedite and maximize the potential for microbiome research to translate into meaningful discovery and tangible clinical applications.

Relative and Quantitative Characterization of the Bovine Bacterial Ocular Surface Microbiome in the Context of Suspected Ocular Squamous Cell Carcinoma

“…The taxonomic diversity of ASVs between samples (beta-diversity) is evaluated through various pairwise distance metrics such as weighted and unweighted UniFrac analysis [ 25 ]. The ocular surface is a relatively low biomass environment, and methods routinely utilized for 16S rRNA gene sequencing relative abundance analysis require various modifications to be suitable for this application [ 25 , 26 , 27 ].…”

Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis

“…Variability in sample collector and lack of standardization of specific technique for sample collection due to patient temperament and ulcer stability could all have played a role in differing yields and affected the accuracy of the study results. As recently stated in a paper by Scott et al, written protocols and standardization of aspects of NGS studies, including DNA extraction, will be important to the reproducibility and significance in future research, 56 and therefore, our lack of a specific and standardized protocol for sample collection beyond order of swab collection, number of swabs collected, and location sampled is a limitation of this study. However, the impact on our results is likely limited as these factors should have influenced all samples in a similar manner and does not necessarily explain the difference between bacterial and fungal results.…”

“…While there was variability among samples of time from collection to shipment and processing, there was no significant difference in storage times found between positive and negative bacterial samples (p = .217) or positive and negative fungal samples (p = .884), indicating that storage time did not alter our results. As this was a clinical study, NGS samples were stored in the refrigerator instead of at the −80 degrees Fahrenheit that is currently recommended for DNA samples to prevent the degradation of DNA 51,56 and mailed at room temperature in the shipping box provided by the laboratory, following their instructions. This was considered most feasible in clinical practice; however, further studies that investigate the storage of NGS samples at lower temperatures, or shipping with or without dry ice, are warranted.…”

The respiratory microbiota and its impact on health and disease in dogs and cats: A One Health perspective

“…Additionally, potential contamination associated with sampling methods will have more impact on the composition of microbial communities in samples with lower biomass, as compared to samples with a larger biomass such as feces 37 . For this reason, it is of the utmost importance that reagents and samples taken from bronchoscopes and other instruments are included in the analysis as controls 42 . Other limitations associated with logistics include high cost of sample processing (especially for a small number of samples), limited availability of the technology, variable number of 16S copies per bacteria, and required expertise to analyze and interpret the data.…”

“…37 For this reason, it is of the utmost importance that reagents and samples taken from bronchoscopes and other instruments are included in the analysis as controls. 42 Other limitations associated with logistics include high cost of sample processing (especially for a small number of samples), limited availability of the technology, variable number of 16S copies per bacteria, and required expertise to analyze and interpret the data. Lastly, clinicians are limited by our still nascent appreciation of these microbial communities.…”