Dataset Information

Challenges and insights in the exploration of the low abundance human ocular surface microbiome.

ABSTRACT:

Purpose

The low microbial abundance on the ocular surface results in challenges in the characterization of its microbiome. The purpose of this study was to reveal factors introducing bias in the pipeline from sample collection to data analysis of low-abundant microbiomes.

Methods

Lower conjunctiva and lower lid swabs were collected from six participants using either standard cotton or flocked nylon swabs. Microbial DNA was isolated with two different kits (with or without prior host DNA depletion and mechanical lysis), followed by whole-metagenome shotgun sequencing with a high sequencing depth set at 60 million reads per sample. The relative microbial compositions were generated using the two different tools MetaPhlan3 and Kraken2.

Results

The total amount of extracted DNA was increased by using nylon flocked swabs on the lower conjunctiva. In total, 269 microbial species were detected. The most abundant bacterial phyla were Actinobacteria, Firmicutes and Proteobacteria. Depending on the DNA extraction kit and tool used for profiling, the microbial composition and the relative abundance of viruses varied.

Conclusion

The microbial composition on the ocular surface is not dependent on the swab type, but on the DNA extraction method and profiling tool. These factors have to be considered in further studies about the ocular surface microbiome and other sparsely colonized microbiomes in order to improve data reproducibility. Understanding challenges and biases in the characterization of the ocular surface microbiome may set the basis for microbiome-altering interventions for treatment of ocular surface associated diseases.

SUBMITTER: Herzog EL

PROVIDER: S-EPMC10505673 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Publications

Challenges and insights in the exploration of the low abundance human ocular surface microbiome.

Herzog Elio L EL Kreuzer Marco M Zinkernagel Martin S MS Zysset-Burri Denise C DC

Frontiers in cellular and infection microbiology 20230901

<h4>Purpose</h4>The low microbial abundance on the ocular surface results in challenges in the characterization of its microbiome. The purpose of this study was to reveal factors introducing bias in the pipeline from sample collection to data analysis of low-abundant microbiomes.<h4>Methods</h4>Lower conjunctiva and lower lid swabs were collected from six participants using either standard cotton or flocked nylon swabs. Microbial DNA was isolated with two different kits (with or without prior ho ...[more]

PMID: 37727808

Similar Datasets

Project description:PurposeThe aim of this study is to provide a reference frame to allow the comparison and interpretation of currently published studies on 16S ribosomal ribonucleic acid amplicon sequencing of ocular microbiome samples using different DNA extraction protocols. Alongside, the quantitative and qualitative yield and the reproducibility of different protocols has been assessed.MethodsBoth eyes of 7 eligible volunteers were sampled. Five commercially available DNA extraction protocols were selected based on previous publications in the field of the ocular surface microbiome and 2 host DNA depletion protocols were added based on their reported effective host DNA depletion without significant reduction in bacterial DNA concentration. The V3-V4 region of the 16S rRNA gene was targeted using Illumina MiSeq sequencing. The DADA2 pipeline in R was used to perform the bio-informatic processing and taxonomical assignment was done using the SILVA v132 database. The Vegdist function was used to calculate Bray-Curtis distances and the Galaxy web application was used to identify potential metagenomic biomarkers via linear discriminant analysis Effect Size (LEfSe). The R package Decontam was applied to control for potential contaminants.ResultsSamples analysed with PowerSoil, RNeasy and NucleoSpin had the highest DNA yield. The host DNA depletion kits showed a very low microbial DNA yield; and these samples were pooled per kit before sequencing. Despite pooling, 1 of both failed to construct a library.Looking at the beta-diversity, clear microbial compositional differences - dependent on the extraction protocol used - were observed and remained present after decontamination. Eighteen genera were consistently retrieved from the ocular surface of every volunteer by all non-pooled extraction kits and a comprehensive list of differentially abundant bacteria per extraction method was generated using LefSe analysis.ConclusionHigh-quality papers have been published in the field of the ocular surface microbiome but consensus on the importance of the extraction protocol used are lacking. Potential contaminants and discriminative genera per extraction protocol used, were introduced and a reference frame was built to facilitate both the interpretation of currently published papers and to ease future choice - making based on the research question at hand.

Dataset Information

Challenges and insights in the exploration of the low abundance human ocular surface microbiome.

Purpose

Methods

Results

Conclusion

Publications

Challenges and insights in the exploration of the low abundance human ocular surface microbiome.

Similar Datasets

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