DNA extraction protocol impacts ocular surface microbiome profile

Abstract

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.

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

“…In a recently published paper by Delbeke et al., 2023, the authors could not generate sequencing libraries for 16S rRNA sequencing when using DNA extraction kits containing a host DNA depletion step ( Delbeke et al., 2023 ). Our data suggests that the OSM can be characterized without the use of 16S rRNA sequencing even on samples where host DNA has been removed during DNA extraction, reinforcing the use of whole-metagenome shotgun sequencing in OSM research.…”

HIF‑1 and macrophage activation signalling pathways are potential biomarkers of invasive aspergillosis

“…The original expression levels from the GSE78000 dataset were standardised using log (1+x). The dissimilarity indices of the standardized samples were calculated using the Bray Curtis method, using the ‘vegdist’ tool ( https://github.com/vegandevs/vegan/ ) ( 17 ). The distance matrix of the dissimilarity index was calculated using the PCoA of the software package ‘ape’ ( http://ape-package.ird.fr/ ).…”

Role of the ocular surface microbiome in allergic eye diseases

“…In general, these techniques analyze genetic material from microbes that are extracted, sequenced, and analyzed using computational tools to provide strain-specific information. Although sensitive to errors and biases such as contamination, these techniques have been shown to reliably enable the study of the OSM [9].…”