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Diane Smith, Suzanne Kennedy, Richard Gibbs, Dan Jones, Cintia De Paiva, Stephen Pflugfelder, Joseph Petrosino; Challenges of Microbiome Research on the Ocular Surface. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3380.
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To apply culture-independent techniques for the characterization of bacterial communities colonizing the ocular surface.
Samples were collected from the right inferior tarsus of three subjects with the Opia EYEPRIM® membrane. Genomic DNA extraction was performed with a modified protocol for the MO-BIO PowerLyzer PowerSoil DNA Isolation Kit on each sample as well as on a blank membrane. Amplification of variable regions V1-V3 of the 16S rRNA gene was performed with the 27F and 534R primers used for the Human Microbiome Project (HMP). The BCM Human Genome Sequencing Center performed Roche 454 pryosequencing on pooled amplicons. Data analysis was performed using mothur.
Working with MO-BIO Laboratories, we developed a method for extracting genomic DNA from low-biomass samples without measureable background. Furthermore, the yield was high enough that whole genome amplification (WGA) was not necessary prior to sequencing. 16S rRNA gene sequencing from EYEPRIM® membranes collected from three subjects produced 130 reads on average that were normalized to 22 reads per sample and classified into 5 phyla (Actinobacteria, Bacteroides, Fusobacteria, Proteobacteria, and Firmicutes). Proteobacteria was the most abundant phylum in all 3 subjects, comprising at least 50% of the microbiota. Sequences classified to the genus level revealed genera such as Propionibacterium, Escherichia, and Staphylococcus, which were previously cultured from the ocular surface.
16S rRNA gene sequencing without WGA identified bacterial genera that include those cultured from the ocular surface. Furthermore, additional genera were also identified as members of the ocular surface microbiota and will be confirmed through longitudinal sampling. Though we were able to perform 16S rRNA sequencing to characterize the microbiota of the ocular surface, the sequencing output was not as robust as with other sample types. Pending whole genome shotgun sequencing will maximize the use of these low-yield samples and characterize species and gene content in the ocular surface microbiome.
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