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Nabeel M Shalabi, Alexander I Tuzhikov, Anat Galor, Qunfeng Dong, Alexander U Panchin, Onsiri Thanathanee, Russell Van Gelder, Terrence Patrick O'Brien, Valery Shestopalov; Experimental approaches in the analysis of microbial community in ocular samples. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6288.
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Indigenous microflora can contribute to ocular surface (OS) infections, the major causes of corneal blindness in the US and world-wide. We developed and tested experimental paradigm for the analysis of microbial composition on the human OS using next generation DNA sequencing (NGS).
Samples of cornea epithelial sheaths were collected from post-surgical material; conjunctiva was sampled using Dacron cotton swabs. Swabbing of the bulbar conjunctiva and both fornices was used for OS samples, skin under the eyelid served as non-OS control. Samples were placed into AssyAssure buffer and stored at -80 C° degree. Total genomic DNA (gDNA) was extracted using MO BIO Ultra Clean Microbial DNA or Qiagen QIAamp DNA isolation kits, tested for gDNA quality, concentration and 16S gene abundances and then processed for a16S amplicon library construction, and sequencing. We utilized 454 Roche sequencing of 16S rRNA gene and BRiSK to analyze the OS microbiome. Sequencing reads were processed with MOTHUR, taxonomic assignment for the filtered sequences was performed with RDP II Classifier and a novel TUIT algorithm that utilizes GenBank database. BRiSK analysis was performed using custom built scripts and databases. The reconstruction of ocular surface microbiomes allowed us to analyze diversity, relative abundance and perform between-group comparisons to assess gender- and infection-related changes. Visual comparison of the groups was performed with MEGAN4.
Significant percentage of eyes showed detectable levels f 16S rDNA by 454 and DNA for viruses and other microbiota by BRiSK sequencing. Bacterial composition in OS samples significantly differed from those in skin samples; the composition on healthy OS differed significantly from that on corneas with keratitis. Individual variation was very high, but same-person eyes showed high level of concordance in microbiome composition. Between gender differences were insignificant. BRiSK results corroborated well with 16S sequence data and were instrumental in detecting phage, fungal and protozoan species.
We have developed and successfully tested experimental protocol for collecting, processing and analysis of microbiota in the human OS samples using NGS. Our procedure allows reliable sample collection and processing for NGS-based analysis of metagenome and microbiome composition.
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