Ocular Surface Microbiome Sample Collection and Processing
STERILE, LYSIS
Before each conjunctival swab, one drop of proparacaine was instilled in both eyes. A sterile solid-core nylon swab (DNA/RNA Shield Swab collection kit; Zymo, Orange, CA, USA) was rotated in the inferior fornix of the left eye, then the right eye, and placed into a bead-beating tube containing 750 µL DNeasy lysis buffer (DNeasy PowerSoil Pro DNA extraction kit; Qiagen, Hilden, Germany) and stored at 4°C until further processing. Several blank swabs with the same proparacaine, swabs, and tubes were performed in room air without a participant present as controls.Genomic DNA was extracted using the DNeasy PowerSoil Pro DNA extraction kit according to the manufacturer's instructions. For each sample, the V1-V3 hypervariable region of the 16S rRNA gene was amplified using primers 27F (5ʹ AGAGTTTGATCCTGGCTCAG 3ʹ) and 534R (5ʹ ATTACCGCGGCTGCTGG 3ʹ) with barcodes to allow multiplex deep sequencing as described previously.