16S rRNA sequencing of tear film microbiota from FS-LASIK patient F8 (preoperative)
The V3-V4 hypervariable region of the 16S rRNA gene was amplified with primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 806R (5'-GGACTACHVGGGTATCTAAT-3') via a two-round PCR strategy to incorporate sample-specific barcodes. Amplicons were purified with AMPure XP beads, quantified by Qubit fluorometry, and pooled at equimolar concentrations for paired-end sequencing (2 x 250 bp) on the Illumina NovaSeq 6000 platform.