2.3. DNA isolation and metagenomic shotgun sequencing
The DNA isolation and metagenomic shotgun sequencing of the AC samples was performed as previously described.10,12 In brief, DNA was extracted from each conjunctival sample using the MasterPure Complete DNA and RNA Purification Kit (Epicentre) according to the manufacturer’s instructions. Sequencing libraries were prepared using the VAHTS Universal DNA library Prep Kit for Illumina (Vazyme, Nanjing, China) and quantified by qPCR using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA). Paired-end 2×150-bp sequencing was performed on a NovaSeq 6000 instrument (Illumina, San Diego, CA, USA). Three negative blank controls, which had reagents from DNA extraction through sequencing, were processed along with the AC samples.