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.