2.3 Sequence data processing
Primer and adaptor sequences were trimmed using cutadaptimplemented in trimmomatic (27), setting a minimal Phred score of
25. The two fragments of the paired-end reads were merged usingfastq-join with default parameters and processed with the
Quantitative Insights into Microbial Ecology (QIIME2 build 8_2020)
pipeline. In details, the Divisive Amplicon Denoising Algorithm 2
(DADA2) was used to denoise the reads (28), that were subsequently
clustered into operational taxonomic units (OTUs) and taxonomically
classified according to the bacterial Greengenes v.13.8 (29) and fungal
UNITE version 7.0 reference databases (30). OTUs which were present in
over 80% of samples with a minimum abundance of 0.01% were classified
as “core OTUs”. Core OTUs were grouped into three categories, based on
their presence in HC, in VKC or in both groups.
QIIME2 pipeline was used for generating the rarefaction curves and for
calculating the α diversity metrics (diversity of species within an
ecosystem) including the Shannon diversity, Simpson index, observed
number of OTUs and phylogenetic diversity.
Weighted and unweighted UniFrac distances, Bray-Curtis and Jaccard
dissimilarity between samples were calculated and used for investigation
of β diversity (diversity of species between ecosystems) through
plotting principal coordinate analysis (PCoA). To determine the factors
potentially influencing the conjunctival microbiome, bacterial and
fungal communities were compared between groups while controlling for
the other variables (age, sex, type of VKC, IgE sensitization, type of
feeding, previous presence of atopic dermatitis, etc.).