Alpha and beta diversity
Alpha diversity was assessed based on species richness in addition to species evenness and richness, to determine the baseline differential characteristics of the EV microbiome in the control and asthma EBC samples (Figure 1A ). The asthma group showed significantly higher alpha diversity (p <0.05) for all four measurements. The observed OTUs and Chao1 estimates showed greater species richness in the asthma EBC samples: Chao1 showed a more moderate increase compared to the observed OTU values. Similarly, the Shannon and Simpson diversity indices both demonstrated higher alpha diversity measures in the asthma group than those in the healthy control group, with the most dramatic increase seen in the Shannon diversity index.
PCoA (based on Bray-Curtis distance of all control and asthmatic patient samples) yielded a primary principal coordinate (PCo1) of 20.13%, while the secondary principal coordinate (PCo2) yielded a value of 8.1% (Figure 1B ). Generally, EBC samples belonging to the healthy control group were clustered below 0.0 along the PCo1 axis and were evenly distributed along the PCo2 axis. Although there were several asthma patient samples in the same cluster of the control group, several asthmatic samples were tightly clustered around the 0.3 value on the PCo1 axis and 0.0 on the PCo2 axis, with only a few control samples included in the cluster. Diversity assessment for the eosinophilic and non-eosinophilic asthmatic patients’ EBC samples yielded no significant differences in any of the four alpha diversity metrics or the PCoA plot for beta diversity (p >0.05) (Figure S1 ).