Clustering analysis of background, supraglotic, and BAL microbiota patterns. (A) Heat map of unsupervised hierarchical clustering of most abundant OTUs at a genus level (relative abundance ≥5% in any sample) in background and supraglottic samples. Background microbiome (sterile saline and saline through bronchoscope, see Additional file 1: Figure S1 for details) is enriched with Staphylococcus, Propionibacterium, and Corynebacterium, while the supraglottic microbiome is enriched with Prevotella and Streptococcus. (B) PCoA analysis based on weighted UniFrac distances clustered background samples separated from supraglotic samples. (C) Heat map of unsupervised hierarchical clustering of BAL samples. Never-smokers are indicated with green labels and smokers with blue labels. Dendrogram shows deep cleft that identified two major BAL microbiomes: one characterized by high relative abundance of Staphylococcus, Propionibacterium, and Corynebacterium which we called PneumotypeUN and a second with high relative abundance of Prevotella, Veillonella, and Streptococcus (pneumotypeSCT). (D) PCoA analysis based on weighted UniFrac distances differentiate the same BAL samples in the same two well-defined clusters (never-smokers in green dots and smokers in blue dots).