*3.2. Diversity of the Oral Microbiome*

A total of 10 samples were sequenced, and 60,371 reads in total were obtained. Of all the reads, 39,013 reads (64.6%) passed quality filtering (low-quality sequence reads and a minimum length of 300 bp) and remained. Both the BS and control groups contained 28,142 and 10,871 reads with an average length of 472 and 465 bp, respectively (Supplementary Table S1). The microbial abundance based on the read number was significantly higher in the BS group than the control group (Figure 1A, *p* < 0.05). OTUs were identified by pairwise alignment to the EzTaxon database at a 3% distance. Totals of 15 phyla, 28 classes, 45 orders, 80 families, 145 genera and 423 OTUs (species) were identified in these samples. The BS group contained 348 OTUs in total (without duplication), whereas the control group had 293 OTUs. The alpha diversity of the microbiome was estimated by Simpson and Shannon indices, indicating that the diversity in the BS group was significantly higher than in the control group (Figure 1B,C, *p* < 0.05).

**Figure 1.** Diversity of the oral microbiome of the subjects. Abundance (**A**), Simpson index (**B**) and Shannon index (**C**) were compared between the black stain and control groups. Significance was determined by *t*-test (*p* = 0.05).
