*3.2. Taxonomic Identification and Operational Taxonomical Unit (OTU) Assessment of Diversity*

An average of 18,621, 19,051, 19,952, 18,136, and 17,402 sequence reads was generated from the supragingival, subgingival, apical abscess, coronal pulp chamber, and root canal samples, respectively, after filtering (Table 2). Operational taxonomical units (OTUs), which is an operational definition of a species or group of species often used only when DNA sequence data are available, were utilized as a measure of diversity [17]. In the present study, OTUs were defined at the 3% divergence (97% similarity) threshold using the average neighbor clustering algorithm. The average numbers of OTUs were 165, 199, 185, 56, and 65, respectively. There were 3–4 times more OTUs in the perio group than in the endo group. Within the supragingival plaque and subgingival plaque, the MIM mandibular right first molar (ID 46-1 and 2) showed the lowest number of OTUs and the non-MIM teeth (ID Nor-1 and 2) showed higher than average OTUs.

The α-diversity in the samples was assessed by the Ace, Chao 1, and Shannon diversity indices at a 3% distance range (Table 2). Diversity is an indicator of richness (abundance) and evenness (composition) in the samples. The average Ace index was 185.62, 236.09, 215.64, 78.97, and 111.86, respectively. The average Chao 1 index was 181.92, 230.68, 224.18, 77.72, and 83.9, respectively. The average Shannon index was 3.47, 3.58, 3.24, 2, and 2.2, **Group Sample ID Taxonomic Identification** <sup>α</sup>**-Diversity Number of Final Reads Number of OTUs \* Ace Chao 1 Shannon** Nor-1 18092 183 201.84 193.62 3.52 **Perio** 11-1 19109 155 181.94 184 3.32 16-1 18517 161 178.61 171.12 3.71 26-1 18293 196 219.54 206.50 3.67 36-1 18317 193 212.38 206.04 3.88 46-1 19395 104 119.42 130.25 2.74 **Average 18621 165 185.62 181.92 3.47** Nor-2 19134 206 250.28 243.27 3.70 11-2 18492 262 319.99 304.78 3.84 16-2 19032 185 217.45 216.95 3.54 26-2 18528 201 213.69 207.56 4.01 36-2 19629 206 238.47 248.50 3.58 46-2 19491 133 176.67 163.00 2.78 **Average 19051 199 236.09 230.68 3.58** 46-3 19952 185 215.64 224.18 3.24 **Endo** 16-4 17033 52 67.61 61.43 2.01 36-4 19238 59 90.32 94 1.98 **Average 18136 56 78.97 77.72 2.00** 16-5 16233 62 73.68 69.80 2.23 36-5 18570 68 150.03 98.00 2.17 **Average 17402 65 111.86 83.9 2.2**

**Table 2.** Taxonomic identification and assessment of α-diversity.

groups.

respectively. Diversity showed a similar pattern to the OTUs between and within the

\* OTUs: operational taxonomical units.

#### *3.3. Taxonomic Identification of the Oral Microbiome*

A total of seven major bacterial phyla (*Spirochaetes, Firmicutes, Fusobacteria, Actinobacteria, Proteobacteria, Bacteriodetes*, and *Saccharibacteria\_TM7*) were identified at a higher than 1% frequency in all the plaque samples (Figure 5A). The perio group showed an average of 5.7 phyla. Only sample 36-2 showed seven phyla, including *Spirochaetes*. Seven samples showed six phyla and six samples showed five phyla. The endo group showed an average of 3.5 phyla and sample 36-4 showed two phyla.

Figure 5B shows that at the genus level, *Veillonella, Streptococcus,* and *Leptotrichia* were present at 5% frequency in almost all the perio group samples. *Parvimonas, Peptostreptococcus, Atopobium*, and *Dialister* were present in all endo group samples. Other genera were found in several samples in relation to the habitat or tooth. *Aggregatibacter actinomyces* were not identified.

Figure 5C shows the analysis of the endo group samples at a frequency of 1%. *Bulleidia* was found in all four samples. *Fusobacterium, Mogibacterium, Prevotella,* and *Olsenella* were present in three samples. *HQ441590\_g* was found in two samples, while *Shuttleworthia, Alloprevotella, Eubacterium\_g10, Selenomonas*, and *Oribacterium* were present in one sample.

**Figure 5. Relative abundance of oral bacteria at the phylum and genus levels.** (**A**) Relative abundance of major bacterial phyla with >1% frequency in all samples. (**B**) Relative abundance of major bacterial genera with >5% frequency in all samples. (**C**) Relative abundance of major bacterial genera with >1% frequency in the endodontic samples.
