• **Endo group**

<sup>1</sup> Coronal pulp chamber and <sup>5</sup> root canal: The extracted tooth was cleaned with 30% hydrogen peroxide [16]. Aseptic techniques such as sterile burs were used to access the pulp space. Bacteriological samples of the pulp chamber were collected immediately after crown access. Pulp remnant and infected dentin were collected using a sterilized spoon excavator and sterilized paper points were inserted to absorb the remaining fluid containing microorganisms. In the root canal, two sequential new paper points were placed at the same level and utilized to soak up the fluid in the canal. Each paper point was held in position for 30 s. Sterilized endodontic files were then used to collect the infected root canal dentin severally. Only the tip area was collected into the tube by cutting.

**Figure 4. Sampling of normal and MIM teeth.** Perio group: - supragingival plaque, - subgingival plaque, and - apical abscess. Endo group: - coronal pulp chamber and -root canal.



\* The crown and root were separated during the extraction; \*\* Samples were taken but DNA extraction failed due to DNA degradation or insufficient amount.

> The samples were placed in sterile 1.5 mL microcentrifuge tubes and frozen for storage at −80 ◦C. DNA was extracted from the clinical samples using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions. The concentration and purity of the DNA samples were determined using an ND-1000 NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) by measuring the absorbance at 260 and 280 nm.

## *2.4. PCR Amplification and Illumina Sequencing*

PCR amplification of the extracted DNA was performed using primers targeting regions between V3 and V4 of the 16S rRNA gene. For bacterial amplification, 341F primers (5 TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3 ; the target region primer) and 805R (5 -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA G-GACTACHVGGGTATCTAATCC-3) were used. Amplifications were conducted under the following conditions: initial denaturation at 95 ◦C for 3 min, followed by 25 cycles of denaturation at 95 ◦C for 30 s, primer annealing at 55 ◦C for 30 sec, and extension at 72 ◦C for 30 s, with a final elongation at 72 ◦C for 5 min. Secondary amplification for attaching the Illumina Nextera barcode was then performed using the i5 forward primer (5 AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-3 ; X indicates the barcode region) and i7 reverse primer (5 -CAAGCAGAAGACGGCATACGAG

AT-XXXXXXXXAGTCTCGTGGGCTCGG-3). The conditions for the secondary amplification were similar to the earlier one except that the amplification cycles were set to 8.

The PCR products were confirmed using 2% agarose gel electrophoresis and visualized by the Gel Doc system (BioRad, Hercules, CA, USA). The amplified products were purified via the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). Equal concentrations of purified products were pooled together and short fragments (non-target products) were removed with the Ampure bead kit (Agencourt Bioscience, MA, USA). The quality and size of the products were assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixed amplicons were pooled, and sequencing was conducted at Chunlab, Inc. (Seoul, Korea) using the Illumina MiSeq Sequencing System (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.
