## 2 Materials and Methods
### Sampling  
The samples were MIM teeth and the non-MIM teeth were the controls. The MIM samples included four permanent first molars and maxillary central incisors as one sample, and the non-MIM teeth including the maxillary left premolars and mandibular right premolars as one sample.  
The oral microbiome was obtained from five habitats around the teeth in the two groups. The perio group included 1 a supragingival plaque, 2 subgingival plaque, and 3 an apical abscess, and the endo group included 4 a coronal pulp chamber and 5 a root canal (Figure 4). The perio samples were collected before the extractions, and the endo samples were collected after the extractions. All samples are listed in Table 1. The sample identification (ID) numbers refer to the two-digit FDI notations.  
* **Period group** 1 Supragingival plaque: The patient was requested not to brush her mouth 24 h prior to sample collection. Supragingival dental plaque samples were collected by rubbing a sterile cotton swab across the cervicobuccal area of the teeth twice or three times under pressure [13].
* 2 Subgingival plaque: The subgingival plaque samples were collected by inserting sterile endodontic paper points (sized 30; two paper points per site) into the gingival sulci or periodontal pocket for 10 s right after isolation and supragingival plaque removal [14].
* 3 Periapical abscess: The periapical abscess sample was obtained via aspiration. The swollen area was aspirated with a syringe fitted with a 16-gauge needle and expressed into a sterile vial [15].
* **Endo group** 1 Coronal pulp chamber and 5 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  
Figure 3: **Extracted teeth affected with MIM.** (**A**) The maxillary right first molar (ID 16) showed twisted and fused roots after cleaning. (**B**) The maxillary left first molar (ID 26) showed a fractured crown and root with granulation tissue and an abnormal calcified surface between the crown and root. (**C**) The mandibular right first molar (ID 46) showed a small canal (red arrow) on the fractured surface. (**D**) The mandibular left first molar (ID 36) showed abundant granulation tissue and pulp necrosis in the pulp chamber.  
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.  
The samples were placed in sterile 1.5 mL microcentrifuge tubes and frozen for storage at \(-80\)\({}^{\circ}\)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.