Quantitative polymerase chain reaction (qPCR) for 16S rRNA gene quantification was performed on the extracted bacterial DNA, as described elsewhere [12], with THUNDERBIRD® SYBR® qPCR Mix (Toyobo Co., Ltd., Osaka, Japan) and 16S rRNA primers (forward: 5′-ACTGAGAYACGGYCCA-3′ and reverse: 5′-CTGCTGGCACGDAGTTAGCC-3′) on an AriaMX Real-Time PCR System (Agilent Technologies, Inc., CA, USA). Then, equal concentrations of the resultant amplified DNA solutions containing the 16S rRNA gene were used to amplify the V3–V4 hypervariable region, to better capture the microbial composition of the skin [30], with KAPA HiFi HS ReadyMix (F. Hoffmann-La Roche, Ltd., Basel, Switzerland) and primers with MiSeq-compatible overhang sequences (forward: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′ and reverse: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAAKCC-3′). After cleaning up the polymerase chain reaction (PCR) products using AMPure XP magnetic beads (Beckman Coulter Inc., CA, USA), indexing PCR was performed for all samples using a Nextera XT Index Kit v2 (Illumina Inc., CA, USA) followed by AMPure XP cleaning. The concentration of each product was measured by means of a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, MA, USA), and equimolar amounts of the products were mixed to create the final library solution.

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