title: 16S rDNA of Homo sapiens: Skin sample
design: The samples were thawed on ice after removal from 80 C, and microbial DNA was extracted using the E.Z.N.A. Soil DNA Kit (Omega Bio-Tek, GA, USA). Sample DNA integrity and quality were assessed using agarose gel electrophoresis and the NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) before the amplification reaction. Then ,icrobial profiling analysis targeted the V3 and V4 regions of the 16S rRNA gene. Universal primers (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT) were used to analyze microbial community diversity and structure in oral and skin tissues of different individuals and populations. Polymerase chain reaction amplification was conducted with TransStart FastPfu DNA polymerase (TransGen Biotech, Beijing, China), with negative control samples included. Any sample group with bands in the negative control amplification was excluded from subsequent experiments. The amplified products were then purified and recovered using Vazyme VAHTS DNA Clean Beads (Vazyme, Nanjing, China). Subsequently, gene libraries were prepared with the TruSeq Nano DNA LT Library Prep Kit (Illumina, CA, USA), and their quality inspected with the Agilent High-Sensitivity DNA Kit (Agilent, CA, USA). Libraries were quantified using the Quant-it PicoGreen dsDNA Assay Kit on the Promega QuantiFluor fluorescence quantification system (Promega Corporation, Wisconsin, USA), with a required concentration above 2 nM for qualification. Qualified sequencing libraries were diluted in a gradient, mixed in equimolar amounts, and denatured with NaOH for 5 min. Following library loading into the MiSeq Reagent Kit V3 (778 cycles, Illumina, USA), paired-ended sequencing was performed on the Illumina MiSeq instrument (Illumina, CA, USA) at Shanghai Personal Biotechnology Co., Ltd (Shanghai, China).