skin_before_16S_3
The total skin microbial DNA was obtained through the Fecal Genome DNA Extraction Sequencing primer were removed from de-multiplexed raw sequences using cutadapt (v1.9). Then, Pairedend reads were merged using FLASH(v1.2.8). The low-quality reads (quality scores<20), short reads (<100bp),and reads containing more than 5% N records were trimmed by using the sliding-window algorithm method in fqtrim (v 0.94). Quality fltering was performed to obtain high-quality clean tags according to fqtrim. Chimeric sequences were fltered using Vsearch software (v2.3.4). DADA2 was applied for denoising and generating amplicon sequence variants (ASVs) . The sequence alignment of species annotation was performed by QIIME2 plugin feature-classifier, and the alignment database was SILVA and NT-16S. Alpha and beta diversities were calculated using QIIME2, Relative abundance was used in bacteria taxonomy. The wilcox test was used to identify the differentially abundant genus, and signifcances were declared at P<0.05. LDA effect size (LEfSe, LDA  3.0, P value < 0.05) was performed using nsegata-lefse. Other diagrams were implemented using the R package(v3.4.4) Kit(AU46111-96, BioTeke,China)according to the manufacturers instruction manual.The DNA was quantified by Qubit (Invitrogen, USA). Total DNA was amplified by PCR using the universal primer 341F/805R(341F: 5-CCTACGGGNGGCWGCAG-3; 805R: 5-GACTACHVGGGTWTCTAATCC-3) . The PCR amplification conditions were pre-denaturation at 98  C for 30 s,denaturation at 98 C for 10 s, annealing at 54 C for 30 s, extension at 72 C for 45 s and 32 cycles. The final extension was at 72 C for 10 min. The PCR product was purifed using AMPure XP Beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantifed using Qubit (Invitrogen, USA). Qualifed PCR products were evaluated using an Agilent 2100 Bioanalyzer (Agilent, USA) and Illumina library quantitative kits (Kapa Biosciences, Woburn, MA, USA), which were further pooled together and sequenced on an Illumina NovaSeq 6000 ( PE250 ), provided by LC-Bio Technology Co., Ltd., Hangzhou, China.
SUB16359362
aa3