title: 16s rRNA sequence of health people
design: 16S rRNA/18SrRNA/ITS genes of distinct regions(16S V4/16S V3/16S V3-V4/16S V4-V5, 18S V4/18S V9, ITS1/ITS2, Arc V4) were amplified used specific primer(e.g. 16S V4: 515F- 806R, 18S V4: 528F-706R, 18S V9: 1380F-1510R, et. al ) with the barcode. All PCR reactions  were carried out with 15 L of Phusion High-Fidelity PCR Master Mix (New England Biolabs);  2 M of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted  of initial denaturation at 98 for 1 min, followed by 30 cycles of denaturation at 98 for 10 s,  annealing at 50 for 30 s, and elongation at 72 for 30 s. Finally 72 for 5 min.Mix same volume of 1X loading buffer (contained SYB green) with PCR products and  operate electrophoresis on 2% agarose gel for detection. PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with Qiagen Gel Extraction Kit(Qiagen, Germany).Sequencing libraries were generated usingTruSeq DNA PCR-Free Sample Preparation Kit  (Illumina, USA) following manufacturer's recommendations and index codes were added. The  library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent  Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq platform  and 250 bp paired-end reads were g