16S rDNA amplicon sequencing of Homo sapiens: fecal sample from patient receiving stent-based diversion technique (SDT), preoperative baseline
16S rRNA genes of distinct regions (16SV3-V4) were amplified used specific primer with the barcode. All PCR reactions were carried out with 15 L of Phusion High-Fidelity PCR Master Mix (New England Biolabs); 0.2M 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 10s, annealing at 50 for 30 s, and elongation at 72 for 30s and 72 for 5 min. The PCR products were purified using magnetic bead purification. Samples were mixed in equidensity ratios based on the concentration of PCR products. After thorough mixing, the PCR products were detected and target bands were recovered. Sequencing libraries were generated and indexes were added. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.