16s rDNA sequencing of human metagenome: duodenal mucosa
Dual-indexed universal primers 341F (CCTACGGGNGGCWGCAG) and 802R (GACTACHVGGGTATCTAATCC; Illumina, USA) were utilized for the two-steps PCR amplification of the V3V4 hypervariable regions of the 16s rRNA gene (16S/ITS Nextera two-step PCR kit, Illumina Inc., USA), according to the Manufacturers Instruction. Briefly, the first-step PCR was carried on amplifying the V3V4 region of the 16s rRNA gene. The resulting PCR amplicons were then used for the second-step PCR for further amplification and inclusion of indexes (barcodes) as well as the Illumina sequencing adaptors. Cycling conditions were initial denaturation at 95C for 3 min, followed by 20 cycles (for the first-step PCR) or 15 cycles (for the second-step PCR) of denaturation at 98C for 20 s, annealing at 56C for 30 s and elongation at 72C for 30 s, one cycle of final elongation at 72C for 5 min and a final cooling step to 10C. The resulting PCR products were quantified by fluorescence spectroscopy (Quant-iT PicoGreen dsDNA Assay Kit, ThermoFisher, USA), pooled in equimolar amounts and then purified with Agencourt AMpure-XP magnetic beads (Beckman Coulter, USA). The final library containing all the pooled samples was sequenced with version 3 of MiSeq Reagent Kit, 2  300 bp output, on a MiSeq desktop sequencer (Illumina, USA). One negative control (350 L of sterile PBS) was included and subjected to the same procedures as the samples.