Bacteria_AM400-ZX01-0101-16SV3V4-M02
The V3-V4 region of the bacterial16S rRNA gene was amplified with the common primerpair (Forward primer, 5'- ACTCCTACGGGAGGCAGCA-3'; reverse primer, 5'- GGACTACHVGGGTWTCTAAT-3') combined with adapter sequences andbarcode sequences.PCR amplification was performed in a total volume of 50 l, which contained10 lBffer, 0.2lQ5 High-Fidelity DNA Polymerase, 10 lHigh GC Enhancer, 1 ldNTP, 10Mof each primerand 60 ng genome DNA. Thermal cycling conditions were as follows: an initial denaturationat 95 C for 5 min,followed by 15cycles at 95 C for 1 min, 50 C for 1 min and 72 C for 1 min, with a final extension at 72 C for 7 min.The PCR products from the first step PCR werepurified throughVAHTSTM DNA Clean Beads.A second round PCR was then performed in a 40lreactionwhich contained 20 l2Phsion HF MM, 8 lddH2O, 10Mof each primer and 10lPCR products from the first step. Thermal cycling conditions were as follows: an initial denaturationat 98 C for 30s, followed by 10 cycles at 98 C for 10s, 65 C for 30s min and 72 C for 30s, with a final extension at 72 C for 5 min. Finally, all PCR products were quantified by Quant-iT dsDNA HS Reagentand pooled together.High-throughput sequencing analysis of bacterial rRNA genes was performed on the purified, pooled sample using the Illumina Hiseq 2500 platform(2250 paired ends)