AMPLICON of pharynx: guttate psoriasis
Polymerase chain reaction (PCR) amplification of target gene was performed using general primers V3-V4. The primers also contained the Illumina 5'overhang adapter sequences for two-step amplicon library building, following manufacturer's instructions for the overhang sequences. The initial PCR reactions were carried out in 50 L reaction volumes with 1-2 L DNA template, 200 uM dNTPs, 0.2 uM of each primer, 5X reaction buffer 10uL and 1U Phusion DNA Polymerase (New England Biolabs, USA). PCR conditions consisted of initial denaturation at 94 C for 2 min, followed by 25 cycles of denaturation at 94 C for 30 s, annealing at 56 C for 30 s and extension at 72 C for 30 s, with a final extension of 72 C for 5 min. The second step PCR with dual 8-base barcodes was used for multiplexing. Eight cycle PCR reactions were used to incorporate two unique barcodes to either end of the 16S amplicons.  Cycling conditions consisted of one cycle of 94 C for 3 min, followed by eight cycles of 94 C for 30 s, 56 C for 30 s and 72 C for 30 s, followed by a final extension cycle of 72 C for 5 min. Prior to library pooling, the barcoded PCR products were purified using a DNA gel extraction kit (Axygen, USA) and quantified using the FTC -3000 TM real-time PCRFunglyn Shanghai. The PCR products from different samples were indexed and mixed at equal ratios for sequencing on the illumina platform at TinyGen Bio-Tech (Shanghai) Co., Ltd.