ITS2-Stool-HomoSapiens
PCR amplification was carried out in two steps: First-round amplification. Primers used for DNA fungal amplification were ITS1 (5TCC GTA GGT GAA CCT GCG G 3), and ITS4 (5TCC TCC GCT TAT TGA TAT GC 3) (Ferrer). A 15-l PCR mixture containing 12,5 l of AmpliTaq Gold 360 Master Mix, 1 l of 360 GC Enhancer (4% v/v), 0,2 M of each primer, and 5 l of DNA template (Applied Biosystems) was used. The PCR cycle condition was: initial denaturation at 95C for 10 min; 35 cycles of 95C 30 s, 55C 30 S and 72C 1 min; and a final extension at 72C for 7 min. Second-round amplification, used ITS86 (5GTG AAT CAT CGA ATC TTT GAA C 3), and ITS4 primers (Ferrer). Semi-nested PCR amplification mixture contained 3 l of first-round product in 30 l of PCR reaction mix with 15 l of AmpliTaq Gold 360 Master Max, 1,2 l of 360 GC Enhancer (4% v/v), 0,2 M of each primer (Applied Biosystems). The cycling program was the same as before. Then, 20 l of second-round product were partially digested , bar-codes were ligated to the amplicons, and the product was purified using the Agencourt AMPure XP beads (Beckman Coulter; Pasadena, CA, USA) according to the manufacturers protocol. Sequencing was performed using Ion 510 & Ion 520 & Ion 530 Kit  Chef (Thermo Fisher Scientific, Carlsbad, CA, USA) on the Ion GeneStudio S5 platform (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturers instructions
SUB15922653
MF36