Illumina MiSeq of fungi: human gut
Both ITS region 1 and ITS region 2 were amplified with the universal primers used two different annealing temperatures (52°C and 55°C) and three replicated PCRs for each annealing temperature. All amplicons that obtained from each replicated PCR and at both annealing temperature were pooled together for each ITS1 and ITS2 PCR. Then the total amplicons from both ITS PCRs (ITS region 1 and ITS region 2) were pooled and sequenced by MiSeq technology.