PCR amplification and next generation sequencing (NGS, Illumina MiSeq)
Polymerase chain reaction (PCR) amplification of ITS hypervariable regions (6F-4R), was performed using the extracted DNA as a template for amplifying an internal fragment of the ITS gene. Primers ITS 3 (5’-GCA TCG ATG AAG AAC 132 GCA GC- 3’), (Macrogen, Seoul, Korea) and ITS 4 (5’-TCC TCC TAT TGA TAT GC-3’), (Macrogen), joined to a multiple identifier sequence (Illumina, Seoul, Korea). For each sample, amplicons were generated in several duplicate PCRs using mixtures (25 μl) containing 25 pmol of each primer, 1x KAPA HiFi Hotstart Ready Mix (Kapa Biosystems, Wilmington, MA USA) and 10 ng of the DNA template. The PCR program consisted of an initial denaturalization stage at 95° C for 3 min, 25 denaturation cycles at 95° C for 30 s, primer annealing at 55° C for 30 s and an extension at 72° C for 30 s, followed by one step final heating at 72° C for 5 min. Amplicons of each treatment were grouped to reduce PCR variability and purified using Ampure accounts XP (Beckman Coulter, IN USA) according to the manufacturer’s instructions. After PCR cleaning, the Illumina sequencing adapters were joined by a second stage of PCR using the Nextera XT index kit (Illumina Inc, Sand Diego, CA, USA). The mixture contained Nextera Index Primers 1 and 2 (5 µl), 2 KAPA HiFi Hot Start Ready Mix (25 µl), DNA (5 µl) and PCR grade water (10 µl) for a total volume of 50 µl. The PCR program in this step consisted of an initial denaturation step at 95ºC for 3 minutes, followed by 8 denaturation cycles at 95°C for 30 s, a primer annealing at 55ºC for 30 seconds an extension at 72ºC for 30 seconds, and a final step at 72ºC. for 5 min. Amplicons were cleaned as described above, and libraries were quantified using Qubit (Invitrogen, CA, USA). The samples were combined in equimolar quantities (4 nM each) and were sequenced on a Miseq platform (paired-end sequencing 2 × 300, considering 2 × 150,000 reads/sample) of Illumina in Macrogen (Seul, South Korea) in accordance with the manufacturer’s instructions.