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.