DNA extraction and PCR
Genomic DNA was extracted from each soil sample (0.25 g) using DNeasy
PowerSoil HTP 96 DNA extraction kit (QIAGEN, Germantown, MD, USA). For
each sample, the fungal ITS2 gene was amplified using the primer set
5.8S-FUN/ITS4-FUN modified for the Illumina platform by fusing CS1 and
CS2 linker primers for forward and reverse primers, respectively
(Tayloret al. 2016).
Polymerase chain reactions (PCR) were conducted with 50 μL assays
composed of 25 μL of KAPA HiFi Master Mix, 1.5 μL of each primer (10
μM), 17 μL of PCR-grade water, and 5 μL of a genomic DNA template (10 ng
μL−1) for each sample. The following thermal profile
was used: initial denaturation and enzyme activation step of 95°C for 3
min, followed by 30 cycles of 98°C for 20s, 58°C for 15s, and 72°C for
15s, with a final extension of 72 °C for 1 min. Qualities of the PCR
products were evaluated by agarose gel electrophoresis. Additional
rounds of PCR were performed to fuse CS1/CS2 linker primers to the
indices and adapters (an initial denaturation and enzyme activation at
95°C for 10 min, followed by 15 cycles at 95°C for 15s, 60°C for 30s,
and 72°C for 60s with a final extension at 72°C for 10 min) before
Illumina MiSeq PE250 sequencing at Génome Québec (Montréal, Québec,
Canada). The Illumina sequences were deposited at NCBI (accession
numbers SAMN27069543 - SAMN27069726).