2.2. Metagenomic DNA extraction, amplicon sequencing and
bioinformatics
Metagenomic DNA was extracted from 0.5 g of sample using Qiagen DNeasy
PowerSoil Pro Kit (Carlsbad, CA, USA). The Internal Transcribed Sequence
1 ribosomal region (ITS1) and hypervariable region V4 of 16S ribosomal
gene were targeted to assess the fungal and prokaryotic community
composition, respectively. The ITS1 region was amplified using barcoded
primers ITS1F/ITS2, suitable for shorter read length (Smith & Peay,
2014), while for the V4 region of 16S, barcoded F515/R806 primer set was
used according to Caporaso et al. (2012). PCR reactions consisted of 1
μL of each primer, 12.5 μL of Taq DNA Polymerase (Thermo Fisher
Scientific Inc., Waltham, MA, USA), 9.5 μL of nuclease-free water
(Sigma–Aldrich, St. Louis, MO, USA) and 5 ng of DNA for a total volume
of 25 μL and occurred in an automated thermal cycler (BioRad, Hercules,
CA, USA),. The ITS1 locus and V4 region were amplified according to
Coleine et al. (2021). Amplicons were quantified by a Qubit dsDNA HS
Assay Kit (Life Technologies, Carlsbad, CA, USA) and then pooled.
Paired-end sequencing (2 × 300 bp) was carried out on an Illumina MiSeq
platform at the Edmund Mach Foundation (San Michele all’Adige TN,
Italy). Demultiplexed ITS and 16S sequence datasets were processed using
AMPtk (Palmer et al., 2018) v.1.5.1 software. Briefly, barcodes/indexes
and primer sequences were removed from raw data. Reads were subjected to
quality trimming to a maximum of 250 bp, discarding those shorter than
100 bp; sequencing artefacts were dropped by using USEARCH v.9.1.13 with
default parameters (Edgar, 2010). Sequence quality filtering was
performed with the expected error parameter of 0.9 (Edgar & Flyvbjerg,
2015); the cleaned reads were merged and clustered at 99% similarity
using VSEARCH (Rognes et al., 2016) v.2.15.1, with DADA2 (Callahan et
al., 2016). Global singletons and rare taxa (<5 reads) were
skipped as likely false positives due to sequencing errors (Lindahl et
al., 2013). Finally, taxonomic identification was performed with hybrid
database SINTAX/UTAX (Edgar, 2010).