DNA Extraction, PCR, Sequencing and Bioinformatics
DNA extractions were completed in six batches where soil samples were
processed within one week of the sample collection. DNA extractions were
done using the Qiagen DNeasy PowerLyzer Soil Kit (Qiagen, Hilden,
Germany) following the manufacturer’s instructions and DNA
quantification was completed using the QuantiFluor dsDNA System
(Promega, Madison, WI, USA) and Quantus Fluorometer (Promega, Madison,
WI, USA). The 16S rRNA gene region was targeted using the 27F and 519R
primer set (Lane, 1991), PCR-amplified, and sequenced on the Illumina
MiSeq platform at the Australian Genome Research Facility (AGRF;
Adelaide, SA, Australia). Raw FASTQ files are available at
https://figshare.com/s/7cd385ad91893d99ea5a.
FASTQ files were processed through the QIIME 2 bioinformatics pipeline
(Bolyen et al., 2019) in a conda environment (Continuum Analytics). Due
to poor quality reverse reads, only the forward reads were used. TheCutadapt plugin was used to remove primer sequences and quality
check the raw sequences (Martin, 2011). The Figaro tool was used
to identify the optimal trimming lengths (Sasada et al., 2020) for
forward reads. These were then trimmed according to the Figarooutput using DADA2 (Callahan et al., 2016). Amplicon sequence
variants (ASVs) were assigned using the SILVA version 138.1 rRNA
database (Glöckner et al., 2017; Yilmaz et al., 2014; Quast et al.,
2013) via the q2-feature-classifier (Bokulich et al., 2018) and
classify-sklearn naïve Bayes taxonomy classifier.