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.