16S amplicons of vineyard soil
Amplification and sequencing of 16S, ITS2 and COI barcodes were performed for the analysis of bacterial, fungal, and non-fungal eukaryotes respectively following Giraldo-Perez at al. (2021). The V3-V4 domain of bacterial 16S rRNA gene was amplified using primers Bakt_341F (5-CCTACGGGNGGCWGCAG-3) and Bakt_805R (5-GACTACHVGGGTATCTAATCC-3; Herlemann et al., 2011). The Internal Transcribed Spacer 2 (ITS2) region of fungal 18S rRNA gene was amplified using primers ITS3F (5-GCATCGATGAAGAACGCAGC-3) and ITS4R (5-TCCTCCGCTTATTGATATGC-3; White at al., 1990). The mitochondrial Cytochrome C Oxidase subunit I gene (COI) of eukaryotes was amplified using mlCOIintF (5- GGWACWGGWTGAACWGTWTAYCCYCC-3; Folmer et al., 1994) and jgHCO2198R (5-TAAACTTCAGGGTGACCAAAAAATCA -3; Geller et al. 2013) primers. Illumina adapters were added to all primers and each barcode from each sample was amplified in triplicate with KAPA HiFi HotStart ReadyMix DNA polymerase (Roche) and then combined. Negative PCR controls (blanks) included in all PCR batches revealed the polymerase was contaminated with an unspecified mixture of bacterial DNA. All sequences that were present in the negative control were subsequently removed from all samples with a post-sequencing bioinformatics removal step using microDecon R package (McKnight et al., 2019). PCR products were cleaned using AMPure XP (Agencourt) and quality-checked using Qubit dsDNA HS Assay kit and Agilent 2100 Bioanalyzer High Sensitivity DNA kits (Santa Clara, CA, USA). The University of Auckland Genomics centre conducted Illumina paired-end library preparation, cluster generation and 600 cycles of 2 x 300bp paired-end sequencing run on Illumina MiSeq.