Soil Microbiome DNA Extraction and Sequencing

The rhizosphere DNA was extracted from 0.5 g sample using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) and DNA samples were randomized across plates. The bacterial V4 region of the 16S rRNA gene was amplified using the protocol described by Lundberg et al. (2013) (link). The universal primer pair 515F and 806R was used to generate bacterial-derived 16S rRNA amplicons. PNA PCR clamps were used to reduce host organelle contamination. The fungal ITS2 region was amplified using the universal primers ITS3/KYO2 and ITS4 (Toju et al., 2012 (link)). All primers were modified to include Illumina adapters¹. Each 25 μl reaction contained 12.5 μl of HiFi HotStart Ready Mix (KAPA Biosystems, Woburn, MA, United States), 1.0 μl of each primer (10 μM), 2.5 μl of DNA template (5 ng/μl), and 8.0 μl PCR-grade water. PCR amplifications (performed in triplicate for each sample) consisted of a 3 min denaturation at 95°C; 25 cycles of 30 s at 95°C, 30 s at 55°C and 30 s at 72°C; and 5 min at 72°C. Samples were cleaned using the AMPure beads XP purification system (Beckman Coulter, United Kingdom) and sequenced on the Illumina MiSeq platform at the Fundación FISABIO (Valencia, Spain) facility using a 2 × 300 nucleotide paired reads protocol.