PCR and amplicon sequencing

To prepare libraries for 16S-v4 rRNA gene sequencing, 0.4 µL of forward and 0.4 µL of reverse primers 515f and 806r, respectively, were used (Apprill et al. 2015; Parada et al. 2016). For ITS1 sequencing, 0.4 µL of forward primer ITS1f and 0.4 µL of reverse primer ITS2 were used (Smith and Peay 2014). Both PCR protocols for 16S and ITS amplification also utilized 5 µL per reaction of DreamTaq Hot Start PCR Master Mix (Thermoscientific). PCRs for the amplification of 16S genes also included 0.15 µL of 100 µM of peptide nucleic acids (PNA) per reaction to suppress the amplification of the 16S sequences of chloroplasts and mitochondria (Lundberg et al. 2013). We used 2.5 µL of template DNA for each reaction, for a total volume of 10 µL per reaction. The 16S PCR included an initial 2-min denaturing cycle, followed by 27 cycles of denaturing for 20 s, PNA annealing for 5 s, primer annealing at 20 s and an extension for 50 s, at 95 °C, 78 °C, 52 °C and 72 °C, respectively. This was followed by a final 10-min extension at 72 °C. The ITS1 PCR included a 2-min denaturing cycle at 95 °C, followed by 27 cycles of denaturing for 20 s, primer annealing for 20 s and an extension for 50 s at 95 °C, 50 °C and 72 °C, respectively. This was followed by a 10-min extension at 72 °C. Both the 16S and ITS plates then underwent a second PCR to attach Illumina adapters with indexes. For this PCR, we used 0.8 µL of 10 µM combined forward and reverse barcoded primers with P5 and P7 Illumina adaptors. Each sample had a unique combination of eight base pairs and a binding site for annealing to amplicon sequences. This indexing PCR also utilized 5 µL per reaction of DreamTaq Hot Start PCR Master Mix, 0.15 µL of 100 µM of PNA and 1 µL of template DNA for a total volume of 10 µL. This PCR step began with a 2-min denaturing cycle, followed by 8 cycles of denaturing for 20 s, PNA annealing for 5 s, primer annealing at 20 s and an extension for 50 s, at 95 °C, 78 °C, 52 °C and 72 °C, respectively. This was followed by a 10-min extension at 72 °C. 10 µL of each reaction was pooled, keeping bacteria and fungi separate. Each pool was normalized using the ‘Just-a-Plate’ kit (Charm Biotech). DNA was quantified using QuantiFluor reagents and a Promega Quantus fluorometer. Pools were combined in equal molarity and sequenced on the Illumina platform Novaseq 6000 at 250 bp PE.