Soil Fungal Community Profiling by Illumina Sequencing

We extracted DNA from the biofertilizer and soil samples using the Qiagen DNeasy PowerSoil Kit by following the manufacturer’s protocol. A negative extraction control was included with each set of extractions. We used the universal gene primer ITS86F and ITS4 [32 (link)] to amplify the large subunit ITS2 region and 5.8S gene of fungal taxa. We performed duplicate PCR reactions for each soil sample, and included negative extraction controls and negative PCR controls. The 20 μl individual PCR assays consisted of 10 μl of 2x Phusion Hot Start II HF Master Mix, 400 nM reverse primer, 400 nM forward primer, and 2 μl DNA template. PCR conditions were 98°C for 30 s, followed by 25 cycles of 98°C for 10 seconds, 58°C for 20 seconds, 72°C for 30 seconds and a final extension (72°C for 5 m). We performed index PCR, adding nextera-style i5 and i7 adaptors to the PCR amplicons to uniquely identify each sample. We performed post-PCR clean-ups between each PCR reaction using [33 (link)] Speed-beads (in a PEG/NaCl buffer). We quantified the molarity of each final library-prepped sample, and then pooled samples in equimolar amounts [34 ]. We used Illumina MiSeq High-Throughput sequencing (2 x 300 bp kit) to characterize the soil fungal communities of each sample.