Bacterial Community Profiling by 16S rRNA Amplicon Sequencing

Total DNA was extracted from the soil using the FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, United States). To the soil in the Lysing Matrix E tube, sodium phosphate buffer and MT buffer were added, and it was homogenized with 2 × 30 s rotations at 6,000 rpm in a Precellys tissue homogenizer (Bertin, Frankfurt a. M., Germany). Further extraction steps were performed as described in the manufacturer’s protocol. Genomic DNA from soil was eluted in 60 μL of nuclease free water and purified twice using Agencourt MPure XP beads (Beckman-Coulter, Krefeld, Germany). The purified genomic DNA was used in a two-step PCR procedure to amplify the V4-V7 region of the bacterial 16S rRNA gene (primers 799F–1192R, Supplementary Table 1) (Bodenhausen et al., 2013). In the first step, the gene fragment was amplified for each sample in triplicates in a 25 μL reaction volume (Agler et al., 2016). Each reaction contained 0.2 μM of each primer, GoTaq Reaction Buffer (1.5 mM MgCl₂), PCR Nucleotide Mix (0.2 mM each dNTP), 1.25 U GoTaq G2 DNA polymerase (Promega, Walldorf, Germany), 0.3% bovine serum albumin, 4 ng template DNA and nuclease-free water. The first PCR amplification was performed as follows: 2 min at 94°C; 25 cycles of 15 s at 94°C, 25 s at 55°C, 45 s at 72°C. The products were purified by electrophoresis in 1.2% agarose gels. The bands at 500 bp were excised and the DNA purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). DNA was finally eluted in 20 μL nuclease free water. The purified products were used for a second PCR, which was performed in the same way but with only 15 cycles. In the second PCR, barcoded primers containing Illumina adaptors (B5-F; B5-1 to B5-64; Supplementary Table 1) were used. The PCR products were also purified by electrophoresis (see above). The DNA concentration of the purified PCR products was determined by fluorometry (QuantiFluor ONE Dye, Quantus Fluorometer, Promega, Walldorf, Germany), and a sequencing library was created by pooling 30 ng of each amplicon. The library was purified twice with Agencourt MPure XP beads.