16S rDNA in soils
DNA was isolated from 0.25-0.35 g of soil according to the manufacturers protocol of the DNeasy PowerSoil kit (Qiagen, the Netherlands). Prior to DNA amplification, the extracts were purified from inhibitors using the Zymo inhibitor removal kit (Zymo Research corp., USA; Clancy et al., 2001; Stevenson F.J., 1982). The V4 region of 16s rRNA gene was amplified using 515F and 806r primers (Caporaso et al., 2011), amended with Illumina Nextera labels (Illumina Inc; San Diego, CA, USA) during the first PCR. Each 25 l reaction mixture contained 1 l of the sample, 0.5 M of each forward and reverse primer, 1 x PCR buffer, 200 M dNTPs and 1 U Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA). PCR conditions were as follows: initial denaturation at 98C for 60 s, followed by 25 cycles of: denaturation at 98 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 30 s; and an additional extension of 72 C for 10 minutes. A second PCR was performed, adding index adapters to each extremity of the PCR products. For this, we used dual barcoded primers with Illumina adapters (2.5 l of 50 x diluted PCR products template and 0.1 M of each primer). The conditions for the second PCR were: 98C for 60 s, 12 cycles: at 98 C for 10 s, 63 C for 30 s, 72 C for 30 s; and a final step at 72 C for 5 min. PCR products were run on an agarose gel to confirm successful PCR amplification and successful amplicons were normalized and purified from primers and primer-dimers using the SequalPrep Normalization Plate Kit (ThermoFisher Scientific). The samples were then pooled into a single library and purified using QIAquick Gel Extraction Kit (Qiagen, Venlo, the Netherlands). The library was quantified with qPCR (KAPA Library Quantification Kits, Kapa Biosystems, Wilmington, MA, USA) and sequenced on an Illumina Miseq using 2  300 cycles of paired-end sequencing.