Amplification of 16S rRNA Gene for Metagenomic Analysis

A set of primers adapted to massive sequencing for the Illumina technology was used to amplify the 16S rRNA gene (V3-V4 hypervariable region^{46 (link)}) from the metagenomic DNA in a PCR reaction. PCR reactions were performed with 30 ng of metagenomic DNA, 200 μM of each of the four deoxynucleoside triphosphates, 400 nM of each primer, 2.5 U of FastStart HiFi Polymerase, and the appropriate buffer with MgCl₂ supplied by the manufacturer (Roche, Mannheim, Germany), 4% of 20 g/mL BSA (Sigma, Dorset, United Kingdom), and 0.5 M Betaine (Sigma). Thermal cycling consisted of initial denaturation at 94 °C for 2 minutes followed by 35 cycles of denaturation at 94 °C for 20 seconds, annealing at 50 °C for 30 seconds, and extension at 72 °C for 5 minutes. Amplicons were combined in a single tube in equimolar concentrations. The pooled amplicon mixture was purified twice (AMPure XP kit, Agencourt, Takeley, United Kingdom) and the cleaned pool requantified using the PicoGreen assay (Quant-iT, PicoGreen DNA assay, Invitrogen). Subsequently, a sequencing library was prepared under a pair-end configuration following manufacturer’s instructions, and sequencing on the Illumina MiSeq platform was performed at Life Sequencing SL facilities (Valencia, Spain). Sequencing statistics and rarefraction curves are shown in Supplementary Table S1 and Supplementary Figure S2, respectively.