Metagenomic DNA Amplification for Sequencing

A set of primers adapted to massive sequencing for the Ion Torrent platform (Lifetechnologies) were used to capture 16S (modified from a previous study42 (link)) and 18S (modified from a previous study43 (link)) rRNA from the solar-panel DNA extraction 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, sequencing on the Ion Torrent platform was performed at LifeSequencing S.L. (Valencia, Spain).