16S rRNA gene amplicon sequencing of Bacteria, 50cm
To prepare the library for Ion Torrent sequencing, the pooled and extracted PCR fragments were amplified using 100 ľl of Platinum PCR SuperMix High Fidelity (Life Technology, USA), 5 ľl of Library Amplification Primer Mix and 25 ľl of unamplified purified PCR product. Amplification was performed with initial denaturation at 95 °C for 5 minutes, followed by 5 cycles of denaturation at 95 °C for 15 seconds, primer annealing at 58 °C for 15 seconds and elongation at 70°C for 1 minute. The library was purified using an Agencourt AMPure XP kit (Beckman Coulter, Inc., USA). The concentrations and fragment lengths of the libraries were determined with a Bioanalyzer 2100 and a High Sensitivity DNA kit (Agilent Technologies, USA). The obtained libraries were diluted to a final concentration of 26 pM, and the different barcoded libraries were pooled in equimolar amounts before sequencing. For Ion Torrent sequencing, the library fragments were attached to Ion Sphere particles using an Ion One Touch Instrument and Ion PGM Template OT2 400 Kit (Life Technologies, USA) according to the manufacturers instructions. After enrichment of the template-positive Ion Sphere Particles using the Ion One Touch ES (Life Technologies, USA), the samples were loaded on an Ion 316 v2 Chip.