16s rRNA V1_V2: faecal specimen from human origin
Samples were bead-beaten for mechanical lysis at 6500 rpm for 30 seconds twice in a MagNA Lyser Instrument (Roche, Mannheim, Germany). After incubation with lysozyme and Proteinase K, enzymes were deactivated at 95°C for 10 minutes and DNA purification was performed according to instructions from the Magna Pure DNA isolation kit III (Bacteria, Fungi). PCR amplification was performed with the target specific primers 27f and 357r and 2 ľl of total DNA extract were used for a 25 ľl PCR reaction in triplicates containing 1 x Fast Start High Fidelity Buffer (Roche, Mannheim, Germany), 1.25 U High Fidelity Enzyme (Roche, Mannheim, Germany), 200 ľM dNTPs (Roche, Mannheim, Germany), 0.4 ľM bar-coded primers and PCR-grade water (Roche, Mannheim, Germany). The final library was quantified using a Quantus Fluorometer device (Promega, Mannheim, Germany) and loaded to an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) using a high-sensitivity DNA assay according to manufacturers instructions for quality control.