T0_WATER10
16S library preparation using the primers 335Fc and 769Rc (Dorn-In et al., 2015). Amplification: 98C for 5 min; 98C for 10 s / 60C for 30 s / 72C for 30 s (28 cycles); 72C for 5 min. PCR reaction mixture (total 25 ul); 12.5 ul of 2 x NEB Next High Fidelity Master Mix, 0.5 ul of each primer(10 pmol), 2.5 ul of 3% BSA, 5 ng of template DNA, an adequate amont of DEPC-treated water. Sample purification: MagSi-NGS  extraction kit (Magtivio B.V., Geleen, Netherlands), the ratio of MagSi-NGS  beads to PCR products is adapted to 0.8. Indexing PCR: Nextera XT Index Kit v2, Amplification: 98C for 5 min; 98C for 10 s / 55C for 30 s / 72C for 30 s (8 cycles); 72C for 5 min. Sample purification: mentioned above. Products are pooled equimolar to a final concentration of 4 nM and sequenced using the MiSeq Reagent kit v3 (600 cycles) for paired end sequencing.