title: 16S rRNA gene seq: Pig gut
design: Targeted amplification of V1-V3 regions of the bacterial 16S rRNA gene. PCR reaction (25 mL) contained 10 ng template DNA, Platinum¨ High Fidelity buffer (x1), dNTP (400 uM of each), MgSO4 (1.5 mM) and Platinum¨ Taq DNA polymerase High Fidelity (1U) and barcoded library adapters (400 nM). V1-V3 primers: 27F AGAGTTTGATCCTGGCTCAG and 534R ATTACCGCGGCTGCTGG. Thermocycler settings: Initial denaturation at 95űC for 2 min, 30 cycles of 95űC for 20 s, 56űC for 30 s, 72űC for 60 s and final elongation at 72űC for 5 min. PCR reactions were run in duplicate for each sample and pooled afterwards. Purification of the amplicon libraries was performed using the Agencourt AMPure XP bead protocol (Beckman Coulter, USA). Individual libraries were quantified with Quant-iT HS dsDNA assay (Life Technologies, USA) and quality checked on a Tapestation 2200 (Agilent, USA). Libraries were pooled in equimolar concentrations, and diluted to 4 nM. The library pool was sequenced using  MiSeq reagent kit v3 (2x300 PE).