title: NPO_category_05
design: The V3 and V4 regions of the 16S rRNA gene were amplified using primers compatible with the Nextera Index Kit (Illumina) (NXt_V3-V4_F 5’-TCGTCGGCAGC GTCAGATGTGTATAAG AGACAGCCTAYGGGRB GCASCAG-3’ and NXt_V3-V4_R 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNNGGGTATCTAAT-3’; adapters in bold). PCR reactions containing 12 μl AccuPrimeTM SuperMix II (Life Technologies, CA, USA), 0.5 μl of each primer (10 μM), 5 μl of genomic DNA (~10 ng/ul), and nuclease-free water to a total volume of 20 µl were run on a SureCycler 8800 (Agilent, CA, USA). Cycling conditions applied were: Denaturation at 95°C for 2 min; 35 cycles of 95°C for 15 s, 55°C for 15s and 68°C for 40 s; followed by final elongation at 68°C for 5 min. To incorporate primers with adapters and indexes, PCR reactions contained 12 μl Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA, MA), 2 μl corresponding P5 and P7 primer (Nextera Index Kit), 2μl PCR product and nuclease-free water for a total volume of 25 μl. Cycling conditions applied were: 98°C for 1 min; 12 cycles of 98°C for 10 s, 55°C for 20 s and 72°C for 20 s; elongation at 72°C for 5 min. The amplified fragments with adapters and tags were purified using AMPure XP beads (Beckman Coulter Genomic, CA, USA). Prior to library pooling clean constructs were quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and mixed in approximately equal concentrations to ensure even representation of reads per sample followed 250 bp pair-ended MiSeq (Illumina) sequencing performed according to the instructions of the manufacturer.