16S rRNA gene amplicons of activated sludge: sample 25
DNA was extracted from samples using the FastDNATM 2 mL SPIN Soil Kit (MP Biomedicals, CA, USA. Genomic DNA clean-up was done with the Genomic DNA Clean & Concentrator-25 (Zymo Research, USA) following the suppliers protocol. Amplicons were generated using the primer set targeting the bacterial V1-V3 region of the 16S rRNA gene (27F: AGAGTTTGATCCTGGCTCAG and 534R: ATTACCGCGGCTGCTGG). The PCR products purification was done following the Agencourt AMpure XP bead protocol (Beckmann Coulter, CA, USA) with a 1.8 beads solution to PCR product volume ratio. A second stage PCR was performed to add the adapters and dual-index barcodes according to Illuminas (CA, US) recommendations. Equimolar concentrations of the amplicon libraries (4 nM) were pooled and sequenced on a MiSeq (Illumina, CA, US) using the MiSeq Reagent kit v3 (2 x 300 paired end).