16S rRNA gene amplicon sequencing of mainstream partial nitritation activated sludge bioreactors: BONCAT, experimental SBR, sludge pre-treat microcosm
16S rRNA gene fragments were pre-amplified using an initial round of 15-cycles of PCR with non-barcoded 515F (5'-GTGYCAGCMGCCGCGGTAA-3') and 926R (5'-CCGYCAATTYMTTTRAGTTT-3') primers (Parada et al., 2016) targeting the V4-V5 hypervariable region of the 16S rRNA gene. The resulting non-barcoded PCR products were then amplified using barcoded 515F and 926R primers. Amplified barcoded PCR products were pooled at equimolar concentrations and sequenced on an Illumina MiSeq in paired-end 300bp mode.