454 of Bacteria and Crenarchaeota: Active AOB and AOA
The V4V5 hypervariable regions of the bacterial 16S rRNA genes (Escherichia coli positions 515907) were amplified using the barcode-tagged primer set F515 (GTGCCAGCMGCCGCGG) and R907 (CCGTCAATTCMTTTRAGTTT). The PCR products of each sample were pooled togetherand purified using an Agarose Gel DNA purification kit (TaKaRa,Dalian, China). An equal amount of PCR product for each samplewas combined into a single tube and pyrosequencing wasperformed on a Roche FLX 454 pyrosequencer.