16S rRNA-Seq of total bacteria: aquaculture tank
The 16S rRNA gene was amplified with the primers 515F (5-GTG CCA GCM GCC GCG GTA A-3) and 806R (5-GGA CTA CHV GGT WTC TAA T-3) that target the hypervariable V4 region. The 50-L PCR mixture contained 25 L of 2  Premix Taq (Takara Biotechnology, Dalian Co. Ltd., China), 1L of each primer (10 M), 3L of template DNA (20 ng/L). All mixtures had ddH2O added to a volume of 50L and PCR were performed on a BioRad S1000 PCR thermal cycler (Bio-Rad Laboratory, CA, USA). The thermocycling steps were as follows: 94 for 5 min, followed by 30 cycles at 94 for 30 s, 52  for 30 s, 72  for 30s and a final extension step at 72  for 10 min.