title: The hypervariable region (V3-V4) of bacterial 16SrRNA gene of WanJiang river, Anhui Province
design: The hypervariable region (V3-V4) of bacterial 16SrRNA gene in water DNA was amplified by PCR using universal primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHV GGGTATCTAATCC-3') [32]. The 5' ends of the primers were tagged with specific barcods per sample and sequencing universal primers.PCR amplification was performed in a total volume of 25 L reaction mixture containing 25 ng of template DNA, 12.5 L PCR Premix, 2.5 L of each primer, and PCR-grade water to adjust the volume. The PCR conditions to amplify the prokaryotic 16S fragments consisted of an initial denaturation at 98  for 30 seconds; 32cycles of denaturation at 98  for 10 seconds, annealing at 54  for 30 seconds, and extension at 72  for 45 seconds; and then final extension at 72  for 10 minutes. The PCR products were confirmed with 2% agarose gel electrophoresis. Throughout the DNA extraction process, ultrapure water, instead of a sample solution, was used to exclude the possibility of false-positive PCR results as a negative control. The PCR products were purifyied by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit( Invitrogen, USA). The amplicon pools were prepared for sequencing and the size and quantity of the amplicon library were assessed on Agilent 2100 Bioanalyzer (Agilent, USA) and with the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. The libraries were sequenced on NovaSeq PE250 platform.