16-S gene sequencing of wastewater metagenome
The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F: 5'- ACTCCTACGGGAGGCAGCA-3' and 806R: 5'- GGACTACHVGGGTWTCTAAT-3' . Both the forward and reverse 16S primers were tailed with sample-specific Illumina index sequences to allow for deep sequencing. The PCR was performed in a total reaction volume of 10 l: DNA template 5-50 ng, forward primer (10M) 0.3 l, reverse primer (10M) 0.3 l, KOD FX Neo Buffer 5 l, dNTP (2 mM each) 2 l, KOD FX Neo 0.2 l, and finally ddH2O up to 20L.After with initial denaturation at 95 C for 5 min, followed by 20 cycles of denaturation at 95 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 40 s, and a final step at 72 C for 7 min. The amplified products were purified with Omega DNA purification kit and quantified using Qsep-400.The amplicon library was paired-end sequenced (2250) on an Illumina novaseq6000.