title: MiSeq of activated sludge: R2 at the end of phase II
design: DNA was extracted using a TIANamp Soil DNA Kit (Tiangen Biotech Inc., China) following the manufacturers protocol. DNA samples were quantified using a Qubit 2.0 Fluorometer (Invitrogen, USA). After that, the V3-V4 region of the bacterial 16S ribosomal RNA genes were amplified by PCR (initial denaturation at 95 C for 3 min, followed by 24 cycles at 94 C for 5 s, 57 C for 90s, and 72 C for 10 s and a final extension at 72 C for 5 min) using forward primers containing the sequence CCTACGGRRBGCASCAGKVRVGAAT and reverse primers containing the sequence GGACTACNVGGGTWTCTAATCC designed by Genewiz (Suzhou, China). Purified PCR products were quantified by Qubit 2.0 Fluorometer (Invitrogen, USA) and then they were sent to Genewiz Co., Ltd. in Suzhou for pair-end sequencing using the Illumina MiSeq sequencing platform (Illumina, USA).