title: sequence of bacteria
design: Specific PCR pruducts was send to Sangon Biotech(Shanghai,China) Co.Ldt for sequencing library preparation and sequencing.Briefly, Library preparation was performed by two step PCR. First round PCR reaction was set up as follows: DNA (10 ng/l) 2l; amplicon PCR forward primer mix (10 M) 1l; amplicon PCR reverse primer mix (10 M) 1l; 2PCR Ready Mix 15 l (total 25l)(Kapa HiFi Ready Mix). The plate was sealed and PCR performed in a thermal instrument (BIO-RAD, T100TM) using the following program: 1 cycle of denaturing at 98 C for 5 min, then 8 cycles of denaturing at 98 C for 30 s, annealing at 60 C for 30 s, elongation at 72 C for 30 s, and a final extension at 72 C for 5 min. Finally hold at 4The PCR products were checked using electrophoresis in 1 % (w/v) agarose gels in TBE buffer (Tris, boric acid, EDTA) stained with SYBR Green I and visualized under UV light. Then we used AMPure XP beads to purify the amplicon product. After that, the second round PCR was performed. PCR reaction was set up as follows: DNA (10 ng/l) 2l; universal P7 primer with index(10 M) 1l; P5 primer with index(10 M) 1l; 2 PCR Ready Mix 15 l (total 30l)(Kapa HiFi Ready Mix). The plate was sealed and PCR performed in a thermal instrument (BIO-RAD, T100TM) using the following program: 1 cycle of denaturing at 98 C for 3 min, then 5 cycles of denaturing at 94 C for 30 s, annealing at 55 C for 20 s, elongation at 72 C for 30 s , and a final extension at 72 C for 5 min. Then we used AMPure XP beads to purify the amplicon product. The libraries were then quantified and pooled. Paired-end sequencing of the library was performed on the NovaSeq6000 model (Illumina, San Diego, CA).