Miseq pe2500

The extraction of bacterial DNA from fresh sample was determined by the method of Li [25] . In brief, Phusion®High-Fidelity PCR Master Mix (New England Biolabs) was used to carry out PCR reactions, following the manufacturer's instructions. The primer 515 F and 907 R was chosen to amplify the V4-V5 region of 16S rRNA gene. The PCR amplicons were then sequenced by using an Illumina MiSeq PE2500 platform at Novogene Company (Beijing, China). After sequencing, paired reads were merged using FLASH (V 1.2.7), and ltered by QIIME. The uparse method was employed to assign operational taxonomic units (OTUs) to the 16S rRNA at a cutoff level of 3% on the Usearch software platform (version 7.1). Based on OTUs results, the alpha indices were calculated with QIIME (Version 1.7.0) and displayed with R software (Version 2.15.3).

Bacterial 16S rDNA V3-V4 regions were selected for microbial diversity detection, and the DNA samples were sent to Beijing Baimaike Gene Technology Co., Ltd. for sequencing using the Illumina MiSeq PE2500 high-throughput sequencing platform. A PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) was used to extract the DNA. The primers for the ampli cation of bacterial 16S rDNA V3-V4 were 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3'). The PCR system included 12.5 μL 2×Taq PCR MasterMix, 3 μL BSA (2 ng/L), 2 μL primer (5 μM), 2 μL primer, and 5.5 μL ddH 2 O. The reaction parameters were predenaturation at 95 °C for 5 min; denaturation at 95 °C for 45 s, annealing at 55 °C for 50 s, and elongation at 72 °C for 45 s, repeated for 32 cycles. The original sequence was uploaded to the NCBI SRA database after 10 min extension at 72 °C.