title: D50_8#7
design: The V4 highly variable region of the 16S rRNA gene was amplified using primers (forward primer: 5-barcode-AYTGGGYDTAAAGNG-3; reverse primer: 5-TACNVGGGTATCTAATCC-3). The barcode is a 7 bp oligonucleotide used to discriminate the samples. There were 9 replicates for each group and a total of 45 samples were included in the experiment. The PCR program was as follows: 98 C for 30 sec; followed by 25 cycles of 98 C for 15 sec, 50 C for 30 s, 72 C for 30 s; and 72 C for 5 min. The target PCR fragment was purified through gel electrophoresis. Subsequently, end repair, A tailing and adaptor ligation were performed in turn to construct sequencing library. The libraries were sequenced for 300 bp at both ends using a Miseq system from Illumina (San Diego, CA, USA).