16S rRNA
Extraction of bacterial DNA was performed from fecal samples by QIAamp Fast DNA Stool Mini Kit as previous reported (Wu et al., 2016). The freshly extracted DNA was purified by 0.5% melting point agarose gel followed by phenolchloroform-butanol extraction. The V3-V4 region of the bacterial 16S rRNA gene from each DNA sample was amplified using the bacterial universal primers (forward primer: 5  -ACTCCTACGGGRSGCAGCAG-3  ; reverse primer: 5  -GGACTACVVGGGTATCTAATC-3  ).The PCR amplification products were purified through AxyPrep DNA Gel Extraction Kit (Axygen, USA), eluted in 30  l water, and aliquoted into three PCR tubes. DNA quality and quantity were assessed by the ratios of 260/280 nm and 260/230 nm, and final DNA contents were quantified with a Qubit? dsDNA HS Assay Kit (Invitrogen, USE). Finally, we used of bacterial DNA amplicons from each fecal sample for 2 * 250 bp paired-end sequencing based on the Illumina Hiseq 2500.