DNA was extracted from the feces and liver using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the protocol for isolation of DNA. Illumina MiSeq sequencing and general data analyses were performed by a commercial company (Majorbio Bio-Pharm Technology, Shanghai, China). Because of initially low bacterial DNA concentrations in some samples, a nested PCR was applied to increase specificity and amplicon yield [73 (link), 74 (link)]. The V3–V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30s for annealing at 55 °C, 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. The resulted PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s protocol. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw fastq files were demultiplexed, quality-filtered by Trimmomatic, and merged by FLASH with the following criteria: (a) The reads were truncated at any site receiving an average quality score < 20 over a 50-bp sliding window. (ii) Primers were exactly matched allowing two nucleotide mismatching, and reads containing ambiguous bases were removed. (iii) Sequences whose overlap longer than 10 bp were merged according to their overlap sequence. Operational taxonomic units (OTUs) were clustered with 97% similarity [75 (link)] cutoff using UPARSE (version 7.1 http://drive5.com/uparse/ ) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm (http://rdp.cme.msu.edu/ ) against the Silva (SSU123) 16S rRNA database using a confidence threshold of 70%.
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