Total microbial DNA was extracted from fecal samples using a commercial stool DNA extraction kit (Qiagen, Germany, No. 51504) as the manufacturer’s protocol. All DNA samples were kept at −20°C until sequencing. The V4-V5 hypervariable region of the 16S ribosomal RNA gene was selected for amplification from DNA samples. The universal primers used were F515 (5′-GTGCCAGCMGCCGCGG -3′) and R907 (5′-CCGTCAATTCMTTTRAGTTT -3′) which also carried an eight-base unique sequence (a so called barcode) for each sample [24 ]. PCR reactions were run and amplicons sequenced as described previously [22 (link)].
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