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Microbial DNA was extracted from the frozen fecal samples using a TIANamp Stool DNA kit (DP328, Tiangen Biotech, Beijing, China) according to the manufacturers instructions. The DNA quality and concentration were measured by agarose gel electrophoresis and a Qubit fluorometer (Life Technologies, Waltham, MA) before downstream high-throughput sequencing. For 16S rRNA sequencing, variable regions 3 and 4 of the 16S rRNA gene were amplified using the 241F and 805R primers. The purified v3,v4 amplicons were sequenced on a HiSeq2500 platform (Illumina, San Diego, CA) with paired-end 250 bp sequencing. |
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