title: 16-S rRNA metagenomic sequencing of gut microbiome
design: DNA extractionDNA extraction was carried out in a clean bench (Heraguard ECO Clean Bench, Thermo Scientific). Total DNA of feces was extracted using the Qiagen Stool Mini Kit according to the manufacturers instructions (Qiagen, Germany). The 16SrRNA gene hypervariable region V3 was amplified using barcoded PCR primers (V3-FCCAGACTCCTACGGGAGGCAGV3-RCGTATTACCGCGGCTGCTG ). The PCRs included approximately 5~10 ng DNA template, 3mol of each primer in 20 L volume of TopTaq buffer containing 2 units TopTaq DNA polymerase (Qiagen, Germany). After denaturation at 94 C for 20s, the PCR amplification was carried out for 30 cycles using the following parameters: 2 min 94 C pre-denaturation, 60 C annealing for 20 s, 72 C extension for 30s, and held at 72C for 10min. DNA concentration and purity was evaluated on 1% agarose gels. DNA was diluted to 1 ng/l using sterile water.16S rRNA gene sequencing and quality controlPCR products were purified with GeneJET Gel Extraction Kit (Thermo Fisher Scientific, USA). Sequencing libraries were generated using Illumina TruSeq DNA PCR-Free Library Preparation Kit (Illumina, USA) following manufacturers recommendations and index codes were added. The library was sequenced on an Illumina HiSeq platform, and the paired-end reads were merged using FLASH software (V1.2.7, http://ccb.jhu.edu/software/FLASH).