Illumina NovaSeq 6000 sequencing; Differences in gut microbiota and its metabolic function among different fasting plasma glucose groups in Mongolian population of China
Fresh feces of volunteers were collected, frozen and stored in -80℃ for a few months. The total DNA from every fecal sample was extracted by Magnetic Soil And Stool DNA Kit of TIANGEN BIOTECH (BEIJING) CO., LTD, then constructed into a library and sequenced by Illumina PE150. This process was completed by the Wuhan metware Biotechnology Co., Ltd.There are mainly two methods in QC for DNA samples:(1) DNA degradation degree and potential contamination was monitored on 1%agarose gels. (2) DNA concentration was measured using Qubit® dsDNA Assay Kit inQubit® 2.0 Flurometer (Life Technologies, CA, USA). OD value is between 1.8~2.0,DNA contents above 1ug are used to constructlibrary.  A total amount of 1μg DNA per sample was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB,USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, the DNA sample was fragmented by sonication to a size of 350bp,then DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. At last, PCR products were purified (AMPure XP system) and libraries were analysed for size distribution by Agilent2100 Bioanalyzer and quantified using real-timePCR.