以16S rRNA测序为基础,比较直接煮沸法与商业试剂盒法提取DNA的差异.docVIP

  Comparison of direct boiling with commercial kits for extracting fecal microbiome DNA with Illumina sequencing 5 10 15 20 25 of 16S rRNA tags# PENG Xin, ZHOU Hongwei, DENG Guanhua, JIANG Yunxia, WANG Yu, ZHANG Guoxia** (Department of Environmental Health, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong, China,510515) Abstract: High cost-efficiency and throughput are major advantages for determining metagenomic 16S rRNA tag sequences using next generation sequencing (NGS) techniques. These methods have significantly changed our view of microorganisms in fields of human health and environmental science. However, DNA extraction using commercial kits has become the major bottleneck because of its high cost and time consuming shortcomings. In the present study, we evaluated the determination of fecal microbiome using a direct boiling method compared to 5 different kinds of commercial methods, e.g. Qiagen and MO BIO kits. The PCoA analysis using UniFrac distances and the clustering result showed that direct boiling with a wide range of feces concentration had similar bacterial communities to most of the commercial kits, except the MO BIO method. The fecal concentration for boiling affected the estimation of α-diversity indices, but the results were generally comparable for boiling and commercial methods. The OTUs determined by direct boiling showed highly consistent frequencies with those determined by most of the commercial methods. Even for the MO BIO kit with an obviously different community structure, most of the OTUs determined at high to medium levels by the MO BIO kit were also determined in the direct boiling results with high confidences. Our present study suggested that direct boiling could be used to determine the fecal microbiome, and would significantly reduce the cost and improve the efficiency of the sample preparation for studying gut microbiome diversity.an) Key words: Microbiome; feces;DNA extractio