Comparison of direct boiling with commercial kits for extracting fecal microbiome DNA with Illumina sequencing of 16S rRNA tags
首发时间:2013-01-23
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)
keywords: Microbiome feces DNA extraction Illumina microbial diversity
点击查看论文中文信息
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
摘要:利用新一代测序技术进行宏基因组16S rRNA的检测,具有通量高、性价比好等优点,这些技术显著改善了我们对人体健康以及环境科学领域微生物的认识。然后,商业DNA提取试剂盒由于其成本高、耗时长的特点,已逐渐成为主要的瓶颈。因此,在本研究中,我们选用了直接煮沸法以及五种不同种类的商业试剂盒,比如Qiagen和MO BIO,来测定并评估粪便中的微生物组。利用Unifrac距离所得到的PCoA分析与聚类的结果显示,直接煮沸法中大部分粪便浓度与除MO BIO外的其余试剂盒法拥有相似的细菌群落结构;同时,直接煮沸法的粪便浓度影响着α多样性指数的评估,但其结果与试剂盒法基本相似;另一方面,直接煮沸法与商业试剂盒法所检测到的OTU呈现了高度的一致性,即使是群落结构差异最为显著的MO BIO试剂盒,所检测到的OTU中的中、高丰度部分,在直接煮沸法中也能获得。本研究表明,直接煮沸法可用于检测粪便中的微生物组,并且能够在肠道微生物组多样性研究中,显著降低成本和提高工作效率。
关键词: 微生物组 粪便 DNA提取 Illumina 测序 微生物群落
论文图表:
引用
No.****
同行评议
共计0人参与
勘误表
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
评论
全部评论0/1000