宁都黄鸡粪便基因组DNA提取方法比较及盲肠微生物组成分析

Comparison of Genomic DNA Extraction Methods and Cecal Microbial Composition Analysis of Ningdu Huang Chicken Feces

要获得高质量的测序结果,一个重要的前提是提供高质量的DNA样品。该研究旨在比较4种样本预处理方法和目前国内通用的3种试剂盒对于盲肠内容物基因组DNA提取的质量,并通过扩增子测序结果分析宁都黄鸡盲肠菌群组成及其多样性,为进一步探索肠道微生物在宁都黄鸡生长中的作用,乃至未来禽微生态制剂的研发奠定基础。结果表明:QIAGEN、BioTeke或BIOMIGA公司试剂盒提取的基因组DNA用于扩增子测序,所得肠道微生物菌群Beta多样性分析结果无显著差异(P>0.05),而Shannon指数和Simpson指数组间差异显著(P<0.05),提示采用不同试剂盒可能对高通量测序分析结果产生影响。宁都黄鸡肠道微生物组成丰富,共检测到21个门、50个纲、80个目、134个科、268个属,相对丰度较高的菌群是拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、广古菌门(Euryarchaeota)和变形菌门(Proteobacteria),相对丰度较高的前3个属分别是甲烷杆菌属(Methanobrevibacter)、理研菌科RC9肠菌群(Rikenellaceae RC9 gut group)和拟杆菌属(Bacteroides)。宁都黄鸡盲肠菌群组成特征再次证明了宿主系统发生地位对肠道菌群结构起到的重要影响作用。

An important prerequisite for ensuring high-quality sequencing results is to provide high-quality DNA and RNA samples.The purpose of this study was to compare the quality of genomic DNA extracted from intestinal faeces by four sample pretreatment methods and three kinds of kits commonly used,and further analyze the composition and diversity of cecal flora of Ningdu Huang chicken by amplicon sequencing.This study could lay a basis for further exploration on the role of intestinal microbes in the growth of Ningdu Huang chicken,and even for the future development of avian microecological preparations.The results showed that when the genomic DNA extracted by QIAGEN,BioTeke or BIOMIGA kit was used for amplicon sequencing,there was no significant difference(P>0.05)in Beta diversity analysis of intestinal microbial flora,but there was significant difference between Shannon index and Simpson index among groups(P<0.05),suggesting that the use of different kits may have an effect on the results of high throughput sequencing analysis.The intestinal microflora of Ningdu Huang chicken was rich.A total of 21 phyla,50 classes,80 orders,134 families,and 268 genera were detected.The relatively abundant microflora belonged to Bacteroidetes,Firmicutes,Euryarchaeota and Proteobacteria,while the first three genus with high relative abundance were Methanobrevibacter,Rikenellaceae RC9 gut group and Bacteroides.The composition characteristics of cecum flora in Ningdu Huang chicken once again proved that the host systematic occurrence position played an important role in the construction of intestinal flora.