三种DNA提取方法对半滑舌鳎肠道菌群高通量测序结果的影响

doi:10.19640/j.cnki.jtau.2022.03.006

天津农学院学报 ›› 2022, Vol. 29 ›› Issue (3): 29-35.doi: 10.19640/j.cnki.jtau.2022.03.006

三种DNA提取方法对半滑舌鳎肠道菌群高通量测序结果的影响

葛仑华, 江博赟, 孙敬锋^通信作者, 吕爱军, 胡秀彩, 陈丽梅

天津农学院水产学院,天津 300392

收稿日期:2020-07-24 出版日期:2022-09-30 发布日期:2022-10-10
通讯作者: 孙敬锋（1976—）,男,教授,博士,主要从事水产动物疾病防治研究工作。E-mail：sun_jf@163.com。
作者简介:葛仑华（1997—）,男,硕士在读,主要从事鱼类疾病与免疫学研究。E-mail：654430388@qq.com。
基金资助:
国家自然科学基金项目（31972840）; 天津市自然科学基金项目（19JCZDJC34600）; 天津市企业科技特派员项目（18JCTPJC64700,19JCTPJC60100）

Effects of different DNA extraction methods on structural analysis of intestinal microbiota from Cynoglossus semilaevis based on high-throughput sequencing

Ge Lunhua, Jiang Boyun, Sun Jingfeng^{Corresponding Author}, Lü Aijun, Hu Xiucai, Chen Limei

College of Fisheries, Tianjin Agricultural University, Tianjin 300392, China

Received:2020-07-24 Online:2022-09-30 Published:2022-10-10

摘要/Abstract

摘要： 本研究旨在分析不同DNA提取方法对半滑舌鳎（Cynoglossus semilaevis）肠道菌群结构分析结果的影响,并了解健康半滑舌鳎肠道菌群结构特征。通过溶菌酶结合超声波裂解法（CLU）、Zirmil-beating细胞破碎方法（ZBC）和QIA商业试剂盒三种方法提取半滑舌鳎肠道菌群基因组DNA,采用Illumina MiSeq高通量测序分析三种方法提取的样品DNA,比较其菌群结构特征和多样性。结果显示,三种方法提取的样品经检测共得到7 516个OTU,而且鉴定的优势菌门一致,主要包括变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、螺旋菌门（Spirochaetae）、拟杆菌门（Bacteroidetes）。通过Alpha多样性指数分析,CLU、ZBC、QIA方法对应的Chao1指数分别为5 668、5 868、4 090;Simpson指数分别为0.021、0.022、0.018,QIA方法的Chao1和Simpson指数均低于其他两种方法。基于UniFrac距离矩阵进行Beta多样性分析显示CLU和QIA方法提取的样品之间的微生物群落结构和物种丰富度更相似。本研究表明不同方法提取的DNA经高通量测序分析鉴定到的主要优势菌群类别一致,但对一些丰度较低的菌群类别鉴定时存在偏差。

关键词: 高通量测序, DNA提取方法, 半滑舌鳎, 肠道菌群, 组成结构

Abstract: The purpose of this study was to analyze the effects of different DNA extraction methods on structural analysis of intestinal microbiota from C. semilaevis based on high throughput sequencing and comprehensively learn about the structural characteristics of its intestinal flora. In this study, three DNA extraction methods including combination of lysozyme and ultrasonic lysis（CLU）, Zirmil-beating cell disruption（ZBC）and QIA commercial kits were used to extract the genomic DNA of intestinal microflora from healthy C. semilaevis. The DNA samples were sequenced on an Illumina MiSeq high-throughput sequencing platform, and the structural characteristics and diversities of the intestinal microflora were analyzed. The results showed that a total of 7 516 OTUs were obtained across all samples extracted by the three DNA extraction methods. The identified dominant bacterial taxa were similar among these samples. At Phylum level, the dominant bacterial taxa included Proteobacteria, Firmicutes, Spirochaetae and Bacteroides. Through Alpha diversity index analysis, the corresponding Chao1 indexes of CLU, ZBC and QIA methods were 5 668, 5 868 and 4 090 respectively, and the Simpson index were 0.021, 0.022 and 0.018 respectively. The Chao1 and Simpson indexes of QIA methods were all lower than those of other methods. Beta diversity analysis based on the UniFrac distance matrix showed that the microbial community structure and species richness were more similar between the CLU and QIA samples. These results indicated that these three different DNA extraction methods reached the similar results in the major dominant intestinal microflora, based on the investigation using high-throughput sequencing analysis. However, biases occurred in the identification results of some less abundant intestinal microflora. The results providereference for research methods of the diversity and structure of fish intestinal microflora, especially the DNA extraction strategy, and contribute to comprehensively expounding the structural characteristics of intestinal microflora of C. semilaevis.

Key words: high-throughput sequencing, DNA extraction method, C. semilaevis, intestinal microflora, composition structure

中图分类号:

S917.1

葛仑华, 江博赟, 孙敬锋, 吕爱军, 胡秀彩, 陈丽梅. 三种DNA提取方法对半滑舌鳎肠道菌群高通量测序结果的影响[J]. 天津农学院学报, 2022, 29(3): 29-35.

Ge Lunhua, Jiang Boyun, Sun Jingfeng, Lü Aijun, Hu Xiucai, Chen Limei. Effects of different DNA extraction methods on structural analysis of intestinal microbiota from Cynoglossus semilaevis based on high-throughput sequencing[J]. Journal of Tianjin Agricultural University, 2022, 29(3): 29-35.

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http://xuebao.tjau.edu.cn/CN/Y2022/V29/I3/29

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三种DNA提取方法对半滑舌鳎肠道菌群高通量测序结果的影响

Effects of different DNA extraction methods on structural analysis of intestinal microbiota from Cynoglossus semilaevis based on high-throughput sequencing

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