不同DNA提取方法引起鲤鱼肠道菌群多样性分析结果的偏差  

Deviation of Analysis Results of Intestinal Flora Diversity of Carp Caused by Different DNA Extraction Methods

作　　者：王浩伊 江博赟 孙敬锋 吕爱军 胡秀彩 Wang Haoyi;Jiang Boyun;Sun Jingfeng;Lv Aijun;Hu Xiucai(Tianjin Key Lab of Aqua-ecology and Aquaculture,Fisheries College of Tianjin Agricultural University,Tianjin,300384)

机构地区：[1]天津农学院水产学院,天津市水产生态及养殖重点实验室,天津300384

出　　处：《基因组学与应用生物学》2020年第8期3461-3467,共7页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31272692);天津市企业科技特派员项目(18JCTPJC64700);天津市水产产业技术体系创新团队建设项目(ITTFRS2017014)共同资助。

摘　　要：为了评价两种不同DNA提取方法引起的鲤鱼肠道菌群结构分析偏差。本研究分别以氧化锆珠破壁法(ZBC)和使用QIAamp Fast DNA Stool Mini Kit试剂盒法(QIA)3次重复提取鲤鱼肠道总DNA,检测DNA含量、纯度,进一步采用Illumina Hi Seq测序平台,利用高通量测序技术处理,来比较分析QIA组和ZBC组的菌群种类和结构偏差。结果表明,从QIA和ZBC样品分别获得(67045±10777.22)条和(79041±8168.68)条有效序列,QIA组样品OTU(Operational taxonomic unit)数量和Shannon多样性指数均显著高于ZBC组,且QIA组样品间OTU重复性优于ZBC组样品间。鲤鱼肠道菌群在门水平上排名前十的有:变形菌门(Proteobacteria),梭杆菌门(Fusobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Firmicutes),放线菌门(Actinobacteria),芽单胞菌门(Gemmatimonadetes),绿弯菌门(Chloroflexi),浮霉菌门(Planctomycetes),异常球菌-栖热菌门(Deinococcus-thermus)和疣微菌门(Verrucomicrobia)。分析得到ZBC法提取到的DNA浓度高,特有的OTU数目多;QIA法的测序结果稳定,菌群多样性和物种丰富度均较高。高通量测序结果表明两种方法提取鲤鱼肠道微生物DNA,其菌群多样性存在偏差,但两种方法均能有效提取到鲤鱼肠道DNA,可为鲤鱼肠道菌群的研究提供参考。The aim of this study was to study the biases from two DNA extraction methods of carp intestine microbiota revealed based on the Illumina HiSeq platform.We extracted the total DNA of intestinal microbiota from Cyprinus carpio L with Zirmil-beating Cell Disruption method,and the QIAamp Fast DNA Stool Mini Kit.DNA quality was evaluated based on the absorbance ratios of 260/280 nm by NanoDrop.Then Illumina HiSeq high-throughput sequencing was used to examine the intestinal bacterial communities following PCR amplification of 16S rDNA V4 region.The average sequence reads obtained from QIA and ZBC samples were 67045±10777.22 and 79041±8168.68 respectively.After resampling at the same depth of 43979 reads,the operational taxonomic unit(OTU)number and Shannon diversity index of QIA samples were significantly higher than those of ZBC samples.By contrast,the reproducibility of OTU among QIA replicates was higher than that among ZBC replicates.The dominant phyla of ZBC and QIA samples were identical,including Proteobacteria,Fusobacteria,Bacteroidetes,Firmicutes,Actinobacteria,Gemmatimonadetes,Chloroflexi,Planctomycetes,Deinococcus-Thermus,and Verrucomicrobia.And the extracted DNA concentration of ZBC is higher,and the unique OTU numbers are large;and the sequel QIAamp Fast DNA Stool Mini Kit is stable.Significant biases on community structure of carp intestinal microbiota were detected which originated from DNA extraction.However,both methods can effectively extract the intestinal DNA of carp,and could provide reference for research intestinal flora of carp.

关 键 词：鲤鱼 DNA提取 肠道菌群 高通量测序 

分 类 号：S917.4[农业科学—水产科学]