基于二代测序的多重竞争性PCR方案的建立及应用

发布时间：2018-04-21 14:54

本文选题：二代测序 + 多重竞争性PCR　；参考：《东华大学》2017年硕士论文

【摘要】：复杂性状的系统遗传学研究中,分析差异表达基因,可从中获取基因转录,基因调控,信号转导通路及其相互联系等相关信息;进而揭示基因在时间、空间上的表达模式。常用的技术有传统的竞争性PCR、实时荧光定量PCR、基因芯片表达谱以及近年来蓬勃发展的基于二代测序平台的各种基因表达分析技术等,研究者常常根据具体实验需求而选取适当的技术。本研究结合多重竞争性PCR和Ion proton二代测序平台,开发了一种针对大样本量的目的基因差异表达分析方案,并应用于野生来源1号染色体小鼠血脂调控相关系统遗传学研究中,为小鼠血脂异常这一复杂性状研究提供了丰富的基因表达数据。基于二代测序的多重竞争性PCR技术包含如下关键点:1)竞争性模板的构建,采用单碱基突变的特异性逆转录引物,通过逆转录反应快捷制备竞争性模板;2)PCR扩增效率标准曲线,竞争性内参模板和目标模板以不同比例梯度混合,通过标准曲线校正引物实际扩增效率带来的定量偏差;3)多重PCR均一性的调整,针对同管反应不同的扩增子,调整特异性引物浓度比例,采用实时荧光定量PCR方式进行定量,每个产物对应一个通用性上游引物和一个特异性下游引物,从而保证多个扩增子间的均一性;4)Ion proton平台测序,基于该平台的数据特点,本研究开发了一套匹配检测基因、分离不同的竞争性模板和reads数目统计分析的完整流程,可快速准确统计结果。首先用8个基因作为小试,对该技术方案的可行性进行评估,结果显示:1)标准曲线拟合度系数R2均大于0.98,优于real-time PCR的拟合度(0.97-0.99),可更精确的校正引物扩增效率偏差;2)计算3个实验重复的标准方差SD表明,该方案和real-time PCR具有相同的稳定性(SD0.05);3)检测2倍基因表达差异能力,该方案和real-time PCR结果有良好的一致性(R20.98),证明该方案优秀的灵敏度和准确度。基于上述结果,将该方案应用于172个小鼠样本113个基因差异表达分析中,发现在不同品系中的小鼠检测基因有显著的表达差异,提示转录水平的差异可能与血脂水平差异相关,为本课题组今后的系统遗传学研究奠定了数据基础;同时,检测113个扩增子的均一性,90%以上reads差异在30倍以内,解决了扩增子测序中过量测序(over-sequencing)和低表达转录本测序深度不足的难题,将目标片段测序成本大大降低。本研究建立的基于二代测序的多重竞争性PCR技术,具有优秀的稳定性,高灵敏度和高准确度,在小鼠血脂调控相关系统遗传学研究中的应用证明了其低廉的成本,以及快捷的后续数据分析。在今后复杂性状疾病的系统遗传学研究中,基于二代测序的多重竞争性PCR一定可以成为众多研究者的热门选择技术。
[Abstract]:In the systematic genetics of complex traits, the differential expression genes can be analyzed to obtain related information such as gene transcription, gene regulation, signal transduction pathways and their interrelationships, and thus reveal the expression patterns of genes in time and space. The commonly used techniques include traditional competitive PCRs, real-time fluorescent quantitative PCRs, microarray expression profiles and various gene expression analysis techniques based on the second-generation sequencing platform. Researchers often select appropriate techniques according to specific experimental needs. In this study, combined with multiple competitive PCR and Ion proton second-generation sequencing platforms, a novel gene differential expression analysis scheme for large sample size was developed and applied to the genetic studies on lipid regulation in mice with chromosome 1 from wild origin. It provides abundant gene expression data for the complex study of dyslipidemia in mice. The multiplex competitive PCR technique based on second generation sequencing consisted of the following key points: 1) Construction of competitive template. Using specific reverse transcription primers of single base mutation, the competitive template was quickly prepared by reverse transcription reaction to prepare the standard curve of amplification efficiency of competitive template. The competitive internal reference template and the target template were mixed with different proportion gradients, and the quantitative deviation caused by the actual amplification efficiency of the primers was corrected by standard curve to adjust the homogeneity of multiple PCR. The ratio of specific primer concentration was adjusted, and real-time fluorescent quantitative PCR was used to quantify each product. Each product was matched with a universal upstream primer and a specific downstream primer, so as to ensure the homogeneity of multiple amplifiers on the proton platform. Based on the data characteristics of the platform, a set of matching detection genes was developed to separate different competitive templates and a complete process of reads number statistical analysis, which can quickly and accurately calculate the results. The feasibility of the technique was evaluated using eight genes as a pilot test. The results showed that the fitting coefficient R2 of the standard curve was greater than 0.98, which was better than that of real-time PCR (0.97-0.99g), which could more accurately calibrate the amplification efficiency deviation of primer. This scheme has the same ability as real-time PCR to detect the difference of 2 times gene expression. It has good agreement with the result of real-time PCR, which proves the excellent sensitivity and accuracy of the scheme. Based on the above results, 113 differentially expressed genes were analyzed in 172 mouse samples. The results showed that there were significant differences in the expression of the detected genes in different strains of mice, suggesting that the difference of transcription level may be related to the difference of blood lipid level. The results provided a data basis for the future systematic genetics research of our group, and the difference of reads among 113 amplifiers was less than 30 times, and 90% of the homogeneity of the three amplifiers was less than 30 times. The problem of excessive sequencing over-sequencing in Amplifier sequencing and insufficient sequencing depth of low expression transcripts were solved, and the cost of target fragment sequencing was greatly reduced. The multiplex competitive PCR technique based on second generation sequencing has excellent stability, high sensitivity and high accuracy. It has been proved to be low cost by its application in the genetics of mouse blood lipid regulation system. And quick follow-up data analysis. In the future, multiplex competitive PCR based on second-generation sequencing will be a hot choice for many researchers in the field of systemic genetics of complex diseases.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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