基于单细胞靶向测序探究基因碱基突变的方法  

A method for reliable detection of genomic point mutations based on single-cell target-sequencing

作　　者：赵利楠 王娜 杨国良[2] 苏现斌 韩泽广 Linan Zhao;Na Wang;Guoliang Yang;Xianbin Su;Zeguang Han(Key Laboratory of Systems Biomedicine(Ministry of Education),Shanghai Center for Systems Biomedicine,Shanghai Jiao Tong University,Shanghai 200240,China;Department of Urology,Renji Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200127,China)

机构地区：[1]上海交通大学,系统生物医学研究院,系统生物医学教育部重点实验室,上海200240 [2]上海交通大学医学院附属仁济医院泌尿科,上海200127

出　　处：《遗传》2020年第7期703-713,共11页Hereditas（Beijing)

基　　金：上海交通大学科技创新专项资金项目(编号:2019TPA09);国家自然科学基金青年项目(编号:81802806)资助。

摘　　要：分析基因碱基突变是探究肿瘤细胞克隆演化的研究方法之一。目前,常取组织不同区域的群体细胞测序,基于群体水平的基因碱基突变频率等信息绘制出肿瘤的克隆演化过程。但是,该方法易遗失低频突变,并且组织群体细胞类型不单一,不能代表特定细胞群体的克隆演化过程。本研究以前列腺基底细胞癌(prostate basal cell carcinoma, BCC)为例,建立了一种探究肿瘤单细胞的基因碱基突变方法,实现了基于单细胞基因碱基突变分析肿瘤细胞的克隆演化过程。首先通过HepG2细胞优化了单细胞全基因组扩增方法,其次用Fluidigm公司的微流控芯片捕获BCC单细胞进行全基因组扩增,然后通过外显子组测序获得SCUBE3和MST1L基因碱基突变信息。通过单细胞靶向扩增和Sanger测序,成功在BCC单细胞中检测到SCUBE3和MST1L基因碱基突变信息。本研究建立的方法为肿瘤细胞的克隆演化研究提供了一种可靠的技术方案。The analysis of genomic point mutations is one of the research strategies to explore the clonal evolution of tumor cells.At present,clonal evolution of tumor cells is mainly determined by bulk sampling and sequencing of different sections of the tumor.Since this approach analyzes a mixture of different cell types,it may not accurately represent the clonal evolution of specific tumor cell populations and likely miss low frequency mutations,especially when the sequencing depths are not sufficient.To address this issue,we have developed a strategy to analyze genomic point mutations from prostate basal cell carcinoma(BCC)tissues at single-cell resolution.Firstly,we optimized the single-cell whole genome amplification procedure with HepG2 cells.Then the single cells from BCC tissue were captured by a microfluidic chip of Fluidigm and processed for whole-genome amplification.Both SCUBE3 and MST1L genomic mutations were obtained by whole exome sequencing.Finally,we examined the genomic mutations through single-cell targeted amplification and Sanger sequencing.The established method successfully reconfirmed the mutations of SCUBE3 and MST1L in BCC at single cell level.The strategy established in this study could provide a useful tool for determining the clonal evolution of tumor cells based on genomic mutations at single-cell resolution.

关 键 词：肿瘤细胞 基因碱基突变 克隆演化 单细胞 

分 类 号：R346[医药卫生—基础医学]