一种新型线粒体DNA深度测序方法的准确性评估  

Accuracy evaluation of a novel method for mitochondrial DNA deep sequencing

作　　者：朱琳[1] 林颖[2] 朱玉青 黄明涛 梁栋[2] 胡平[2] 许争峰[2] 张军[1] ZHU Lin;LIN Ying;ZHU Yuqing;HUANG Mingtao;LIANG Dong;HU Ping;XU Zhengfeng;ZHANG Jun(State Key Laboratory of Reproductive Medicine,Nanjing Medical University,Nanjing 211166,Jiangsu;Department of Prenatal Diagnosis,Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University,Nanjing Maternity and Child Health Care Hospital,Nanjing 210004,Jiangsu,China)

机构地区：[1]南京医科大学生殖医学国家重点实验室,南京211166 [2]南京医科大学附属妇产医院&南京市妇幼保健院产前诊断中心,南京210004

出　　处：《临床检验杂志》2023年第3期167-170,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然基金面上项目(31871445)。

摘　　要：目的 与基于长片段PCR(lrPCR)的传统mtDNA测序方法比较,评估本课题组前期研究开发的一种新型非PCR依赖的mtDNA深度测序方法的可靠性。方法 同时采用新方法和传统方法对10例产前筛查孕妇外周血样本进行平行试验,对比两种方法所得结果,结合人群数据库(MitoMAP和mtDB)信息,评估新方法的准确性。结果 10例样本中共检出380个变异,其中351个变异同时被两种方法检出,29个变异仅被单一方法检出。对29个不一致变异中出现于2个或2个以上样本的8个变异进一步分析发现,2个仅被新方法重复检出的变异在人群数据库中存在记录;而6个仅传统方法重复检出的变异在人群数据库中未见记录。结论 与传统基于lrPCR的mtDNA测序方法相比,新方法针对同质性变异及高异质度变异的检测能力相当,而针对低异质度变异检测时具有更高的准确性。Objective To evaluate the accuracy of a novel PCR-independent mtDNA deep sequencing method developed in previous studies compared with the traditional mtDNA sequencing based on long fragment PCR(lrPCR)method.Methods Ten peripheral blood samples were simultaneously subjected to parallel experiments using the both methods:novel and traditional lrPCR-based NGS(next gene sequence)method.The sequencing results obtained from the two approaches were compared,and the accuracy was evaluated by combining the information from 2 population databases(MitoMAP and mtDB).Results A total of 380 variants were detected in the 10 samples.Of these,351 were detected by the both methods,and 29 were detected exclusively by single method.The 8 variants in the 29 inconsistent variants which appeared in 2 or more samples were further analyzed.The results showed that 2 variants which were only repeatedly detected by the new method were recorded in the population database.However,none of the 6 variants detected only by the traditional method were recorded in the population databases.Conclusion Compared with the traditional lrPCR-based NGS method,the newly developed method has exhibited a similar detection capability for both of homoplasmic and high-level heteroplasmic variantsThe new method exhibited higher accuracy for low heterogeneity variation detection and has shown advantages of higher accuracy in detection for low level of heteroplasmic variants.

关 键 词：线粒体分离纯化 长片段PCR 线粒体DNA 高通量测序 异质性 异质度 变异 

分 类 号：R446[医药卫生—诊断学] R394[医药卫生—临床医学]