两种台式高通量测序平台应用于癫痫患者基因突变检测的对比研究  

Comparison of two benchtop next generation sequencing platforms for mutation screening in patients with epilepsy

作　　者：司锘[1] 王蓉蓉[1] 张俐俐 李小玲 汪涵[1] 卢超霞[1] 柳青[3] 张学[1] Si Nuo;Wang Rongrong;Zhang Lili;Li Xiaoling;Wang Hart;Lu Chaoxia;Liu Qing;Zhang Xue(McKusick- Zhang Center for Genetic Medicine, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing 100005, China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院,医学分子生物学国家重点实验室,麦库西克-张孝骞协和遗传医学中心,北京100005 [2]中国医科大学细胞生物学系,沈阳110122 [3]中国医学科学院北京协和医院神经科,北京100073

出　　处：《国际遗传学杂志》2018年第1期1-6,共6页International Journal of Genetics

基　　金：中国医学科学院医学与健康科技创新工程（2016-I2M-1-002）;北京市科技计划（Z151100003915078）;国家高技术研究发展计划863计划（2015AA020407）

摘　　要：目的 应用 Illumina MiSeq 和Ion Torrent PGM 两种台式高通量测序平台,使用同样的序列捕获系统,结合两个平台各自配备的标准化试剂及软硬件设备,对癫痫患者进行突变检测,对比两个平台在使用成本、操作难易度、突变检测效果等方面的表现,从而针对癫痫多基因诊断 panel 优化检测流程、指导测序平台选择.方法 获取患者知情同意后采集30名癫痫患者外周血,提取基因组 DNA.其中8名患者 DNA 采用 Ion XpressTMPlus gDNA Fragment Library Preparation,另22名患者基因组 DNA 采用 Illumina TruSeq DNA LT sample prep kit 进行文库制备,之后使用 NimbleGen 定制序列捕获系统对428个目的基因的编码区进行富集.构建完成的文库分别采用 Ion Torrent PGM 和 Illumina MiSeq 测序平台测序.Ion Torrent PGM 平台产出数据用Ion Torrent Suite 3.2软件进行序列比对及SNPs 和Indels 提取,Illumina MiSeq 平台产出数据用 BWA-MEM 和 Samtools 软件进行序列比对和变异提取.结果 Illumina MiSeq 平台测序每次运行最多可完成12个样品的测序,总数据量超过5 Gb,平均测序深度达137 ×,靶区域覆盖度为96.4%,平均每个样本提取2056 ±9.042个变异,从样本制备到数据产出周期约6天.Ion Torrent PGM 平台一张318 v2芯片可得到1.4 Gb 数据量,8个样本平均测序深度达70 ×,靶区域覆盖度为96.1%,平均每个样本提取3028 ±678.9个变异,从样品制备到数据产出周期约6天.PGM 平台使用成本低于 MiSeq,而 MiSeq 平台操作易用性及测序质量优于PGM 平台.结论 靶序列捕获结合下一代测序技术可以实现对癫痫致病基因的批量快速检测,两种台式高通量测序仪均可满足检测需求,Ion Torrent PGM 平台应用灵活、成本经济.Illumina MiSeq 平台单次运行产出数据大,数据准确性高,适用于大批量样品检测.Objective We performed mutation screening in patients with epilepsy by using two benchtop next-generation sequencing platforms, Illumina MiSeq and Ion Torrent PGM.The same target capture system was used, together with standard reagents, software and hardware for each of the plat-forms.Here we compared their performance and discussed the advantages and disadvantages of each se -quencer, optimized the process of mutation screening and provided suggestions for sequencing platform se -lection.Methods The peripheral blood of 30 patients with epilepsy was collected after obtaining informed consent, and genomic DNA was extracted.Ion XpressTMPlus gDNA Fragment Library Preparation kit and Illumina TruSeq DNA LT sample prep kit were used for library preparation in 8 and 22 samples, respec-tively.Coding region sequences of 428 target genes were enriched by NimbleGen target capture system and sequenced either on Illumina MiSeq or Ion Torrent PGM.Output data from PGM were analyzed by Ion Torrent Suite 3.2 for alignment and variants extraction, while data from Miseq were analyzed by BWA-MEM and Samtools.Results Up to 12 samples could be sequenced on Illumina Miseq per run, with to-tal throughput more than 5 Gb, 137 x mean coverage sequencing depth, 96.4% capture efficiency rate on target regions and 2056 ±9.042 variants detected per sample, and the whole test period lasted for 6 days.Comparatively, up to 8 samples per 318 v2 chip could be sequenced on PGM, with total through-put about 1.4 Gb, 70 x mean coverage sequencing depth, 96.1% capture efficiency rate on target re-gions and 3028 ±678.9 variants detected per sample,and the test period was 6 days.Conclusion Targeted sequence capture combined with next generation sequencing could be an efficient tool for rapid mutation detection in epilepsy.All two benchtop next-generation sequencers evaluated here could generate usable sequences; however there are some differences between the qualities of their data.Ion Torrent PGM is more flexible and cost effective, while Illumina MiS

关 键 词：台式高通量测序平台 MiSeq测序仪 PGM测序仪 

分 类 号：R742.1[医药卫生—神经病学与精神病学] R440[医药卫生—临床医学]