二代测序原理与技术(IonProton)

1|IonTorrentbyLifeTechnologies™ProprietaryandConfidentialIonProton™Sequencer——基研生物国内率先引进二代半测序平台2|IonTorrentbyLifeTechnologies™ProprietaryandConfidentialIonProtonSystemIonProtonSystem的测序原理3|IonTorrentbyLifeTechnologies™ProprietaryandConfidential芯片就是测序仪半导体测序扩展迅速、反应灵敏符合半导体行业摩尔定律通用试剂与测序芯片自由组合测序速度快最快12小时测序时间-----后光学原理4|IonTorrentbyLifeTechnologies™ProprietaryandConfidentialIonPI™ChipIonPI™芯片适用于全外显子组或全转录组测序IonPII™芯片适用于人类全基因组测序165Mwells10Gbfor2Exomes目前实测：60-80MFilteredReads4hours660Mwells20Xhumangenome目前实测：240-320MFilteredReadsRunsinHoursIonPII™Chip5|IonTorrentbyLifeTechnologies™ProprietaryandConfidential测序原理简单----后光学原理直接检测氢离子，无需荧光检测6|IonTorrentbyLifeTechnologies™ProprietaryandConfidential高通量芯片内进行测序反应RothbergJ.M.etalNaturedoi:10.1038/nature10242SensorPlateSiliconSubstrateDrainSourceBulkdNTP∆pH∆Q∆VSensingLayerH+4种dNTP依次流过Ion芯片DNA聚合反应产生的氢离子的直接检测每个碱基的检测只需要几秒7|IonTorrentbyLifeTechnologies™ProprietaryandConfidential芯片示意图8|IonTorrentbyLifeTechnologies™ProprietaryandConfidential1.依次掺入四种碱基9|IonTorrentbyLifeTechnologies™ProprietaryandConfidential2.碱基配对结合10|IonTorrentbyLifeTechnologies™ProprietaryandConfidential3.释放氢离子11|IonTorrentbyLifeTechnologies™ProprietaryandConfidential4.读取模版碱基12|IonTorrentbyLifeTechnologies™ProprietaryandConfidential5.连续两个相同碱基结合13|IonTorrentbyLifeTechnologies™ProprietaryandConfidential6.读取模版的连续两个相同碱基14|IonTorrentbyLifeTechnologies™ProprietaryandConfidential7.芯片上几十万、几百万甚至上千万个DNA片段同时读取序列信息15|IonTorrentbyLifeTechnologies™ProprietaryandConfidential7.芯片上几十万、几百万甚至上千万个DNA片段同时读取序列信息16|IonTorrentbyLifeTechnologies™ProprietaryandConfidential新一代Ion半导体高通量测序平台原理及其特点IonProtonSystem的特点17|IonTorrentbyLifeTechnologies™ProprietaryandConfidential一天之内完成从样本制备到测序数据产生全部流程IonProton™OneTouch2SystemIonProton™SequencerProton™TorrentServerIonKits18|IonTorrentbyLifeTechnologies™ProprietaryandConfidential测序速度快，随到随测制备测序文库油包水相扩增数据分析收集纯化磁珠上机测序2小时5小时12小时0.5小时测序流程介绍19|IonTorrentbyLifeTechnologies™ProprietaryandConfidentialCriteriaProtonHiseq2000测序原理电位检测原理光学检测原理单端测序读长200bp（还有提升空间）100bp数据准确性单次测序原始数据准确性99.9%Hiseq2000公开的招标参数上描述单次测序原始数据准确性为99.9%实验时间1天慢速模式：11天快速模式：40小时数据通量PI芯片：8-16G(2个RNA样本即可开机测序)PII芯片：32-64G慢速模式：300G(60个RNA样本即可开机)快速模式：最高120GIonProton与Hiseq2000技术指标比较20|IonTorrentbyLifeTechnologies™ProprietaryandConfidential二代测序与芯片技术的比较每个小格是一簇长度为60bp长度探针，每个探针鉴定一个基因的表达很亮，如果一个基因表达量高，那么该小格所释放的荧光强度越高。一条基因一根探针60bp整个基因中，仅以与这60bp探针互补杂交到的基因表达量来代表整个基因的表达量。基因芯片21|IonTorrentbyLifeTechnologies™ProprietaryandConfidential二代测序与芯片技术的比较二代测序通过测序技术直接获得这些基因的序列片（Reads）。一个基因表达量越高，该基因测到的Reads数量也越多。一条基因一条Reads100bp22|IonTorrentbyLifeTechnologies™ProprietaryandConfidential新一代Ion半导体高通量测序平台原理及其特点数据处理23|IonTorrentbyLifeTechnologies™ProprietaryandConfidentialRawData预处理在Mapping之前得到干净的Reads初步过滤去接头去除低质量序列随机Blast去除过短片段去除PCR-Duplication24|IonTorrentbyLifeTechnologies™ProprietaryandConfidential新一代Ion半导体高通量测序平台原理及其特点数据后期处理Mapping率分析统计结果Persequencequalityscores25|IonTorrentbyLifeTechnologies™ProprietaryandConfidential新一代Ion半导体高通量测序平台原理及其特点数据后期处理26|IonTorrentbyLifeTechnologies™ProprietaryandConfidentialMapping回reference序列的reads数量RPKM(ReadsPerKilobasesperMillionreads)RPKM=Mapping回referece序列的reads数量该基因外显子长度TotalExonReadsExonLengthXMappedReads(million)RPKM27|IonTorrentbyLifeTechnologies™ProprietaryandConfidential差异可变剪接筛选可变剪接方式1可变剪接方式228|IonTorrentbyLifeTechnologies™ProprietaryandConfidential可变剪接示意图Isoform1Isoform2Experiment1Experiment2Exp1Exp229|IonTorrentbyLifeTechnologies™ProprietaryandConfidentialThankYou!