Ion-PGM介绍

8Proprietary&Confidential•快速：半导体传递电信号；•无复杂的光路系统传导光学信号；无昂贵的CCD实时拍照和大库存的图像采集•高效和廉价：在天然的DNA延伸过程中进行实时检测（其他平台都需复杂的级联反应）•一张测序芯片上可同时进行数百万到上亿的测序反应314芯片1M孔；316芯片6M孔；318芯片11M孔；P1芯片150M孔RothbergJ.M.etalNature2011v475SensorPlateSiliconSubstrateDrainSourceBulkdNTPTocolumnreceiver∆pH∆Q∆VSensingLayerH+IonTorrent™半导体测序技术简介每个孔下方有一个传感器，记录每次反应的信号值9Proprietary&ConfidentialIonPGMTM二代测序流程PrepareLibraryClonalAmplificationDNA/RNADataAnalysisLibraryPreparation(文库构建）DNASequencing（测序）IsolatePositiveIonSphere™ParticlesDataAnalysis（数据分析）LoadChipandSequenceTemplatePreparation（模板扩增）10Proprietary&ConfidentialPGT121类广泛中和抗体的发现1,798HIV-1infectedindividuals18eliteneutralizersIAVIdonors17,36,39,84ActivatedclonalmemoryBcellsScreeningforbnAbVRC01HQVQLVQSGGQMKKPGESMRISCRASGYEFID.........CTLNWIRLAPGKRPEWMGWLKPRGGAVNY.ARPLQGRVT..MTR.......DVYSDTAFLELRSLTVDDTAVYFCTRGKNCD....YNWDFEHWGRGTPVIVSSVRC02HQVQLVQSGGQMKKPGESMRISCQASGYEFID.........CTLNWVRLAPGRRPEWMGWLKPRGGAVNY.ARPLQGRVT..MTR.......DVYSDTAFLELRSLTADDTAVYYCTRGKNCD....YNWDFEHWGRGTPVTVSSVRC03HQVQLVQSGAVIKTPGSSVKISCRASGYNFRD.........YSIHWVRLIPDKGFEWIGWIKPLWGAVSY.ARQLQGRVS..MTRQLSQDPDDPDWGVAYMEFSGLTPADTAEYFCVRRGSCD..YCGDFPWQYWGQGTVVVVSSVRC-PG04HQVQLVQSGSGVKKPGASVRVSCWTSEDIFER........TELIHWVRQAPGQGLEWIGWVKTVTGAVNFGSPDFRQRVS..LTR.......DRDLFTAHMDIRGLTQGDTATYFCARQKFYTGG..QGWYFDLWGRGTLIVVSSVRC-PG04bHQVQLVQSGSGVKKPGASVRVSCWTSEDIFER........TELIHWVRQAPGQGLEWIGWVKTVTGAVNFGSPNFRHRVS..LTR.......DRDLFTAHMDIRGLTQGDTATYFCARQKFERGG..QGWYFDLWGRGTLIVVSSVRC-CH30HQVQLVQSGAAVRKPGASVTVSCKFAEDDDYSPHWVNPAPEHYIHFLRQAPGQQLEWLAWMNPTNGAVNY.AWQLHGRLT..ATR.......DGSMTTAFLEVRSLRSDDTAVYYCARAQKRGR...SEWAYAHWGQGTPVAVSSVRC-CH31HQVQLVQSGAAVRKPGASVTVSCKFAEDDDYSPYWVNPAPEHFIHFLRQAPGQQLEWLAWMNPTNGAVNY.AWYLNGRVT..ATR.......DRSMTTAFLEVKSLRSDDTAVYYCARAQKRGR...SEWAYAHWGQGTPVVVSSVRC-CH32HQVQLVQSGAAVRKPGASVTVSCKFAEDDDFSPHWVNPAPEHYIHFLRQAPGQQLEWLAWMKPTNGAVNY.AWQLQGRVT..VTR.......DRSQTTAFLEVKNLRSDDTAVYYCARAQKRGR...SEWAYAHWGQGTPVVISA3BNC60HQVHLSQSGAAVTKPGASVRVSCEASGYKISD.........HFIHWWRQAPGQGLQWVGWINPKTGQPNN.PRQFQGRVS..LTR...QASWDFDTYSFYMDLKAVRSDDTAIYFCARQRS......DFWDFDVWGSGTQVTVSS3BNC117HQVQLLQSGAAVTKPGASVRVSCEASGYNIRD.........YFIHWWRQAPGQGLQWVGWINPKTGQPNN.PRQFQGRVS..LTR...HASWDFDTFSFYMDLKALRSDDTAVYFCARQRS......DYWDFDVWGSGTQVTVSS12A12HSQHLVQSGTQVKKPGASVRISCQASGYSFTD.........YVLHWWRQAPGQGLEWMGWIKPVYGARNY.ARRFQGRIN..FDR.......DIYREIAFMDLSGLRSDDTALYFCARDGSGDDT...SWHLDPWGQGTLVIVSA12A21HSQHLVQSGTQVKKPGASVRVSCQASGYTFTN.........YILHWWRQAPGQGLEWMGLIKPVFGAVNY.ARQFQGRIQ..LTR.......DIYREIAFLDLSGLRSDDTAVYYCARDESGDDL...KWHLHPWGQGTQVIVSPNIH45-46HQVRLSQSGGQMKKPGESMRLSCRASGYEFLN.........CPINWIRLAPGRRPEWMGWLKPRGGAVNY.ARKFQGRVT..MTR.......DVYSDTAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGRGAPVTVSSSequencingnewbnAbsDonorbnAbIAVIdonor17PGT121PGT122PGT123PGT124PGT133PGT134DennisBurton&PascalPoignard‟slabsatTSRI,Nature2011中和65-70%HIV-1分离株11Proprietary&ConfidentialPGM检测出所有的PGT121类抗体重链•PGM-derivedHCantibodyomePGT121Hidentity(%)IgHV4-59germlinedivergence(%)PGT123Hidentity(%)PGT133Hidentity(%)10102103104PGT122Hidentity(%)PGT124Hidentity(%)PGT134Hidentity(%)1010210310410102103104PGMsequencingidentifiedclosely-relatedsomaticvariantsforall6heavychainsofthePGT121class,whereas454pyrosequencingonlyfoundsomaticvariantsforPGT121heavychain.•454-derivedHCantibodyomePGT121Hidentity(%)IgHV4-59germlinedivergence(%)PGT123Hidentity(%)PGT133Hidentity(%)PGT122Hidentity(%)PGT124Hidentity(%)PGT134Hidentity(%)101021031041010210310410102103104IAVI1712Proprietary&Confidential重链和轻链的进化树分析13Proprietary&ConfidentialIonPGMTM模板恒温扩增IA-500型试剂盒•快速（2小时）•高效（500-800bp文库）1.Rehydrationbuffer2.ISPs+library3.Primermix4.StartSolution40˚Cfor30minutes1.StopSolution2.2XWash3.ESEnrichment10minsetup15minwash&setup+50minEStime“Cleanroom”“Dirtyroom”14Proprietary&ConfidentialPGMTM的500bp到600bp读长580bpLibrary318chip730bpLibrary316chip600500E.coli15Proprietary&ConfidentialIA500和长序测序试剂显著的提高了测序质量•+26%测序通量的增多•+80bp读长的增加•47%无差距序列–更高的准确度HeavychainLightchain16Proprietary&Confidential无偏5‟RACE免疫组库文库测序需要长的读长~100bp~100bp~300-400bpPGM/454500bpFrontiersinImmunology2013v4,p15‟RACE17Proprietary&ConfidentialPGMTM能够很好地测序cDNA免疫组库文库•从注射疫苗后的志愿者的血样中提取外周血单核细胞(PBMC)•构建B细胞IgG的重链和轻链的5‟RACE文库•IA500恒温模板扩增•每个318芯片负载了三个编码的文库•长序试剂进行300周期的测序•每个样品得到了一到二百万个高质量的序列18Proprietary&Confidential基因特异性引物设计的重要性GoodprimersetPoorprimerset进一步体现了5‟RACE方法的优异性19Proprietary&Confidential免疫组库测序需要配套的数据分析方法•没有标准化的数据分析系统•不同的测序平台需要相应的数据处理方式•数据的过滤应利用已知的胚系区域的主要克隆序列信息•测序分析结果需要进一步的验证，用来完善分析方法•可借鉴的数据分析软件：•IMGT/HighV-Questwebservice:数据处理量低•IgBLAST•iHMMune-align•Decombinator•MiTCR20Proprietary&ConfidentialPGMTM是一种快速高效的免疫组库测序平台•文库的长度可大到800bp•IA500提供了小于2小时的高效恒温模板扩增•长序测序试剂可达到600bp的读长•318芯片可产生约五百万的序列•整个流程已经得到合作伙伴的验证(文稿在印刷或提交阶段）•人的5‟RACEIgG文库（500bp读长）•猴的5‟RACEIgG文库（600bp读长）ForResearchUseOnly.Notforuseindiagnosticprocedures.