染色体非整倍性检测新方法课件

划时代产品划时代产品IonTorrent半导体测序仪半导体测序仪-重新制定分子诊断游戏规则重新制定分子诊断游戏规则SemiconductorSequencingforLife1划时代产品IonTorrent半导体测序仪Semicondu目录测序技术与芯片技术的比较 Ion Torrent测序技术简介 试管婴儿胚胎移植前的染色体异倍性检测PGM在其他医学研究与临床上的应用1234Confidential and ProprietaryDO NOT DUPLICATE目录测序技术与芯片技术的比较 Ion Torrent测序技术2测序技术与芯片技术的比较Confidential and ProprietaryDO NOT DUPLICATE测序技术与芯片技术的比较Confidential and 3Confidential and ProprietaryDO NOT DUPLICATE比较项目比较项目测序技术测序技术芯片技术芯片技术检测原理检测原理直接碱基测定检测（数字信号）间接探针杂交检测（模拟信号）数据库依赖数据库依赖不依赖数据库芯片设计探针必须依赖较成熟的数据库（数据分析也只能做已知基因）交叉杂交干扰交叉杂交干扰无此项干扰芯片探针会有交叉杂交现象，例如片段与探针有几个错配也能杂交，产生假阳性数据分析数据分析持续升级，可随着数据库和研究成果的更新重新进行分析只能依据设计芯片时的数据库进行分析灵敏度灵敏度可随着数据量的提高而提高杂交原理较难提高灵敏度数据准确度数据准确度定量水平（qPCR级别）定性水平（与qPCR结果进行比较时，只看趋势不看具体数值）特异性数据分析特异性数据分析测序能做很多芯片无法实现的数据分析，例如新基因的发现，基因融合等等只能做已知设定的基因的检测覆盖度覆盖度可覆盖全基因组受到探针数目以及扫描仪精度的限制，无法一张芯片对全基因组覆盖。重复一致性重复一致性一致性好，科研论文质量更高一致性差，要是芯片属于不同的批次，或者实验时间隔得比较长，重复性就更差染色体平衡易位染色体平衡易位可以不可能测序技术与芯片技术的比较Confidential and ProprietaryDO NOT DUPLICATEConfidential and Proprietary4Ion Torrent测序技术简介 Confidential and ProprietaryDO NOT DUPLICATEIon Torrent测序技术简介 Confidential5Confidential and ProprietaryDO NOT DUPLICATEBlock DiagramFISH未来的发展趋势原位杂交原位杂交FISHFISHFQ-PCRFQ-PCR荧光定量荧光定量PCRPCRLAMPLAMP等温扩增等温扩增BioChipBioChip生物芯片生物芯片SequencingSequencing测序测序SequencingFQ-PCR测序 分子诊断的金标准核酸提取核酸提取技术与市场的发展趋势Confidential and ProprietaryDO NOT DUPLICATEConfidential and Proprietary67技术简单、成本低通量大、灵活检测速度快Ion Torrent 2.5 generation师出名门师出名门世界首台二代测序已发明人世界首台二代测序已发明人Jonathan Rothberg Jonathan Rothberg（20042004年发布年发布454454测序仪）测序仪）全新发明全新发明*革命性技术革命性技术直接检测氢离子，免去其他二代测序技术由于照相带来的测序直接检测氢离子，免去其他二代测序技术由于照相带来的测序时间长，数据处理压力大和存储压力大的烦恼时间长，数据处理压力大和存储压力大的烦恼真正的真正的2.52.5代测序代测序7Confidential and ProprietaryDO NOT DUPLICATE7技术简单、成本低Ion Torrent 2.5 gen简单的化学8Confidential and ProprietaryDO NOT DUPLICATE简单的化学8Confidential and Propri测序应用小型基因组基因合集基因表达全转录组人类外显子组人类基因组Ion PGMTM测序仪Ion ProntonTM测序仪PI316318PII314314316318314316318318PIPIPIIPIIPI318PIConfidential and ProprietaryDO NOT DUPLICATE测序应用小型基因组基因合集基因表达全转录组人类外显子组人类基9*更快的速度 Fastest Next-Gen Workflow10*更快的速度 Fastest Next-Gen Workfl Ion Dx technology2-4 hour sequencing runs Low cost sequencer and kitsMillions of 200+base pair long reads Field proven robustnessCore product completionLow cost,starting at$399 per runRegulatory filingsBuilds on Lifes IVD experience:7500 Fast Dx&3500Dx20112012Ion PGM 2012开始CE-IVD and 510(k)证书的申请11Ion Dx technologyCore product试管婴儿胚胎移植前的染色体异倍性检测Confidential and ProprietaryDO NOT DUPLICATE试管婴儿胚胎移植前的染色体异倍性检测Confidential12Sample ATEXTSample ATEXTReads mapped to Chromosome%reads/totalReads mapped to Chromosome%reads/total65215608753661348914ChrABA/BB/A125595%105%123466%115%Mapping readsMapping to Hg19CLCbio WorkbenchGenomics Gateway(beta)数据分析数据分析CalculationChromosome per chromosomeMapped reads vs.Total readsRatio Sample A vs.Sample BFull reads set or 150K reads subsetConfidential and ProprietaryDO NOT DUPLICATESample ASample AReads mapped t13TrisomyTrisomy结果解释结果解释(using 150K reads)(using 150K reads)Confidential and ProprietaryDO NOT DUPLICATETrisomyTrisomy 结果解释(using 150K14PGM在其他医学研究与临床上的应用Confidential and ProprietaryDO NOT DUPLICATEPGM在其他医学研究与临床上的应用Confidential 15Samples15 plasma DNA samples isolated from maternal blood Standard SOLiD fragment library construction protocolePCRSequencing performed on an upgraded V2 instrumentEach library deposited into a single spot on a quad array3 fragment libraries/slide.2 slides/instrument run.Bead density:130K/panel:today:400K per panelBioinformatics:Tags of 35 bp were mapped to repeat-masked hg18 reference genomeSD of GR were calculated as following:无创产筛（无创染色体异倍性检测）16Samples无创产筛（无创染色体异倍性检测）1617*Preliminary disease groups&gene list based on OMIM available at Configuration Subject to ChangeDetection of germline variants at 100X depthUse with Ion 316 ChipFor Research Use Only.Not intended for any animal or human therapeutic or diagnostic use.300 genes,10,000 amplicons,*one day单基因病检测Cardiovascular37 genes37 genesDevelopmental 61 genes61 genes30 genes30 genesMetabolic Neuromuscular 81 genes81 genesOther;Immune,cancer,etc 94 genes94 genes1717*Preliminary disease groups 46 genes,739 mutations肿瘤个体化治疗For Research Use Only.Not intended for any animal or human therapeutic or diagnostic use.KRASBRAFEGFRTP53PIK3CACSF1RFGFR2JAK2NRASPTPN11ERBB2SRCFGFR3NPM1RB1CDKN2ARETHNF1ASMAD4GNASPDGFRAMPLJAK3ABL1PTENFLT3STK11SMARCB1KITMETVHLNOTCH1HRASAKT1ALKMLH1FBXW7ERBB4KDRATMCDH1IDH1CTNNB1APCFGFR1SMOConfidential and ProprietaryDO NOT DUPLICATE46 genes,739 mutations肿瘤个体化治疗18ReportReportReportRemaining fwd&rev primers(10,000 rxns)shipped in plates to allow user optimizationRemaining fwd&rev primers(10,000 rxns)shipped in plates to allow user optimizationChip Type Genome Coverage 500X(Somatic)20X(Germline)Ion 3141 kb10 kbIon 31650 kb100 kbIon 318500kb1,000 kbTotal#of amplicons/poolOligos per pool48 3,07296-6,144Ion AmpliSeq 客户定制试剂盒-自由定制待测基因ReportReportRemaining fwd&r19Rapid sequencing,de novo assembly&identification of novel microbial strains.MondayMay 30*Library preparationO104:H4 and HUSC41 samples(reference)strain libraries preparedTuesdayMay 31Sequencing runs0104:H4 amplified and sequenced 2 x 2 runs(Ion 314)WednesdayJune 01 Sequencing runs0104:H4 sequenced3 x 2 runs(Ion 314)ThursdayJune 02 AssemblyDraft Genome identified,Assembled,Submitted and Released from NCBIFridayJune 03 Assay DesignTaqMan Assays Designed*May 22 CEDC reports significant increase in patients with hemolytic uremic syndrome“The biggest advantage of the PGM from my point of view as a public health official is that its speedy,and speed is what is needed at the moment,”Prof.Dr.Med Dag Harmsen,University Hospital Muenster“The PGM takes the shortest time to generate genomic data.”Junjie Qin,BGI MondayMay 30*Library preparationO104:H4 and HUSC41 samples(reference)strain libraries preparedTuesdayMay 31Sequencing runs0104:H4 amplified and sequenced 2 x 2 runs(Ion 314)WednesdayJune 01 Sequencing runs0104:H4 sequenced3 x 2 runs(Ion 314)ThursdayJune 02 AssemblyDraft Genome identified,Assembled,Submitted and Released from NCBIFridayJune 03 Assay DesignTaqMan Assays Designed快速未知病原筛查E.coli Outbreak Characterized using Ion PGM sequencer in 3 days 20Rapid sequencing,de novo asse2121