Non-Invasive-Tumor-Mutation-Detection-of-Cell-Free.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,BACKGROUND,Previous studies have demonstrated the ability to detect tumor DNA mutations by,PCR methodologies,within cell,free urinary DNA(cfDNA)in metastatic cancer patients.,This has opened up the possibility of using a,massively parallel deep sequencing approach,for more global profiling of tumor mutations using cell free urinary DNA from these patients.,Figure 1:Tracking cell free DNA,A)As,cells,in the body,die,each day,B)Genomic DNA is released into the,bloodstream,.,C)The cellular DNA is broken down into,smaller segments,and these are filtered by the,kidney,.,D)These small,stable fragments collect in the,bladder,E)And are excreted into,urine,where Trovagene technology can identify and quantify mutations of interest.,Here we report the development of a,KRAS assay,using cfDNA extracted from urine that enriches for extremely low levels of mutant DNA thereby providing high detection sensitivity.,cfDNA detected in the urine of individuals with cancer has the advantage of being a,non-invasive,patient-friendly,means to determine the mutational status of a cancer.,Tumors harboring KRAS mutations confer a survival and growth advantage to cancer cells and are largely resistant to targeted therapies.,Assay Development,Specifications,o To achieve clinical utility the assay needs to meet the following specifications:,High sensitivity,between 0.010.05%,2)Amplify an ultrashort DNA footprint(30 bp,).,Design,RESULTS:Significance,RESULTS:Patient,RESULTS:Quantification,CONCLUSIONS,o We demonstrate that,massively parallel sequencing can be an effective tool to monitor mutation status of the KRAS gene,in urinary cfDNA.,o The assay is selective and highly specific for all,seven KRAS mutations within KRAS codons 12 and 13.,o Preliminary results show that,mutated KRAS,could be detected in the urine of 8 out of 9 patients whose tumor tissue contained a KRAS mutation.,o Discrepancy of the called nucleotide in 4 of the 8 detectable tumor samples may highlight discrepancies in patient tumor heterogeneity of these samples.,o,Using,massively parallel DNA sequencing,to detect mutations from cellfree urinary DNA has the,potential,to non-invasively monitor metastatic patients for response,non-response and the emergence of resistance mechanisms of molecularly targeted therapies.,基因测序技术,第一代测序,1977,年,Sanger,发明了具有里程碑意义的,末端终止测序法,，同年,A.M.Maxam,和,W.Gilbert,发明了,化学降解法,。,第二代测序,Next-generation sequencing,核心思想是,边合成边测序,（,Sequencing by Synthesis),，即通过捕捉新合成的末端的标记来确定,DNA,的序列，现有的技术平台主要包括,Roche/454 FLX,、,Illumina/Solexa Genome Analyzer,和,Applied Biosystems SOLID system,。,第三代测序,第三代测序技术也叫从头测序技术（,de novo sequence,）建议还是用,Single Molecule Real Time(SMRT)DNA Sequencing,表示）即,单分子实时,DNA,测序,测序设备的垄断和高速度换代,1990,2005,2020,Year,2015,2010,2000,1995,Mb1000,Mb4000,ABI373,ABI377,ABI3130,ABI3730,ABI3730 xl,GA-I,GA-II,Less Than 5 yrs,HiSeq1000/2000,Mb4500,ABI3700,ABI3700 xl,SOLiD,SOLiD2,SOLiD3,5500 xl SOLiD,ABI3130 xl,GA-IIx,5500 SOLiD,20,测序设备发展现状,第一代（稳定需求）,ABi,3130 xL,3730 xL,3500 xL,第三代（即将面市）,Helicos Biosciences,Helicos Genetic Analysis System,Pacific Biosciences,RSSystem,第二代（高速发展）,Roche,Genome Sequencer FLX System,GS Junior System,Illumina,Genome Analyzer IIx,MiSeq,HiSeq 1000,HiSeq 2000,Life Technologies(ABi),5500 SOLiD System,5500 xL SOLiD System,Ion,Torrent PGM,DanaherMotion,Polonator G.007,Complete Genomics,无锡艾吉因生物信息技术有限公司,AG-100,深圳华因康基因科技有限公司,Pstar-1,中科院北京基因组所,/,半导体所,BIGIS-1,BIGIS-4,21,第二代测序,高通量测序技术（,High-throughput sequencing,）,以能一次并行对几十万到几百万条,DNA,分子进行序列测定和一般读长较短等为标志。,根据发展历史、影响力、测序原理和技术不同等，主要有以下几种：,大规模平行签名测序（,Massively Parallel Signature Sequencing,MPSS),聚合酶克隆（,Polony Sequencing,）,454,焦磷酸测序（,454 pyrosequencing,）,Illumina(Solexa)sequencing,ABI SOLiD sequencing,离子半导体测序（,Ion semiconductor sequencing,）,DNA,纳米球测序（,DNA nanoball sequencing,）等。,MPSS,由,Lynx Therapeutics,公司在,90,年代发展的,Massively Parallel Signature Sequencing,MPSS,技术是,“,下一代,”,测序,技术,发展的先驱。,MPSS,是一种基于磁珠（,bead,）和接头（,adaptor,）连接和解码的复杂技术，测定结果短，多用于,转录组,测序，测定基因表达量。,MPSS,测定结果有序列偏好性而易丢失,DNA,中某些特定序列，且操作复杂，已逐渐淡出，被新的方法替代。,Lynx Therapeutics,公司于,2004,年和,Solexa,合并，随后,被,Illumina,收购,。,公司名称,技术原理,技术开发者,商业模式,Apply Biosystems(ABI),基于磁珠的大规模并行克隆连接,DNA,测序法,美国,Agencourt,私人基因组学公司（,APG,）,上市公司：销售设备和试剂获取利润,Illumina,合成测序法,英国,Solexa,公司首席科学家,David Bentley,上市公司：销售设备和试剂获取利润,Roche,大规模并行焦磷酸合成测序法,美国,454 Life Sciences,公司的创始人,Jonathan Rothberg,上市公司：销售设备和试剂获取利润，该产品已经于,2013,年停产。,Helicos,大规模并行单分子合成测序法,美国斯坦福大学生物工程学家,Stephen Quake,上市公司：,2007,年,5,月首次公开募股（,IPO,）（,P.S.,由于该平台准确率太低等原因，该公司已于,2012,年宣告破产）,Complete Genomics,DNA,纳米阵列与组合探针锚定连接测序法,美国,Complete Genomics,公司首席科学家,radoje drmanac,私人公司：投资额为,4650,万美元，该公司于,2013,年被华大基因收购。,高通量测序平台,Illumina/Solexa Genome Analyzer,测序的基本原理是边合成边测序,在,Sanger,等测序方法的基础上，通过技术创新，用不同颜色的,荧光标记,四种不同的,dNTP,，当,DNA,聚合酶合成互补链时，每添加一种,dNTP,就会释放出不同的荧光，根据捕捉的荧光信号并经过特定的计算机软件处理，从而获得待测,DNA,的序列信息。,操作流程,测序文库的构建,（,Library Construction,）,将,DNA,随机片段化,；,两头加上特定的接头（,Adaptor,）,锚定桥接（,Surface Attachment and Bridge Amplification,）,预扩增（,Denaturation and Complete Amplification,）,单碱基延伸测序（,Single Base Extension and Sequencing,）,数据分析（,Data Analyzing,）,flow cell(,玻璃管,),，,8,个,Lane(,无数固,定,单链接头,),；形成,桥状结构,固相桥式,PCR,扩增,；上百万条,成簇分布的双链待测片段,四种荧光标记的,dNTP,，测序峰；,Base Calling,Illumina/Solexa Genome Analyzer,Thank you!,