AB公司基因分型新方法新工具 Linsen Yang 杨林森 Business Development Manager, China Applied Biosystems
AB公司基因分型新方法新工具 Linsen Yang 杨林森 Business Development Manager, China Applied Biosystems [email protected] 欢 迎 参 加 Applied Biosystems 基因分型技术进展讲座 报告题目: AB公司SNP基因分型技术平台与疾病研究技术路线 1. 2. 3. 4. Resequencing: 基因突变和SNP位点的发现 SNPlex： 高通量SNP分析/3130/3130XL/3730/3730XL TaqMan SNP assays： 定量PCR技术平台 SNPbrowser 软件： SNP位点数据库和位点选择软件 报告人: 杨林森 分子生物产品业务发展部经理 美国应用生物系统公司 [email protected] 2 © 2006 Applied Biosystems AB 拥有三大获Nobel 奖的技术 • Dideoxy Sanger DNA Sequencing Chemistry (1975) (Frederick Sanger, 1958, 1980) and 4-color Automatic Sequencer technology; (Hood and Hunkapillers, 1980) Created Celera 1998, completed human genome sequences on 2001! • The matrix assisted laser desorption/ionization (MALDI, 1987) Koichi Tanaka, 2002); and time-of-flight (TOF, 1955) Mass spectrometer;( TY Lee, 1986) Sequencing whole Proteins? Not enough sensitivity yet! • Polymerase Chain Reaction (PCR, 1985);(Kary Mullis, Michael Smith, 1993). Real-Time PCR Technology (AB, 1996) Detection of gene expression from a single cell? 3 © 2006 Applied Biosystems 基因分型定义与研究目的 • 通过对基因变异或多态性的分析来寻找基因组内的功能基因 • 研究目标: 找到与表型相关的基因多态性或突变 (SNP, 插入/ 确失, MNP) • 疾病:致病基因的定位与解析 • 功能与生化机制解析 • 农业研究: 辅助育种 • 群体遗传学 • 病原微生物的基因分型 • 个体化用药/基因诊断 4 © 2006 Applied Biosystems 基因分型技术方法分类 • 疾病基因定位: 发现 • Linkage Mapping Determines the arrangement of genes on chromosomes in conjunction with inherited traits • Candidate Region association studies Tests for associations with SNPs in particular chromosomal intervals • Candidate Gene association studies Tests for associations with SNPs in one or more genes • Whole Genome Association studies Hypothesis-free approach for association study • cSNP功能性SNP直接关联分析： 发现 • 致病基因验证： 验证 用另一批样本或另一种检测平台对定位结果进行验证 • 人群筛查，诊断 对目标疾病的高危人群进行检测分析 5 © 2006 Applied Biosystems Single Nucleotide Polymorphisms (SNPs) 单一碱基序列多态性 A Single Nucleotide Polymorphism (SNP) is a change in the nucleotide present at a single base in a known DNA sequence. ATGCATGCATGCATGCATGC ATGCATGCATACATGCATGC This G/A is a SNP 6 © 2006 Applied Biosystems 采用不同类型的SNP进行两种主要基因分型应用 • SNPs 分型用于致病基因的定位 • Want 10 – 40% minor allele frequency in a given population • Spacing needed is set by LD, which depends on haplotype and population, but is typically 5 – 50kbp • 致病性或影响功能的SNPs/基因突变 – May be quite rare (< 1%) and still affect millions of people – Spacing is defined by the mutational spectrum, which can be quite dense 7 © 2006 Applied Biosystems AB 提供多种技术平台,可进行多种基因分型应用 APPLICATION 高密度突变/SNP 连锁分析或关联分析 候选基因 /侯选区 域关联分析 较大侯选区域/全 基因组关联分析 8 PLATFORM • 3130/3730基因分析仪 • 既分型又可发现新突变 双重目的,分型金标准 • 微卫星STRs,SNPs • 7300/7500/7900 荧光定量PCR仪 • 位点选择灵活,成本低 • 方便 试剂盒全部经过验证 • 基因分型和基因表达分析 • SNPlex System on 3730xl/3130XL基因分析仪 • 低成本 • 超高通量, 位点可选 基因及SNP位点选择: SNPbrowser 3.5 © 2006 Applied Biosystems 欢 迎 参 加 Applied Biosystems 基因分型技术进展讲座 报告题目: AB公司SNP基因分型技术平台与疾病研究技术路线 1. 2. 3. 4. Resequencing: 基因突变和SNP位点的发现 SNPlex： 高通量SNP分析/3130/3130XL/3730/3730XL TaqMan SNP assays： 定量PCR技术平台 SNPbrowser 软件： SNP位点数据库和位点选择软件 报告人: 杨林森 分子生物产品业务发展部经理 美国应用生物系统公司 9 © 2006 Applied Biosystems Resequencing定义：医学测序/比较基因序列分析 • A method that compares sequences obtained from one or multiple DNA samples to a known reference sequence • To identify variations in DNA sequence • To correlate changes with important scientific questions • Why do we feel like dozing after lunch? De novo: •1 base/peak Resequencing: •Mixed bases/peak 10 © 2006 Applied Biosystems Resequencing的重要性 • Cancer Genome project • • • • • 11 (NHGRI -$1.6B) Evolutionary origins Disease susceptibility initiatives (Alzheimers, Diabetes, Asthma, etc.) Wash U, Sanger, Broad, Baylor – new resequencing projects Functional Genomics of “junk” DNA Pharmacogenomics Initiative © 2006 Applied Biosystems Resequencing的应用 Application: Application: Resequencing Resequencing AB AB Systems: Systems: Alternate PCR-Sequencing, Targeted Targeted Seq., Seq., Alternate Names: Names: PCR-Sequencing, •• VariantSEQr™ VariantSEQr™ resequencing resequencing primers primers Boutique Boutique Seq., Seq., Medical Medical Sequencing, Sequencing, Directed Directed Seq. Seq. Description: Comparison of of sequences sequences obtained obtained from from one one Description: Comparison •• AmpliTaqGold® AmpliTaqGold® PCR PCR Master Master Mix Mix & & 9700 9700 •• BigDye® BigDye® Terminator Terminator v3.1 v3.1 or or multiple multiple DNA DNA samples samples to to aa reference reference sequence sequence to to •• 3130, 3130, 3730 3730 series series GA GA analyzers analyzers identify identify variation variation like like SNPs, SNPs, MNPs, MNPs, in-dels. in-dels. •• SeqScape® SeqScape® software software Used To correlate correlate DNA DNA sequence sequence change change with with gene gene Used for: for: To function, function, disease, disease, evolutionary evolutionary origins, origins, pharmacogenomics pharmacogenomics Complementary Complementary technology: technology: CGH CGH (Comparative (Comparative Genomic Genomic Hybridization), Hybridization), SNP SNP Discovery Discovery & & Validation Validation tools. tools. Target Target customers: customers: •• Core Core & & Service Service labs labs (can (can now now provide provide gene-specific gene-specific primers primers to to their their clients) clients) •• Customers &)) Customers of of Core/Service Core/Service Lab Lab (can (can reduce reduce or or eliminate eliminate time time & & labor labor for for primer primer design, design, sequencing sequencing& •• Academic/Clinical/Pharma &)) Academic/Clinical/Pharma researchers researchers (Standard (Standard set set of of primers, primers, better better comparison comparison across across studies, studies, sequencing sequencing& •• Genome Genome Centers Centers (retooling (retooling for for resequencing, resequencing, estimate estimate reseq. reseq. will will replace replace de de novo novo as as primary primary method). method). Available Available Collateral: Collateral: VariantSEQr™ VariantSEQr™ Summary Summary protocol, protocol, myScience myScience && NCBI NCBI VariantSEQr™ VariantSEQr™ web web sites sites (http://www.ncbi.nlm.nih.gov/genome/probe/), AB application application notes notes and and posters. posters. (http://www.ncbi.nlm.nih.gov/genome/probe/), AB 12 © 2006 Applied Biosystems Resequencing技术流程 VariantSEQr Re-sequencing Primers 13 © 2006 Applied Biosystems VariantSEQr™ 定义 • PCR-Sequencing of exons and 5’-UTR regions of human genes. ≈ 420,000 PCR primer pairs for ≈ 16,000 human genes. • The sequence for the primers for the Resequencing Amplicons (RSA) that make up a Resequencing Set (RSS) is available on the NCBI Probe database 14 © 2006 Applied Biosystems VariantSEQr™ 高知识含量试剂产品 Prepare Genomic DNA • 280 genes on AB portal • Available as pre-mixed oligos Perform PCR Amplification Perform DNA Sequencing • Available for NCBI Build 35 • ≈16,000 genes on NCBI • SeqScape® templates downloadable from new AB portal (Feb 2006) • “Non-280” genes not available as a product • mitoSEQr™ (Mitochondrial Resequencing) Perform Electrophoresis 15 Analyze Data • Primers available Jan 2006 • 46 primer sets © 2006 Applied Biosystems mitoSEQr™ Resequencing primers 线粒体DNA • Two Resequencing sets • MitoALL™ - 46 resequencing amplicons for the whole mtDNA. >98% coverage. • MitoCR™ – 9 resequencing amplicons for the control region. 100% coverage. • 5 amplicons shared between the two sets • Wet tested on multiple DNA samples and from many types of tissues (fresh to formalin-fixedparaffin-embedded tissues) • 500 (10 µL) reactions 16 © 2006 Applied Biosystems VariantSEQr™ 详细实验流程 1. PCR Reaction - 10 µl reactions: AmpliTaq Gold® (2X), 5.0μL; 50% UltraPure™ Glycerol, 1.6μL; Forward VariantSEQr primer 1.0μL (0.6μM/μL); Reverse VariantSEQr primer 1.0μL (0.6 μM/μL); Genomic DNA, 1.0μL (5-10 ng/μL). Recommended thermocycling conditions AB 9700: 96ºC 5 min, followed by 40 cycles of 94ºC 30 sec, 60ºC 45 sec, and 72ºC 45 sec. Final extension 72ºC 10 min. Hold at 4ºC. 2. PCR Reaction cleanup – 2 µL ExoSAP-IT® (USB), 37ºC 30 min followed by 80ºC 15min. 3. Sequencing Reaction - 10 µl reactions – BigDye® v3.1, 4.0 µL; M13 forward or reverse primer (3.2 pmol/µL), 1.0 µL; water, 3.0 µL; PCR product, 2.0 µL. Thermocycling conditions AB 9700: 96ºC 1min, followed by 25 cycles of 96ºC 10sec, 50ºC 5sec, and 60ºC 4min. Hold at 4ºC. 4. Sequencing Reaction Cleanup – 2.5µL of 125 mM EDTA, mix, add 30µL of 100% ethanol, mix. Incubate at room temperature for 15min. Centrifuge 2000xg 45min. Remove supernatant. Add 30µL of 70% ethanol, centrifuge 2000xg 15min. Remove supernatant. Air dry. Resuspend in 10µL of Hi-Dye Formamide. 5. Electrophoresis - 3130(xl) - POP-6™; Genetic Analyzer Running buffer with EDTA; 36 cm array; RapidSeq36_POP6_1 run file; KB_3130_POP6_BDTv3.mob; KB.bcp. 3730(xl) - POP-7™; 3730/3730xl Running Buffer with EDTA; 36 cm array; RapidSeq36_POP7_1; Run file; Mobility file KB_3730_POP7_BDTv3.mob; KB.bcp 6. Data Analysis - Import SeqScape template and sample files into a new project. Click “Analyze”. 17 Review and examine variants. Create Mutation/Genotyping Report. © 2006 Applied Biosystems Release of VariantSEQr™ to NCBI 18 © 2006 Applied Biosystems New NCBI Probe Database • Genome-wide Resequencing primer sequences • More than 400,000 primer pairs for nearly 16,000 Human Genes • Now Available through NCBI ProbeDB http://www.ncbi.nlm.nih.gov/genome/probe 19 © 2006 Applied Biosystems VariantSEQr™ on NCBI 20 Type in VariantSEQr and gene name © 2006 Applied Biosystems ABI 基因分析仪大家族 相同软件和试剂平台 3700 310 1 21 3130 3130XL 4 16 3730 3730xl 48 96 © 2006 Applied Biosystems Applied Biosystems--- 20年研发制造经验，书写测序仪历史！ 生产规模 3730(xl) 1986年 全球第一台 370 373 测序仪 377 3100-avant , 3100 3130,3130xl 中、高通量 310 基础应用 1996：全球第一台毛细管测序仪 22 © 2006 Applied Biosystems 新一代基因分析仪： 3130／3130XL的特色 • 灵活： 一种胶Pop-7支持基因测序和片段分析的各种应用，多用途基因分析仪 • Like the multi-application plate feature and the use of same polymer across any capillary length. • • • • • • 23 分析速度提高一倍：１小时／750碱基 测序读长：大于1000碱基． 维护更方便：无灌胶用的注射泵 24小时无人监控 自动操作，连续自动进样 16-32 倍测序试剂稀释 支持新应用：SNPlex © 2006 Applied Biosystems 3130 and 3730 系列基因分析仪的主要应用 基因序列分析应用：杂合子测序 基因片段分析应用: 5色荧光 •Mutational Profiling •AFLP •Methylation •BAC Fingerprinting •Multi Locus Sequence Typing •tRFLP •Resequencing •Microsatellites •MicroSeq •LOH or Relative Fluorescence Quantitation •De Novo Sequencing •SAGE •SNP Discovery •Genome finishing and contigbuilding •Large template sequencing 24 •SNP Genotyping •Conformational Analysis •SNaPshot •TILLING •SNPlex © 2006 Applied Biosystems 基因分析仪在病原微生物研究和检测中的应用 • 基因序列分析 • 新型病原微生物的全基因组分析 • 病毒分类和耐药性突变 • • 细菌真菌分类 • • • • • HBV,HCV,HIV 16s RNA 或 D2 Region of Fungus rRNA MLST 毒力株和无毒株的序列比较，微生物致病机理！ 混合基因组分析: 基于16s RNA 流行病学确定病源 • 片段分析 • • • 25 病原菌的AFLP指纹图分析 SSCP突变分析 48重SNP分型技术: SNPlex © 2006 Applied Biosystems SNPlex™ Genotyping System SNPlex™ System 高通量SNP分型方法 • 基于基因分析仪的超高通量SNP基因分型技术方法 • 采用Applied Biosystems 3130,3130XL,3730 and 3730xl 基因分析 仪(16, 48 and 96 道毛细管) • 15 minute run time (36 cm capillaries) • One polymer for multiple applications (POP-7) • SNP 位点可自由选择或采用定制位点 • 试剂盒采用通用的多重OLA/PCR 技术和不同长度的ZipChute™ 杂 交探针,电泳迁移率不同 27 © 2006 Applied Biosystems SNPlex™ System技术平台 多重连接酶反应Oligonucleotide Ligation Assay (OLA) Highly specific and robust chemistry 48-plex currently available 客户选择SNP位点 Web-based bioinformatics pipeline for assay design Web-ordered assays shipped directly to customers 通用实验试剂 Ready to use components (buffers, enzymes, and controls) 基因分析仪及主要消耗品 3130XL,3730 and 3730xl DNA Analyzers 16,48 or 96 capillary configuration 全套方法与技术流程 28 Chemistry Guide Laboratory Automation Guide Assay Design Guide 软件 Data Collection GeneMapper Software BioTrekker™ Software © 2006 Applied Biosystems 实验方法主要分3步 Prep genomic DNA Kinase probes and linkers Perform OLA OLA hybridisation Purify by enzymatic digestion Universal PCR Universal PCR Capture on SA-plates Hybridise ZIPChute KitTM Kit ZIPChute Kit Hybridization Wash, Elute & Load 3730 Analyzer Primary Analysis & QC data 29 © 2006 Applied Biosystems SNPlex 基因分型方法特点 • OLA-PCR 方法,高通量分型 • 可做48重检测 • 基因分型成本低:0.06-0.09US$ • 客户可以选择位点，为人／大 鼠／小鼠样本提供生物信息学 支持 • 采用3730XL，3730和3130XL 基因分析技术平台 • 支持1000-5000样本 30 © 2006 Applied Biosystems 详细实验流程 31 © 2006 Applied Biosystems 数据收集和结果分析 32 © 2006 Applied Biosystems SNPlex™ System的主要特点： 3130-3730均可使用 多重检测与自动化流程保证了超高通量低成本SNP分型检测 Achieve >1.5M GTs per week with common robots and methods 48-plex重SNP分析检测灵活性大,满足各种项目的 研究需要 Candidate gene and fine mapping association studies Projects ranging from 100s-1000s SNPs 客户选择位点,网上实验设计可以提供快速而灵活的项目设计 Access to state-of-the-art bioinformatics pipeline over the web to ensure designed assays are specific and robust 基因分析仪=序列分析仪+基因分型仪 Single instrument platform for sequencing and GT’ing 33 © 2006 Applied Biosystems SNPlex 分析通量容易放大 3130 也可以做啦 With the manual protocol using 96-well hybridization plates and no robotics on the 3130xl/3730/3730xl Æ4200 to 12600 genotypes per day With a just Hydra robot using 384-well hybridization plates on the 3130xl/3730/3730xl Æ17500 to 53000 genotypes per day With a fully automated protocol using 384-well hybridization plates on the 3730/3730xl Æup to 450000 genotypes per day *1 pre and 1 post PCR dual 96 well thermal cyclers required (or 2x single 96 well blocks) 34 © 2006 Applied Biosystems 新的SNPlex Master Mix 配方简化了OLA反应步骤 Ligation UNG/Kinase/Ligate Probes Kinase gDNA dried in plate Linkers Kinase Kinase UNG/Kinase/Ligation buffer UNG Master Mix UNG treatment OLA buffer Ligase Plus dATP Cuts 3 hrs off protocol time. Assemble Chilled Cycle Immediately UNG improves robustness of assay 35 © 2006 Applied Biosystems 新产品 – Linkage Mapping Set • This product is available for sale today • contains material for 1 384 well plate (more if dead volume is minimized) PN • • • • 36 4357150C Consists of 3922 SNPs in clusters spaced by genetic distance. This is a key differentiator vs other products with SNPs chosen by physical distance. This also allows for the use of fewer SNPs while still maximizing information content. Customers will be able to download an excel sheet of the LMS SNPs that can be used to select SNPs by chromosome and build custom SNPlex pools Collateral: Poster from ASHG 2005, Webinar on LMS design, Excel sheet Support documentation for Merlin, a free linkage mapping software package © 2006 Applied Biosystems C O N T I N U U M O F D I S C O V E R 37 Y SNPlex™ 基因分型技术平台支持用户在单一3130/3730平台上 完成疾病研究的全部研究工作 ,无与伦比的核心引擎 Microsatellite or SNP based linkage mapping 以寻找含有致病基因的基因区域 SNP-based 精细定位以缩小范围, 找到相关基因 对致病基因进行测序以找到 疾病相关的基因突变 © 2006 Applied Biosystems 用荧光定量PCR进行SNP分型 TaqMan® Gene Expression Assays & TaqMan® Genotyping Assays service Applied Biosystems基于荧光定量PCR技术平台的基因组产品 60万种基因表达分析试剂盒和350万种SNP基因分型试剂盒 SNP Assays Gene Expression Assays-on-Demand products Assays-on-Demand products +1 Regulatory regions Protein coding regions Gene SNP Expression Assays-by-DesignSM Service 39 © 2006 Applied Biosystems TaqMan SNP 基因分型方法 • • • • • • 40 采用TaqMan方法 金标准SNP分型技术 方便的技术流程 位点选择非常灵活 有大量成套试剂或代客合成 采用实时荧光定量PCR方法 © 2006 Applied Biosystems TaqMan SNP Genotyping: 每个反应管内 2 条引物+2条TaqMan® MGB探针 (VIC/FAM) • 16万种 high-confidence assays • TaqMan® MGB probe technology under universal assay conditions 2D Barcoded Tubes 750 rxns 5µL rxn size 41 Assay Information Provided on CD Minor Allele frequency SNP Base Context Sequence Reporter 1 Dye Reporter 2 Dye © 2006 Applied Biosystems e Applied Biosystems Real-Time PCR Systems 7300 7500 7900HT 96 well Block Format 96 well 96 well 384 well Microfluidic Card (GenEx) Reaction Size Applications 42 25 uL 25 uL Genotyping Genotyping Gene Expression Gene Expression +/- Assay +/- Assay 5 uL (384 well) 25 uL (96 well) Genotyping Gene Expr © 2006 Applied Biosystems TaqMan® SNP Assay Results “FAM dye” 2/2 1/2 1/1 “VIC dye” 43 © 2006 Applied Biosystems TaqMan® Assays – Highlights • TaqMan® Assays 已经被认为是基因分型和基因表达分析的金标准 • • • 准确 重现性好 使用方便 • Product lines available for TaqMan® SNP Genotyping Assays • • • • Validated Pre-Designed DME Custom assays • 已经在全球各个领域的客户得到广泛应用 • • • • • 44 Academic Pharma Biotech Agriculture Core labs © 2006 Applied Biosystems TaqMan® SNP 基因分型试剂 • 超过3,500,000种试剂盒已经上市 四种产品可选：Validated, cSNP, Pre-Designed, or DME 最全面完整的SNP分型试剂: SNPs from Celera, NCBI dbSNP, HapMap & other human variation databases • Custom assay designs also available 单管试剂盒 – 直接可以使用! • Genotyping assays contain two primers and two allele-specific probes • VIC dye and FAM dye labeled probes 全部引物探针均在同一条件下事先优化 • All assays run under a single set of conditions • DME is exception 生物信息学支持Bioinformatics applied during assay design • (does not include Custom assays) 严格的生产质量控制 • Mass spec • Quantitation • Functional testing (human targets) • • • • • • 45 © 2006 Applied Biosystems HapMap Project SNPs的重要性 • Validated SNP genotype data for > 3.2 million genome-wide SNPs in 44-60 individuals from: • Yorubans - Ibadan, Nigeria, Africa (YRI) • Caucasian - North & West European ancestry; Utah, USA (CEU) • Japanese - Tokyo, Japan (JPT) • Chinese - Han Chinese; Beijing, China (CHB) • 项目研究目标: To develop a genome-wide haplotype map for identifying haplotype blocks, common haplotypes and haplotype tag SNPs in these populations. • 研究基因及基因多态性对疾病影响的有力工具 HapMap SNPs will be the Golden Standard for association studies 46 © 2006 Applied Biosystems HapMap SNP assays • TaqMan® SNP Genotyping Assays是分析 HapMap SNPs的最灵活的工具 • Customer can quickly search for their SNP(s) of interest, order assay(s), and have results within a few days • We currently offer assays for >2.7 Million TaqMan® Assays for HapMap SNPs • > 2.7 M TaqMan® Assays • > 110,000 Validated Assays • > 2.6 M Pre-Designed Assays • ~ 24,000 nonsynonymous coding SNP assays • Represents ~ 70% of all HapMap SNPs • SNPbrowser™ Software v3.5 includes the complete HapMap SNP set is the easiest way to search HapMap data and get assays for HapMap SNPs • Includes all HapMap MAF information • Includes Linkage Disequilibrium Maps (i.e., genetic association maps), putative haplotype blocks, and haplotype tag SNP selection methods, all calcualated from HapMap SNP genotype data. 47 © 2006 Applied Biosystems TaqMan® SNP Genotyping Assays: 产品分类概况 48 产品名称 产品说明 TaqMan® Validated SNP Genotyping Assays 160,000 population-validated SNP assays (Inventoried) TaqMan® Coding SNP Genotyping Assays 60,000 assays that target gene-coding regions TaqMan® Pre-Designed SNP Genotyping Assays 3,400,000 SNP assays designed in-silico (Made to Order) TaqMan® Drug Metabolism Genotyping Assays 2400 assays that target SNPs in 220 genes known to be part of drug metabolism pathways (Inventoried) Custom TaqMan® SNP Genotyping Assays Custom assays designed around customer’s SNP of interest – any possible SNP, any genome (30,000 Inventoried / 30,000 Made to Order) © 2006 Applied Biosystems TaqMan® Validated 已经验证合成好的SNP Genotyping Assays 主要特点 • • • • >160,000 validated assays ~10 kb spacing across gene regions 包括> 67,000 HapMap SNPs 已获得各人种基因型分析和最小等位基因频率 (45 samples each) • Caucasian • African American • Chinese • Japanese • All assays have MAF > 5% in at least one population • 已经大规模测试保证极高的成功率 • 已经有库存试剂盒，接定单后可以直接发货 49 © 2006 Applied Biosystems TaqMan® 编码区SNP分型试剂盒 主要特点 • ~60,000 assays that target gene coding regions • ~30,000 in inventory • ~30,000 available as pre-designed assays • Includes > 1,700 HapMap SNPs • Importance: SNP appearing in coding regions may have a direct impact on the protein derived from a gene, causing a structural and/or functional or change • • • Known Causative SNP Putative Functional SNP (pfSNP) Non-synonymous SNP (nsSNP) • 直接关联分析: trend to move from surrogate markers in association studies to using potential causative SNPs • Functional Test on assays • Functional testing on 10-20 DNA samples (4 populations) are completed before assays placed into inventory 50 © 2006 Applied Biosystems Coding SNPs的重要性 基因编码区 – coding SNPs的三种类型: CCA ACG AGA GGT GCA TAC CAG AGT Pro Thr Ala Ala Ala Tyr Gly Ser ACT Thr Silent SilentSNP SNP GAA Glu TAG stop Missense MissenseSNP SNP Nonsense NonsenseSNP SNP Synonymous SynonymouscSNP cSNP Non NonSynonymous SynonymouscSNPs cSNPs(nsSNPs) (nsSNPs) No Amino Acid change 51 Amino Acid changes © 2006 Applied Biosystems 功能性 SNPs的定义 • May be “causative” sequence variants • May alter protein structure and function • Includes nonsynonymous cSNPs & regulatory SNPs • Use for direct association analysis • Tend to have low minor allele frequencies (< 10 % MAFs) • Public domain sources have relatively sparse representation of putative functional cSNPs • OMIN, PharmGKB, HGMD, etc. • May be more difficult to design cSNP assays if present in conserved coding sequence domains • Little statistical power to identify linked changes (due to low MAFs; association studies often use higher MAF SNPs) 52 © 2006 Applied Biosystems TaqMan® 药物代谢酶基因分型试剂盒主要特点 • Over 2400 DME Assays are available • • • • • • 53 • Collection includes all of the SNPs represented in the PDAR product line Target SNPs in 220+ genes encoding Drug Metabolism Enzymes • SNPs associated with these genes may influence the rate of drug metabolism within individuals Includes SNPs, InDels, and MNPs Where available, the assays are mapped to allele nomenclature at public allele nomenclature sites Includes novel target SNPs from Applera Sequencing Project Performance tested on 180 unique DNA samples from four human populations • Caucasian, African American, Chinese, Japanese Inventoried and shipped upon order © 2006 Applied Biosystems 药物代谢酶基因 Pharmacogenetics 合作项目 • 美国国立癌症研究所 Core Genotyping Facility (CGF) 和Applied Biosystems 公司合作检测分析药物代谢酶基因的多态性 • CGF 采用Applied Biosystems TaqMan® Drug Metabolism Genotyping 全部2400种试剂盒: • International HapMap samples (N=270) • SNP500Caner samples and additional population control samples (N=382) • Clinical sample set • 数据正在联合分析并将结果会公布在http://SNP500Cancer.nci.nih.gov and elsewhere. 54 © 2006 Applied Biosystems SNP分型试剂产品比较 Feature Validated Coding Pre-Designed DME Custom Key Benefit Highest success rate due to extensive testing Ideal for detection of putative functional SNPs High-density, genomewide marker coverage SNPs in genes targeting drug metabolism pathways Any possible SNP, any genome 160,000 60,000 3,400,000 2400 N/A 112,000 (70%) 24,000 (40%) 2,600,000 (76%) 800 (33%) N/A Human Human Human Human Any • • Number of Assays Number of HapMap SNPs Species Detection of Insertsions and Deletions (InDels) Bioinformatics Evaluation of Target SNP Sequences • • • • Customer dependent Bioinformatics Design of Primers and Probes • • • • • Manufacturing QC Analytical & Functional tests • • • • • Minor Allele Frequency (MAF) Validation • MAF data may be available from HapMap & others MAF data may be available from HapMap & others • MAF data may be available from public sources Small ½ Small ½ Multiple scale Small Medium Large Small Small Medium Large Inventoried ½ Inventoried ½ Made to Order Made to Order Inventoried Made to Order Scale(s) Inventory Status 55 © 2006 Applied Biosystems 基因分型技术平台Genotyping TaqMan SNP Genotyping 56 SNPlex Genotyping System © 2006 Applied Biosystems SNPbrowser™ Software v3.5 SNP位点选择工具 用于多基因病关联分 析 SNPbrowser™ SNP浏览器软件简介 • 免费软件 专门软件可以用来简化疾病关联分析研究时SNP位点 的选择 • 可以显示每一个SNPs 位点在染色体/基因的位置，相邻 SNPs. • 包含超过3百万个SNP位点的详细信息 以及多种SNP位点选择 工具来帮助研究人员挑选SNP . • 既方便Facilitates both SNP位点选择又方便网络订购 Applied Biosystems TaqMan® and SNPlex™ SNP 基因分型试剂. SNPbrowser™ 软件是选择 SNP 的首选工具。 58 © 2006 Applied Biosystems SNPbrowser™ Software v3.5 软件中的SNP试剂数据库 包含从HapMap, Celera RefSNP, and NCBI dbSNP数据库中的SNP数据 1. TaqMan® SNP Genotyping Assays ~ 160,000 Validated SNP Assays (inventoried) ~ 60,000 nonsynonymous coding SNP Assays total (~ 30 k inventoried, ~30 k made to order) > 3 million Pre-Designed SNP Assays 2. SNPlex™ Genotyping System Includes > 5 million pre-screened SNPs for SNPlex™ assays v3.5 版中的全部试剂已和公司网站上的数据库内容同步 59 © 2006 Applied Biosystems SNPbrowser™ Software v3.5包含经过验证的SNP LD Map 数据库 1. HapMap Project LD Map database – Golden Standard - 包含完整的国际HapMap 项目全部验证的SNP基因分型数据，包括3 百万SNPs位 点，44-60 无关个体的基因型，黑人, 白人, 日本人和中国人。 70 % and 90 % of HapMap SNPs covered by robust TaqMan® assays and by SNPlex™ System assays, respectively. 2. Applied Biosystems LD Map database 经过验证的SNP基因分型数据，采用TaqMan® Validated SNP Genotyping 分析 了160,000 SNPs ，180 人，每种人群45 个个体，分别为美国黑人, 白人 Caucasian, 日本人和中国人 2个独立的经过验证的 SNP 基因型数据库 underlie all genetic functions in SNPbrowser™ Software. Linkage disequilibrium (LD) 连锁不平衡 A measure of the nonrandom association between two or more genetic markers on a chromosome. SNPs are in complete LD if they are inherited together (no evidence of recombination between them). 60 © 2006 Applied Biosystems LD Map database selected 位点注解信息 SNPs Blue SNPs – validated in LD Map db Gray SNPS – unvalidated in LD Map db LD/Haplotype blocks Power Genes Exons Locations in physical map SNP set selections 61 Locations in population-specific metric LD map © 2006 Applied Biosystems 标签SNP 位点选择指引 • Tagging SNPs -标签SNP 是一组信息价值大的SNPs ，它们强连锁在一起，共 同遗传，中间无基因重组：strong LD with one another (coinherited; little or no evidence of recombination between markers). • Genotypes of validated SNPs are used to remove SNPs providing redundant genetic information in a selected population and chromosome region. (1) Genotype Correlation - eliminates SNPs with redundant genotypes (2) Pairwise r2 or (3) Haplotype R2 – eliminate SNPs providing redundant genetic information due to strong LD correlation with other SNPs, while maintaining a given pairwise r2 or Haplotype R2 threshold value. 可以减少SNP基因分型的工作量，省时更有效的研究 62 © 2006 Applied Biosystems 下载SNPbrowser™ Software v3.5 from www.allsnps.com • 需要注册以获得一个序列号，以便安装软件 • 已经注册的用户在软件更新时回自动收到一个电子邮件或者打开电脑时自动提示 • Updates to microsite highlight v3.5 new features (also see ReadMe file) SNPbrowser™ Software v3.5中的帮助信息 • Updated Help Text = User Manual (access from Menu Bar) Comprehensive description of all features, including: • LD Map databases and SNP assay sets • SNP annotation and genotype downloads • SNP wizards functions and workflows • Step-by-step ordering of TaqMan® SNP and SNPlexTM Genotyping System assays • FAQs • Literature references • Glossary 63 © 2006 Applied Biosystems AB 提供多种技术平台,可进行多种基因分型应用 APPLICATION 高密度突变/SNP 连锁分析或关联分析 候选基因 /侯选区 域关联分析 较大侯选区域/全 基因组关联分析 64 PLATFORM • 3130/3730基因分析仪 • 既分型又可发现新突变 双重目的,分型金标准 • 微卫星STRs,SNPs • 7300/7500/7900 荧光定量PCR仪 • 位点选择灵活,成本低 • 方便 试剂盒全部经过验证 • 基因分型和基因表达分析 • SNPlex System on 3730xl/3130XL基因分析仪 • 低成本 • 超高通量, 位点可选 基因及SNP位点选择: SNPbrowser 3.5 © 2006 Applied Biosystems
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