高通量测序技术简介.ppt

第五部分：其他类型的芯片,微缩芯片实验室（Lab-on-a-chip) 药物运输芯片 生理功能辅助芯片 纳米芯片,Next Generation Sequencing,Sample fragmentation Library preparation Sequencing reaction Data analysis,Roche 454 焦磷酸测序 Pyrophosphate Sequencing,Illumina Solexa 合成测序 Sequence by Synthesize,ABI SOLiD 连接法测序 Sequence by Ligation,第六部分 高通量测序技术简介,Roche 454 焦磷酸测序 Pyrophosphate Sequencing 基本原理,454 sequencing： Emulsion PCR (emPCR),Mix DNA Library & capture beads (limited dilution),“Break micro-reactors” Isolate DNA containing beads,Generation of millions of clonally amplified templates on each bead No cloning and colony picking,Create “Water-in-oil” emulsion,+ PCR Reagents,+ Emulsion Oil,Perform emulsion PCR,Adapter carrying library DNA,A,B,Micro-reactors,Adapter complement,Enrich,Anneal Seq primer,Centrifuge Step,Load Enzyme Beads,454 sequencing： Deposition of DNA beads into the PicoTiterPlate,Load beads into PicoTiterPlate,Illumina Solexa 合成测序 Sequence by Synthesize 基本原理,Clonal Single Molecule Arrays 单分子克隆,1000 molecules per 1 m cluster 1000 clusters per 100 m square 40 million clusters per experiment,Prepare DNA fragments,Ligate adapters,Attach single molecules to surface,Amplify to form clusters,Reversible Terminator Chemistry 可逆终止反应,All 4 labelled nucleotides in 1 reaction,Sequencing-by-Synthesis (SBS),First base incorporated,Cycle 1: Add sequencing reagents,Remove unincorporated bases,Detect signal,Cycle 2-n: Add sequencing reagents and repeat,1、每轮测序反应加入四种带有荧光标记的dNTP，末端带有可以被去除的阻断基团 2、每轮反应只能整合一个核苷酸，仪器读取相应的荧光信号 3、信号读取结束，用化学方法去除阻断基团，进行下一轮测序反应,T T T T T T T G T ,T G C T A C G A T ,The identity of each base of a cluster is read off from sequential images 根据每个点每轮反应读取的荧光信号序列，转换成相应的DNA序列,Base calling from the raw data,Solexa 测序 Workflow,ABI SOLiD 连接法测序 Sequence by Ligation 基本原理,文库制备：微珠单分子克隆,1024种8碱基探针 4色荧光，4种双核苷酸，每色荧光有256个探针(46),SOLiD 利用探针的连接反应读取模板的DNA序列,连接法测序 （一）,每个探针进行检测的两个碱基后面有三个匹配碱基，因此一条测序引物读取的序列是不完整的,测序引物与adapter退火,探针连接，检测荧光,切除荧光基团,第二轮探针连接，检测荧光,切除荧光基团,连接法测序 （二）,测序引物沿着Adapter移动5次，确保每个位点都被检测,连接法测序 （三）,0位置是Adapter的最后一个碱基，因此只检测一次， 该碱基是进行解码所必须的。,Advantage & disadvantage,454 sequencing 读取长度大，400bp 可以对未知基因组进行从头测序de novo sequencing 当遇到polymer时，如AAAAAA等，荧光强度和碱基个数不成线性关系，判定重复碱基个数有困难 Solexa sequencing 高度自动化的系统 读取片段多，适合进行大量小片段的测序，如microRNA profiling 基于可逆反应，随反应轮数增加，效率降低，信号衰减，读取序列较短，给de novo sequencing 拼接带来困难 SOLiD sequencing 每个碱基读取两次非常高的准确性，特别是对于SNP的检测 灵活的系统，完善的磁珠编码系统，可以进行样品的pooling，分割测序区域 读取长度受连接反应的轮数限制，给de novo sequencing 拼接带来困难,高通量测序的应用,De novo 测序 基因深度测序（genome re-sequencing） 转录组深度测序（transcriptome re-sequencing） Digital expression profiling ChIP-seq Methy-seq,Transcriptome resequencing：,malignant pleural mesotheliomas (MPMs) ：恶性胸膜间皮瘤 pulmonary adenocarcinoma (ADCA)：肺腺癌,Transcriptome characteristics,Solid line： at least one read Dashed line：at least 20 reads,Expression difference between MPM and ADCA sample compare to a lung tissue control,Analysis of percent- age of reads containing known coding region SNVs in the six tissue samples.,SNV： Single Nucleotide Substitution Variant,Digital expression profiling（1）： 人大脑组织与UHR（Universal Human Reference）的表达差异,Digital expression profiling & microRNA re-sequencing：,hESC： human embryonic stem cells EB： embryoid bodies,ChIP-seq（1）： 人一号染色体DNA-蛋白相互作用,ChIP-seq（2）：,Sequenced short reads (typically 2550 bp) from ChIP-Seq experiments are rst mapped onto the reference genome. The mapped reads are then used to estimate statistical parameters, which include the estimation of the average length F of sequenced DNA fragments.,Methy-seq（1）： 肿瘤和MCF7细胞系中 BRCA!启动子区域的甲基化差异,Some highlights: Correlation between ChIP-Seq and his prior SAGE-like method (called GMAT) has r=0.906 However the resolution with ChIP-Seq was dramatically higher. Furthermore, ChIP-Seq was more sensitive and generated less false-negative regions 12,726 genes whose transcription levels are known in CD4+ T-cells were correlated with the histone modifications and 35,961 Pol II binding site islands were identified This cost-effective method produces digital-quality data and should find broad applications in our efforts to understand the contribution of the human epigenomes in gene expression and epigenetic inheritance,Methy-seq（2）：,部分参考文献阅读,Genome re-sequencing van Orsouw N J, Hogers R C, Janssen A, et al. Complexity reduction of polymorphic sequences (CRoPS): a novel approach for large-scale polymorphism discovery in complex genomes. PLoS ONE, 2007, 2(11): e1172 Hillier L W, Marth G T, Quinlan A R, et al. Whole-genome sequencing and variant discovery in C. elegans. Nat Methods, 2008, 5(2): 183188 Transcriptome re-sequencing Mortazavi A, Williams B A, McCue K, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods, 2008, 5(7): 621628 Sugarbaker D J, Richards W G, Gordon G J, et al. Transcriptome sequencing of malignant pleural mesothelioma tumors. Proc Natl Acad Sci USA, 2008, 105(9): 35213526 Digital expression profiling Ruby J G, Jan C, Player C, et al. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell, 2006, 127(6): 11931207 Morin R D, OConnor M D, Griffith M, et al. Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res, 2008, 18(4): 610621 ChIP-seq Johnson D S, Mortazavi A, Myers R M, et al. Genome-wide mapping of in vivo protein-DNA interactions. Science, 2007, 316(5830): 14971502 Robertson G, Hirst M, Bainbridge M, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation an