张扬基因芯片.ppt

Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Company name,*,Click to edit Master title style,内容简介,第一部分 芯片概述,第二部分 几种芯片的应用介绍,第三部分 不同芯片平台的介绍,第四部分 芯片实验中应该注意的问题,第五部分 定制芯片的一个案例介绍,Company name,生物芯片研究基因组学的有力工具,拷贝数改变,Copy Number Changes,Differential Gene Expression,Splice Variant Analysis,体细胞突变,Somatic Mutations,线粒体突变,Mitochondrial Mutations,WG SNP Mapping Arrays,Exon Arrays,3 IVT Expression Arrays,Exon Arrays,Whole Genome Tiling Arrays,Promoter Tiling Arrays,Mito Resequencing,线粒体重测序,Targeted Genotyping Products,CustomSeq Resequencing Arrays,Germline Mutations,WG SNP Mapping Arrays,Protein-DNA interactions,ChIP-on chip,Company name,What whole genome scanning for?,影响表观遗传学的重要因子,SNP,的变化,基因组甲基化,基因拷贝数变化,插入，缺失,采用的技术,Affymetrix SNP,系列芯片（,SNP,CNVs),Agilent CGH,系列芯片,Illumina,Hap,系列芯片,Company name,SNP,与遗传突变,Single Nucleotide Polymorphisms(SNPs),是指在基因组上单个核苷酸的变异,包括置换、颠换、缺失和插入。,在人类基因组中大概每,300,个碱基就有一个,SNP,。,从理论上来看每一个,SNP,位点都可以有,4,种不同的变异形式,但实际上发生的只有两种,即转换和颠换,二者之比为,2:1,。,SNP,在,CG,序列上出现最为频繁,而且多是,C,转换为,T,原因是,CG,中的,C,常为甲基化的,自发地脱氨后即成为胸腺嘧啶。,一般而言,大多数,SNP,对细胞功能无影响，但少数可以使人对疾病易感，或者对药物敏感。,Company name,为什么,SNP,会变化？,Company name,SNP,和肺癌,4,月,3,日,Nature,上发表文章公布了肺癌和遗传疾病的关系。,发现肺癌和编号为,rs1051730,和,rs8034191,的,SNP,位点有关。两个位点都位于,15,号染色体上，影响到多个基因，其中包括三个尼古丁受体基因。,这两个位点的突变使得吸烟者患肺癌几率增加了,80%,。,Company name,Comparative Genomic Hybridization(CGH),BAC1,red,green,red,green,BAC2,probes,target,normal,tumor,Company name,Company name,自闭症,(Autism),和染色体,16p11.2.,的部分缺失,Company name,Company name,采用FISH探针验证结果,Analysis with Fluorescence in Situ Hybridization(FISH).,BAC clone RP11-504I2 was used as a probe for FISH analysis.signal was seen on one chromosome 16(arrow)but not on the other chromosome 16(arrowhead).,Company name,Tiling,的技术原理,Exon B,Exon A,35,bp spacing,Genome,5,bp spacing,35,bp,5,bp,10,bp gap,20,bp overlap,Company name,Tiling array,的应用领域,Mapping regions of transcription,（测定,RNA,转录区域）,Transcription factor binding sites,（测定转录因子在,DNA,上的结合区域）,Sites of DNA methylation,（,DNA,甲基化位点）,Histone Modification,（染色体组蛋白修饰）,Chromosomal origins of replication,（基因复制起点）,RNA binding protein sites,（,RNA,结合蛋白在,RNA,上的结合位点）,Chromosome copy numbers and deletions,（染色体拷贝数及缺失）,Population biology/Evolution,（群体生物遗传及进化）,Company name,Tiling,芯片的使用案例,目的,:,研究拟南芥的表观遗传学和转录多型性。,材料：,Columbia(Col)and Vancouver(Van),研究方法：,通过,Tiling array,研究两者基因组,CCGG,甲基化位点差异。使用识别,CCGG,位点,HpaII,和,MspI,两种限制性内切酶酶切基因组,DNA,其中，,HpaII,在,CCGG,内的胞嘧啶被甲基化的情况下无法识别酶切位点，而,MspI,则可以。,推论：,如果,CCGG,有甲基化，则样本被两种限制性内切酶酶切的片段有差异，通过比较片段的差异可知酶切的位点。,Company name,拟南芥的,Tiling array,实验结果,Company name,Tiling and Tilling,TILLING,即Targeting Induced Local Lesions IN Genomes(定向诱导基因组局部突变技术),是由美国Fred Hutchinson癌症研究中心Steven Henikoff领导的研究小组发展起来的,是一种全新的反向遗传学研究方法.TILLING技术借助高通量的检测手段,快速有效地从由化学诱变剂(EMS)诱变过的突变群体中鉴定出点突变.,Company name,Company name,DNA,甲基化,DNA methylation occurs at 5MC within CpG dinucleotides.CpG frequency 5X less than expected,though to be due to increased mutation at CpGs.Transitions(C-T)occur at 10-40 fold higher than other dinucleotides.CpGs are,not,randomly distributed in the genome.CpGs are methylated in repeats and 3 regions of genes,but,not in CpG islands.,There are estimated to be about 26,000-45,000 CpG islands,many which tend to be located in the promoter regions of housekeeping genes and some tissue specific genes.,Company name,Company name,Company name,DNA is compacted into chromatin by complexing with nucleosomes made up of histone octomers.The histone tails extend out from the nucleosome and can be modified(for example by methylation or acetylation)to etermine the accessibility of DNA for transcription,Company name,各种芯片平台介绍,Affymetrix,芯片平台,Illumina,芯片平台,Agilent,芯片平台,Combimatrix,芯片平台,Beckman SNP stream,平台,Company name,Affymetrix芯片平台,技术成熟，稳定度高，拥有完整的质量控制体系，以及,Netaffx,数据库支持。,是目前探针密度最高的一种芯片，推出时间较早有大量的文章支持。,对实验仪器和实验环境的要求高，试剂，芯片费用较高。,可供选择的芯片种类较多，其中研究外显子的芯片为其所独有。,Company name,预制的基因芯片 芯片滚动杂交仪 全自动芯片洗涤工作站,智能化的分析软件,高分辨率的芯片扫描仪,Affymatrix,基因芯片系统硬件组成,Company name,GeneChip,的操作流程,以真核生物为例,L,L,L,L,L,L,L,L,cDNA,AAAA,总,RNA,的制备,反转录,体外,转录,生物素标记的,cRNA,片段化处理,带标记的,cRNA,片断,35-200 bases,0.5-2 ug/ul,起始用量,5-10ug,（,IVT,）,L,L,杂交混合液的制备,Eukaryotic,Hyb.Control,Control,Oligo B2,杂交,洗涤,扫描,数据分析,Company name,光蚀刻原位合成技术,1.,光蚀刻原位合成,Affymetrix,基因芯片的特点,Company name,2.,高密度的点阵技术,1,个平方厘米的面积至少可排列几十万到一百多万个探针合成区（“点”）,Affymetrix,基因芯片的特点,Company name,Affymetrix,芯片的探针设计原理,5,3,基因序列,11,20,对,25mer,探针,Mismatch,Perfect Match,MM Mismatch,单点突变,Oligo,探针,Company name,基因表达,开放,基因表达,关闭,Oligo arrays,cDNA arrays,多个检测结果可以参考,只有一个点的信号,20 m,150 m,PM,探针,MM,探针,芯片结果准确可靠,Company name,13,400,Signal Intensities:,13,090,Signal Intensities:,同一样本，,不同批次的芯片,Signal Intensities:13,400,Signal Intensities:11,670,不同的样本，,不同批次的芯片,False I/D,(,2 fold),2 fold),85%,Taqman AOD5-10 ng 1-2495%,Taqman Manual5-10 ng 1-2455%,Taqman PDAR5-10 ng 1-24 75%,MGB Eclipse5-10 ng 1-2490%,SNPlex40 ng 24,4895%,SNPStream 5-10 ng 12,4895%,Golden Gate 250 ng 96-153699%,Infinium750 ng 14K 24K98%,Whole Genome,Infinium750 ng 100K,300K,550K98%,GeneChip250 ng(x2)10K,50K,250K 93%,Beckman SNPstream,和其他系统的比较,Company name,Company name,芯片实验中常见的问题,How to prepare my samples?,How to design the experiment,？,Pooling or not?,Which one is better,one dye or two dye?,How many replicates?,How to analysis database?,Company name,How to prepare the samples?,RNA sample for microarray,：,RNA,without degradation,（,Electrophoresis,）,Without enzyme inhibitor,Better not cDNA,Enough amount,（,total RNA 10-20ug),DNA,sample for microarray,：,DNA integrity,Without enzyme inhibitor,Without other Genomic DNA or PCR product contaminate,（,except pooling,samples,）,Company name,RNA,质量的经验判断,28S,18S,普通琼脂糖电泳结果,Agilent 2100,分析结果,Company name,实验的设计,Case 1:,Samples:,Liver tissue from mice treated by cholesterol modifying drugs.,Question 1,:,Find genes that respond differently between the treatment and the control.,Question 2:,Find genes that respond similarly across two or more treatments relative to control.,T1,C,T2,T3,T4,x 2,x 2,x 2,x 2,Company name,Case 2:interaction,Samples:,treated cell lines at 4 time points (30 minutes,1 hour,4 hours,24 hours),Question:,Which genes contribute to the enhanced inhibitory effect of OSM when it is combined with EGF?Role of time?,ctl,OSM,EGF,OSM&EGF,4 times,Company name,Pooling or not?,在大多数的情况下，,pooling,的样本可以用来做为参照或者代表一群有共同特征的样本，从而节省芯片的使用量，尽可能多的获得这个群体的共性。但反过来说，也会丢失样品的个体信息。,As left pictures shows,the RNA expression level has been significantly affected by certain factors,using total RNA amount as pooling control will lead to information lose.,Company name,Some results.,Pooled variance t-test on“individual”data,treated versus controls:,chipA,669 p0.001,594/(669,1948)common to overall analysis,226 p2,221/(226,356)common to overall analysis,chipB,245 p0.001,196(245,743)common to overall analysis,80 p2,77/(80,143)common to overall analysis,Company name,Company name,One dye or two dyes?,One dye means one sample per chip,Two dyes means two samples(control and target)could hybrid in one chip at the same time.,Obiviously,two dyes method will save chips.,But is this really true?,Company name,Comparing one dye with two dyes,Comparing with two dyes,one dye method could got stronger signals.,Each microarray data could be divided or merge into certain group freely.,High,Low,Two dyes,One dye,Company name,我们为什么重复,?,生物,最经常的变异来源,样品准备,方法、试剂和操作者,系统,芯片、仪器、试剂和操作者,Sample 1,Sample 2,Sample 3,High,高,Low,低,Company name,How many replicates?,For Affymetrix systems with the best analysis(see),we find 3 to 5 chips per group gives useful information.,If an exploratory study aims to find large(more than two-fold)differences between two conditions,then a design with three samples per condition is usually adequate.If the aim is to find smaller differences,or almost all of the large differences,then five samples per group are necessary to obtain sufficiently reliable enough estimates of variation among samples within conditions,in order to distinguish true differences between conditions.,Company name,How many replicates?,To do meaningful clustering requires at least 20 samples,and generally many more.The key issue for clustering genes is how many different types of samples there are,because the different conditions expose the correlations in gene regulation.It is not useful to try to cluster genes from only two groups,as is sometimes done,and rarely useful to cluster genes from a study of fewer than five groups.,Company name,定制芯片服务案例,背景：,一个客户要求定制细菌基因组芯片，已经报道的该细菌的全基因组有两条，其中基因有些不同。,客户研究经费有限，不便于全部将基因组增加上去。,客户想研究自己的细菌不同基因的差异表达情况。,Company name,案例一，实验设计,经过Blast比较发现甲菌株和乙菌株之间序列相似度95%以上的基因有4057个。不相似基因分别为315个和90个。,Company name,4057,90,315,甲菌株,乙菌株,Company name,基因相似性比较,我们用这两个菌株跟大肠杆菌基因组做了序列比对，发现两者共有的功能基因1543个，差异基因2604个。,因此定制芯片的点数可以控制在一个经济合理的范围内。,Company name,芯片设计方案,2604,+,315,=,2919,Company name,芯片检测的结果,Company name,Thank You!,