深测序技术介绍.ppt

Sample fragmentation Library preparation Sequencing reaction Data analysis; ;;; ;;;;1;Solexa 测序 Workflow; ;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(4^6);;连接法测序（二）;连接法测序（三）;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 ;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：;Transcriptome characteristics;;Digital expression profiling microRNA re-sequencing：;ChIP-seq（1）： 人一号染色体DNA-蛋白相互作用;ChIP-seq（2）：;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’;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): 183—188 Transcriptome re-sequencing Mortazavi A, Williams B A, McCue K, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Na