高通量测序守技术简介.pptVIP

Next Generation Sequencing Sample fragmentation Library preparation Sequencing reaction Data analysis Clonal Single Molecule Arrays单分子克隆 Reversible Terminator Chemistry可逆终止反应 Sequencing-by-Synthesis (SBS) 连接法测序（一） 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 Digital expression profiling（1）：人大脑组织与UHR（Universal Human Reference）的表达差异 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): 183—188 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): 621—628 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): 3521—3526 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): 1193—1207 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): 610—621 ChIP-seq Johnson D S, Mortazavi A, Myers R M, et al. Genome-wide mapping of in vivo