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【2017年整理】深度与测序技术简介
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
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