深度测序技术简介重点.ppt

专业建库 测序 专业建库 测序 专业建库 测序 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 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
25–50 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）： 第二代测序技术在制备测序文库的时候都需要经过PCR扩增，而这一PCR过程可能引入突变或者改变样品中核酸分子的比例关系。另外，第二代测序的读长普遍偏短，在进行数据拼接时会遇到麻烦。为了克服这样的缺点，业界发展出了以单分子实时测序和纳米孔为标志的第三代测序技术。简介如下： Helicos公司的Heliscope单分子测序仪基于边合成边测序的思想，将待测序列随机打断成小片段并在3末端加上Poly(A)，用末端转移酶在接头末端加上Cy3荧光标记。用小片段与表面带有寡聚Poly(T)的平板杂交。然后，加入DNA聚合酶和Cy5荧光标记的dNTP进行DNA合成反应，每一轮反应加一种dNTP。将未参与合成的dNTP和DNA聚合酶洗脱，检测上一步记录的杂交位置上是否有荧光信号，如果有则说明该位置上结合了所加入的这种dNTP。用化学试剂去掉荧光标记，以便进行下一轮反应。经过不断地重复合成、洗脱、成像、淬灭过程完成测序。Heliscope的读取长度约为30-35 nt，每个循环的数据产出