高通量测序原理和应用精要.ppt

RNA-seq 基因表达差异（DGE） 转录本 DGE DGE Fragment buffer N6 primer mRNA DNA RNase H ds cDNA Superscript III DNA polymerase I 转录本 转录本 RNA-seq在医口中的应用 用于癌驱动基因的筛选 用于融合基因的筛选 用于可变剪接的筛选 用于RNA水平的SNP分析 …… Hiseq 2000 Hiseq 2000 High-seq 2000 Some of our partners on cancer and complex disease studies Welcome to BGI. New campus in Shenzhen: Beijing branch: Hangzhou branch: Shenzhen branch: Thanks ！ 在ff-dNTP中,其中的一个甲基被加入了一个荧光基团. * * * Purpose: To visually demonstrate sequencing by synthesis Details: A visual illustration of 9 cycles of sequencing by synthesis. Following the 20 micron square from the previous slide you can see how the clusters change color each cycle. Looking at the top circled cluster the color changes from green to blue, to red, to green…corresponding to T, G, C, T… Looking at the bottom circled cluster the color is green, green, green, green, green…corresponding to a homopolymer T interrupted by a G (cycle 8)? This is a great moment to indicate our ability to accurately sequence homopolymers (unlike 454). Details are shown in the data description of our BAC sequencing standard later in this presentation. Conclusion: Visual explanation of sequencing by synthesis 仪器：CLUSTER STATION GENOME ANALYZER * * 8oxo-G是物理性切割位点 U是酶切位点 Indexed Sequencing * * * * * * 高通量测序原理和应用 沈 峰 深圳华大基因研究院 Illumina/Solexa genome analyzer C T A G C T A 5’- 3’- …-5’ G T 合成第一个碱基 Cycle 1:按顺序加入反应试剂 清除未反应的碱基和试剂 激发碱基荧光并收集荧光信号 去除阻断基团和荧光基团 Cycle 2-n: 重复前面的步骤 G T C T A G T C T G C T A G A Base Calling 1 2 3 7 8 9 4 5 6 T T T T T T T G T … T G C T A C G A T … The identity of each base of a cluster is read off from sequential images CS负责将待测样品“种植”于检测芯片(Flow Cell)中并进行扩增。 GA负责对芯片内的基因片段进行边合成边测序反应(SBS)，收集碱基信息。 Single-Read Sequencing 单向测序 Paired-End Sequencing 双向测序 Indexed Sequencing 混合样品测序 Single-Read Sequencing Single-Read Sequencing(SR, 单向测序)指只检测基因片段一端的序列信息。 接头 接头 互补接头 基因序列 Paired-End Sequencing Paired-End Sequencing (PE, 双向测序