新一代测序技术的原理 Next-generation sequencing technology overview中英文 教学PPT课件.ppt

SOLiD的特点 通量大 成本低 序列短 Ligase的方式虽然能一定程度避免phasing/pre-phasing，但增加的复杂度也降低了效率 灵活性差，对小数据量测序不适用 Two-base coding 2005 2006 2007 Birthday Principle Pyrosequencing Sequencing-by-Synthesis Sequencing-by-Ligation Roche 454 Genome Analyzer/ Hiseq 2000 ABI SOLiD Next-generation sequencing/Deep sequencing technology main platforms 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 3 Hiseq 2000测序技术流程 制备芯片 模板杂交 桥式PCR扩增 测序引物准备测序 2 测序 测序生成 base calls 3 数据分析 拍照图片 Intensities Reads Alignments 4 DNA打断 末端修复 加A 加接头 纯化 文库制备 1 Fragment DNA 5’ 3’ 5’ + 3’ 5’ 5’ + 5’ 3’ 3’ 5’ 1.End repair T4 polymerase , DNA Pol 1 (Klenow fragment) 3’ 5’po4 po45’ 3’ Phosphorylation T4 polynucleaotide kinase, ATP 3.Ligation of adaptors 3’A po45’ A3’ 5’po4 2.Add 3’ Adenosine with Klenow (3’exo- ) and dATP + DNA insert 5’ 5’ 3’ 3’ SBS oligo P7 反向互补序列 3’T 5’po4 3’ 5’ SBS oligo P7反向互补序列 PCR enrichment 5’ 5’ 3’ 3’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 1st round PCR P7 P5 SBS oligo P7 反向互补序列 P7 3’ PCR enrichment (cont.) 5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 2nd round PCR Insert P7 P5 SBS oligo P7 反向互补序列 P5 Cluster template SBS oligo P7 * * * * * * * * Next-generation sequencing technology overview Agenda 测序技术简史 第一代测序技术 第二代测序技术 第三代测序技术 1 测序技术简史 1950 1960 1970 1980 1990 2000 2010 Development of Sanger Sequencing (1977) Invention of Automated Fluorescent Sequencer (1985) Invention of Capillary Sequencer (1996) Invention of Applied Biosystems Solid System (2007) Invention of Illumina Genome Analyzer System (2006) Invention of 454 GS 20 Sequencer (2005) chemical degradation method by Maxam-Gilbert method (1977) Chemical degradation method by Whitfield (1954) Invention of Heliscope single molecular sequencer Invention of Single molecule real time(SMRT) DNA sequencing Invention of Nanopore single molecular sequencing (Oxford Nanopore corporation) Invention of personal gen