（）
（修饰末端）
（去除未连接上的 接头）（基因组DNA）（基因组DNA小片段） （）
（） （） （纯化连接产物）
Fragment library
Genomic DNA
Mate-paired library
27b
27b
p
p
27b
27b
p
p
Create library of DNA fragments
可逆阻断技术
Illumina Solexa Flowcell
Flowcell
一个 flowcell 包括8个lanes
Lane 1
Lane 8
Image from 1 tile
•Each lane contains multiple tiles – total 100 每个lane上有许多个tiles——共计100个（见上图）
Whitfield (1954)
1970 1980
1990 2000
2010
Development of Sanger Sequencing (1977)
Invention of Capillary Sequencer (1996)
Invention of Automated Fluorescent Sequencer
基因组测序 Genome Analyzer
分析 Analysis Pipeline
•Genomic •mRNA •Small RNA •ChIP-Seq
样品预处理
* Grow Clusters * 5 hours * Or split process in stages * Safe stopping points
* 开始测序 * 序列簇的丰度检测 * 一个循环每个tile 照4张照片 * 2-3天
* 图像分析
?* Bustard: basecalling * Gerald: 序列分析
PCR成簇 5-6h
测序 6-8d
拼接分析
（纯化的基因组DNA）
（基因组DNA小片段 小于800bp）
（具有5’-磷酸末端的粘性片段）
(1985)
Invention of 454 GS 20 Sequencer
(2005)
Invention of Applied Biosystems
Solid System (2007)
Invention of Illumina Genome Analyzer System (2006)
Sanger 测序法原理
Invention of Single
molecule real time(SMRT)
DNA sequencing
Invention of Nanopore
single molecular sequencing
(Oxford Nanopore corporation)
1950 1960
Chemical degradation method by
测序技术基础
罗龙海 2009-03-02
• Sanger测序技术原理 • 第二代测序技术原理 • 第三代测序技术原理
测序技术发展史
chemical degradation method by
MaxamGilbert method (1977)
Invention of Heliscope
single molecular sequencer
•Each tile is imaged four times per cycle – one image per base 每个循环会对每个tile照4次相——每个碱基都会成像
Illumina/on
Cluster 工作站 Cluster Station
1个tile上有20,000个cluster 每个实验可完成3.2-4千万个cluster)
Cluster Generation: Amplification
OH OH
diol
P7
P5
Grafted flowcell
diol diol
* 生成“簇” * 5小时
* Start Sequencing * Cluster Density Evaluation * 4 images per tile per cycle * Run time: 2-3 days
* Firecrest: Image Analysis * Bustard: Basecalling *Gerald: Sequence Alignment
Dr. Fred Sanger
Frederick Sanger was awarded the prize in both 1958 and 1980. He is the fourth person in the world to have been awarded two Nobel Prizes and the only person to receive both in chemistry. "dideoxy" sequencing technique (Sanger et al., 1977)
DNA双脱氧链终止法测序
第二代测序技术
Illumina Genome Analyzer ABI SOLiD Roche 454
454 Soleபைடு நூலகம்a SOLID
二代测序技术
年份
2005
原理
Pyrosequencing
2006
边合成边测序
2007
边连接边测序
第二代测序技术
Illumina Solexa ABI SOLiD Roche 454
Random array of clusters （Cluster的随机排列）
~1000 molecules per ~ 1 um cluster
~20.000 clusters per tile
32-40 million clusters per experiment （1个clustetion序列簇的产生
Prepare DNA fragments
Ligate adapters
Attach single molecules to surface Amplify to form clusters
通过cluster将单分子放大、固定
100um