华大基因 测序技术基础原理
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(ddNTP阻断末端)
OH
OH
diol diol
Cluster Amplification
OH
Denature and Hybridization
SBS3 （变性、杂交） SBS——边合成边测序
Sequencing By Synthesis
边合成边测序
SBS Cycle 1. Incorporation 结合 2. Scan 扫描拍照 3. Cleavage 清洗
40-50 Gb 10 days
2*75 bp Substitution
40-50G / 10天 / 一个RUN
读长：75bp
错误类型：
（可双向）
替换
Illumina solexa ABI SOLiD Roche 454
SOLID 测序技术
AB /SOLID Workflow 工作流程
1. 文库制备 2. Emulsion PCR 3. Beads Enrichment 4. 微珠沉积 5. 连接测序 6. 数据分析
(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
(PNK) 变性和去磷酸作用
OH
Resynthesis of P5 Strand
Sequencing
Second Read （NO.2次 读序）
Denaturation and Hybridization SBS8
Block with ddNTPs
OH
P7 Linearization (fpg)
Illumina Solexa
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)
The identity of each base of a cluster is read off from sequential images
Paired End
Paired End
双末端测序、正反双向测序
（用于组装较大的Gene，一次最多读100bp）
• Sample Preparation 样品前准备
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
Template Hybridization （模板杂交）
diol diol
Whitfield (1954)
1970 1980
1990 2000
2010
Development of Sanger Sequencing (1977)
Invention of Capillary Sequencer (1996)
Invention of Automated Fluorescent Sequencer
Block with ddNTPs
末端ddNTPs 封闭
Denaturation and Hybridization SBS3
Sequencing 测序
Denaturation and Hybridization SBS3
Sequencing First Read
（NO.1次 读序）
OH OH
Denaturation and De-Phosphorylation
Sequencing biochemistry
DNA cluster 、 Reversible terminators 、 Sequencing by Synthesis
形成DNA簇； 可逆阻断技术 边合成边测序（SBS）
Base/Run Time/run
Read length
Dominant error type
• Cluster Generation 分子簇的生成
• Sequence By Synthesis 边合成边测序
Sample Preparation
5’
T
3’
A
5’
T
3’
A
A
3’
T
5’
A
3’
T
5’
加双末端
Grafted FlowCells
OH
OH
diol
P7
P5
OH
8oxo-G
OH
U
8oxoG-P7 U-P5
diol
diol diol
2nd cycle annealing
Cluster Generation: Amplification
diol
diol diol
2nd cycle extension
OH
Periodate Linearization (高碘酸盐，线性化)
Blocking with ddNTP ()
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cluster上有1000个分子
Work Flow：
1.文库制备 2.Emulsion PCR 3.Beads Enrichment 4.微珠沉积 5.连接测序 6.数据分析
序列可以用超声波、机械剪切或酶解等方法，随机或者定向的打断成小片段
* 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
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 Solexa 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
Initial extension
diol diol
1st cycle denaturation （No.1循环：变性）
diol diol
diol diol
1st cycle
annealing （No.1循环：退火）
1st cycle
extension （No.1循环：延伸）
diol diol
2nd cycle denaturation
OH OH
U
P7
P5
Grafted flowcell
U
U
Template hybridization
U
U
Initial extension
U
U
1st cycle Denaturation
U
U
1st cycle annealing
U
U
wk.baidu.com
1st cycle extension
U
U
2nd cycle denaturation
•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/GA Workflow 工作流程
文库构建 Library Preparation
Cluster 工作站 Cluster Station
Detect Signal 检测荧光信号
Cleave Terminator and Dye 去掉末端封闭，染色
Cycle 2-n: Add sequencing reagents and repeat
?
Base Calling
碱基识别
T G C TAC GAT …
1
2
3
4
5
6
7
8
9
TTTTTTTGT…
（）
（修饰末端）
（去除未连接上的 接头）
（基因组DNA文库）
（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
Single Read Periodate Linearization
Paired End
Uracil Specific Excision Reagent (USER) 尿嘧啶特异性识别位点-P5
?formamidopyrimidine glycosylase (fpg) …糖基化酶-P3
Cluster Generation: Initial Extension
DNA片段的文库构建
Cluster Generation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
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
Invention of Single
molecule real time(SMRT)
DNA sequencing
Invention of Nanopore
single molecular sequencing
(Oxford Nanopore corporation)
1950 1960
Chemical degradation method by
U
U
U
2nd cycle annealing
Cluster Generation: Amplification 成簇
25个循环
n=25 total
U
U
U
2nd cycle extension
U
U
Cluster Amplification
P5 Linearization (USER)
胞嘧啶识别位点处切割
* 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）
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
Sequencing By Synthesis 3’ 5’ (SBS)
A
C G
T
C
A
T
G A
T
G
C
T G C T A C G A T A C C C G A T C G A T
5’
Cycle 1: Add sequencing reagents 加入合成所需反应物
First base incorporated 第一个碱基合成上
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）
OH
OH
diol diol
Cluster Amplification
OH
Denature and Hybridization
SBS3 （变性、杂交） SBS——边合成边测序
Sequencing By Synthesis
边合成边测序
SBS Cycle 1. Incorporation 结合 2. Scan 扫描拍照 3. Cleavage 清洗
40-50 Gb 10 days
2*75 bp Substitution
40-50G / 10天 / 一个RUN
读长：75bp
错误类型：
（可双向）
替换
Illumina solexa ABI SOLiD Roche 454
SOLID 测序技术
AB /SOLID Workflow 工作流程
1. 文库制备 2. Emulsion PCR 3. Beads Enrichment 4. 微珠沉积 5. 连接测序 6. 数据分析
(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
(PNK) 变性和去磷酸作用
OH
Resynthesis of P5 Strand
Sequencing
Second Read （NO.2次 读序）
Denaturation and Hybridization SBS8
Block with ddNTPs
OH
P7 Linearization (fpg)
Illumina Solexa
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)
The identity of each base of a cluster is read off from sequential images
Paired End
Paired End
双末端测序、正反双向测序
（用于组装较大的Gene，一次最多读100bp）
• Sample Preparation 样品前准备
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
Template Hybridization （模板杂交）
diol diol
Whitfield (1954)
1970 1980
1990 2000
2010
Development of Sanger Sequencing (1977)
Invention of Capillary Sequencer (1996)
Invention of Automated Fluorescent Sequencer
Block with ddNTPs
末端ddNTPs 封闭
Denaturation and Hybridization SBS3
Sequencing 测序
Denaturation and Hybridization SBS3
Sequencing First Read
（NO.1次 读序）
OH OH
Denaturation and De-Phosphorylation
Sequencing biochemistry
DNA cluster 、 Reversible terminators 、 Sequencing by Synthesis
形成DNA簇； 可逆阻断技术 边合成边测序（SBS）
Base/Run Time/run
Read length
Dominant error type
• Cluster Generation 分子簇的生成
• Sequence By Synthesis 边合成边测序
Sample Preparation
5’
T
3’
A
5’
T
3’
A
A
3’
T
5’
A
3’
T
5’
加双末端
Grafted FlowCells
OH
OH
diol
P7
P5
OH
8oxo-G
OH
U
8oxoG-P7 U-P5
diol
diol diol
2nd cycle annealing
Cluster Generation: Amplification
diol
diol diol
2nd cycle extension
OH
Periodate Linearization (高碘酸盐，线性化)
Blocking with ddNTP ()
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cluster上有1000个分子
Work Flow：
1.文库制备 2.Emulsion PCR 3.Beads Enrichment 4.微珠沉积 5.连接测序 6.数据分析
序列可以用超声波、机械剪切或酶解等方法，随机或者定向的打断成小片段
* 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
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 Solexa 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
Initial extension
diol diol
1st cycle denaturation （No.1循环：变性）
diol diol
diol diol
1st cycle
annealing （No.1循环：退火）
1st cycle
extension （No.1循环：延伸）
diol diol
2nd cycle denaturation
OH OH
U
P7
P5
Grafted flowcell
U
U
Template hybridization
U
U
Initial extension
U
U
1st cycle Denaturation
U
U
1st cycle annealing
U
U
wk.baidu.com
1st cycle extension
U
U
2nd cycle denaturation
•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/GA Workflow 工作流程
文库构建 Library Preparation
Cluster 工作站 Cluster Station
Detect Signal 检测荧光信号
Cleave Terminator and Dye 去掉末端封闭，染色
Cycle 2-n: Add sequencing reagents and repeat
?
Base Calling
碱基识别
T G C TAC GAT …
1
2
3
4
5
6
7
8
9
TTTTTTTGT…
（）
（修饰末端）
（去除未连接上的 接头）
（基因组DNA文库）
（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
Single Read Periodate Linearization
Paired End
Uracil Specific Excision Reagent (USER) 尿嘧啶特异性识别位点-P5
?formamidopyrimidine glycosylase (fpg) …糖基化酶-P3
Cluster Generation: Initial Extension
DNA片段的文库构建
Cluster Generation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
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
Invention of Single
molecule real time(SMRT)
DNA sequencing
Invention of Nanopore
single molecular sequencing
(Oxford Nanopore corporation)
1950 1960
Chemical degradation method by
U
U
U
2nd cycle annealing
Cluster Generation: Amplification 成簇
25个循环
n=25 total
U
U
U
2nd cycle extension
U
U
Cluster Amplification
P5 Linearization (USER)
胞嘧啶识别位点处切割
* 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）
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
Sequencing By Synthesis 3’ 5’ (SBS)
A
C G
T
C
A
T
G A
T
G
C
T G C T A C G A T A C C C G A T C G A T
5’
Cycle 1: Add sequencing reagents 加入合成所需反应物
First base incorporated 第一个碱基合成上
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）