Cluster Generation
Prepare DNA fragments Ligate adapters
Attach single molecules to surface Amplify to form clusters
100um Random array of clusters
~1000 molecules per ~ 1 um cluster ~20.000 clusters per tile
1st cycle extension
2nd cycle denaturation
2nd cycle annealing
Cluster Generation: Amplification
OH
diol diol diol diol
OH
diol
2nd cycle extension
Cluster Amplification
可逆阻断技术
•Each lane contains multiple tiles – total 128 •Each tile is imaged four times per cycle – one image per base
BGI current sequencing capacity
Illumina GA
Invention of Applied Biosystems Solid System (2007) Invention of Illumina Genome Analyzer System (2006)
第二代测序技术
Illumina Genome Analyzer /Hiseq 2000 ABI SOLiD Roche 454
Base/run
Base/day
95G
7G
300G
35G
Illumina/Hiseq 2000 Workflow
Genomic DNA
Mate-paired library
Fragment library
27b p
27b p
27b p
27b p
Create library of DNA fragments
测序种类
Single-Read Sequencing 单向测序 Paied-End Sequencing 双向测序 Indexed Sequencing 混合样品测序
1、Single-Read Sequencing
Cluster Generation: Amplification
OH
OH
diol
Illumina Solexa & Hiseq 2000
Domina nt error type
Substitu tion Substitu tion
Sequencing biochemistry DNA cluster 、 Reversible terminators 、 Sequencing by Synthesis DNA cluster 、 Reversible terminators 、 Sequencing by Synthesis
5’
nnnnGzzz 3’
C-probe
T-probe
5’
5’
nnnnCzzz
nnnnTzzz
universal seq primer
p5’
1µm 1µm beadbead
5’
Adapter Oligo Sequence
Next-generation sequencing technology
Birthday
2005
Principle
Pyrosequencing
2006
Sequencing-by-Synthesis
2007
Sequencing-by-Ligation
第二代测序技术
Illumina Genome Analyzer /Hiseq 2000 ABI SOLiD
1st cycle extension
2nd cycle denaturation
2nd cycle annealing
Cluster Generation: Amplification
n=25 total
U
U U U
U
2nd cycle extension
Cluster Amplification
OH
OH
Periodate Linearization
Blocking with ddNTP ()
Denature and Hybridization SBS3
2、Paied-End Sequencing
Comparison of Flow Cell Adaptors Between SR and PE
Beads collected following emulsion PCR:
P2
P1
P2 Beads with no product Beads with amplified product (~40K PCR products per bead)微珠富集 4.微珠沉积
Cluster Generation: Initial Extension
OH
OH
U
U U U U U U
P7
P5
Grafted flowcell
Template hybridization
Initial extension
1st cycle Denaturation
U
U
U
U
U
U
U
U
U
1st cycle annealing
OH OH
8oxo-G diol
OH
OH
U
P7
P5
8oxoG-P7
U-P5
Single Read Periodate Linearization
Paired End Uracil Specific Excision Reagent (USER)
formamidopyrimidine glycosylase (fpg)
Base/Run
Time/run
Read length
Solexa
95 Gb
13-14 days
2*150 bp
Hiseq 2000
300Gb
7-8 days
2*100 bp
Illumina solexa ABI SOLiD
SOLID 测序技术
AB /SOLID Workflow
Work Flow：
3.微珠富集 4.微珠沉积
5.连接测序
6.数据分析
2. Emulsion PCR
Templates
+
P1-coupled DNA beads ~ 100,000 P1 sites per bead
Start with 2 Billion bead 3.Beads Enrichment 4.微珠沉积 5.连接测序 6.数据分析
Genomic DNA
Mate-paired library
Fragment library
27b p
27b p
27b p
27b p
Create library of DNA fragments
Illumina Hiseq (128)
flowcell
Read length(bp) Cluster Camera
1 flowcell/1 side
2×150 6.2 million/slide 2(G/T, A/C)
2 flowcells/4 sides
2×100 2.6-3.5 million/slide 4(G,T,A,C)
1950
1960
1970
1980
1990
2000
2010
Chemical degradation method by Whitfield (1954)
Development of Sanger Sequencing (1977)
Invention of Capillary Sequencer (1996) Invention of Automated Fluorescent Sequencer (1985) Invention of 454 GS 20 Sequencer (2005)
二代测序技术介绍
周 正
2011-01-09
Sequencing Technology Development
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)
Cycle 2-n: Add sequencing reagents and repeat
T C
G A T
5’
Base Calling
TG C TAC GAT …
1 2 3 4 5 6 7 8 9
TTTTTTTGT…
The identity of each base of a cluster is read off from sequential images
P5 Linearization (USER)
Block with ddNTPs