Single-strands flip over to hybridize to adjacent primers to form bridges. Hybridized primer is extended by polymerase.
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1-8 samples
Fragment DNA Repair ends / Add A overhang
Ligate adapters Select ligated DNA
Hybridize to flow cell Extend hybridized oligos Perform bridge amplification
Perform sequencing on forward strand Re-generate reverse strand
Perform sequencing on reverse strand
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Sample Prep - Resequencing
Hiseq2000
15bp
30bp，50bp， 75bp，100bp
30G 600G
454
750bp
100bp，400bp 0.7G
3730
1000bp X 96 300bp，600bp 0.0001G
测序时间 10天 14天
7小时 2小时
Illumina workflow
❖Sample preparation
•Cluster intensities •Cluster noise
For each tile:
•Corrected cluster intensities •Cluster sequence •Cluster probabilities
For all data:
•Quality Filtering •Sequence Alignment •Run Statistics Visualization
Single molecules bound to flow cell in a random pattern
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Cluster generation: Bridge amplification
Single-strand flips over to hybridize to adjacent primers to form a bridge. Hybridized primer is extended by polymerases.
Surface bound adapter 1
Sequencing primer binding site
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Surface bound adapter 2
Flow cell
Surface of flow cell coated
with a lawn of oligo pairs
Newly synthesized covalently attached to the flow cell surface.
Original template
discard
Newly synthesized
strand
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Cluster generation: Covalently bound spatially separated single molecules
Cluster generation: Bridge amplification
Bridge amplification cycle repeated till multiple bridges are formed
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Cluster generation
.params file
Analysis PC/cluster
data transfer
GA Analysis Pipeline
Image Analysis
Base calling
Sequence Analysis
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For each tile:
Double-stranded bridge is denatured. Result: Two copies of covalently bound singlestranded templates.
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Cluster generation: Bridge amplification
Paired end sequencing
3’ ends are
blocked
Original forward strand is cleaved
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Sequencing reverse strand
Hybridize sequencing
5500xl SOLiD
HeliScope
2 x 150 25_35 25_30
优点 读长最长； 通量高 通量非常高
通量高；试 剂消耗少 通量高
缺点 同聚性错误；仪 器和试剂价格贵 价格贵；后期分 析复杂 读长太短
读长太短
测序平台 测序长度
进化过程
产出
SOLiD
15
30bp
Solexa
150bp X 2
▪ Visualize the data, reports the results
Sequencing process
1 Library prep (~ 6 hrs)
2 Automated Cluster Generation (~ 5 hrs)
1-8 samples
3 Sequencing (~ 46 to 120 hrs)
Bustard
• Base with highest corrected intensity is called
AC
G
T
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C
Gerald
• Filtering removes low quality base calls
GEneration of Recursive Analyses Linked by
primer
Add 4 FlNTP’s + Polymerase
Incorporated Fl-NTP is imaged
X 36 - 50
Terminator and fluorescent dye
are cleaved from the Fl-NTP
CONAdapter sequence
3’ extension
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Cluster generation: Denature double-stranded DNA
Double-stranded molecule is denatured.
Original template is washed away.
Solexa
Flow cell in GAIIx
Image re-analysis pipleline
Instrument PC
Images (.tif)
Lane 1..8
Cycle 1..36
Tile_Cycle_Image_a, Tile_Cycle_Image_c, Tile_Cycle_Image_g, Tile_Cycle_Image_t
dsDNA bridges denatured. Reverse strands cleaved and washed away.
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Cluster generation
… leaving a cluster with forward strands only.
高通量测序技术及原理介绍
童贻刚 军事医学科学院 微生物流行病研究所 tong.yigang@
14
公司 Roche/454 Illumina ABI/SOLiD Helicos
系统名
测序长度
FLX System 200_700
HiSeq 2000/miSeq
Paired end sequencing
Bridge formation
3’ extension
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Paired end sequencing
Double stranded DNA is denatured
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CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Bridge amplification
double-stranded bridge is formed.
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Cluster generation: Bridge amplification
Flow cell
Prism
Fluidics port
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Fluidics port