高通量测序技术及原理介绍
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CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Hybridize fragment & extend
> 50 M single molecules hybridize to the lawn of primers
Bound molecules are then extended by polymerases
Adapter sequence
3’ extension
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Denature double-stranded DNA
Double-stranded molecule is denatured.
Original template is washed away.
Double-stranded bridge is denatured. Result: Two copies of covalently bound singlestranded templates.
▪ 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)
高通量测序技术及原理介绍
tong.
公司 Roche/454 Illumina ABI/SOLiD Helicos
系统名 FLX System
HiSeq 2000/miSeq 5500xl SOLiD HeliScope
测序长度 200_700 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
14
测序平台 SOLiD Solexa Hiseq2000 454 3730
测序长度 30bp 150bp X 2
750bp 1000bp X 96
进化过程
❖Cluster generation
▪ Bridge PCR
❖Sequencing on Genome Analyzer IIx
▪ RTA (Run Time Analysis)
❖ Analysis pipeline
▪ Offline analysis, alignment, SNPs calling, reads counting
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
CONFIDENTIAL – DO NOT DISTRIBUTE
Sample Prep - Resequencing
8 channels
Key to the simplified workflow • Clonal clusters are
generated in a contained environment (need no clean rooms) • Sequencing also performed in the flow cell on the generated clusters
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Bridge amplification
double-stranded bridge is formed.
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Bridge amplification
Newly synthesized covalently attached to the flow cell surface.
Original template
discard
Newly synthesized
strand
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Covalently bound spatially separated single molecules
15bp 30bp，50bp， 75bp，100bp 100bp，400bp 300bp，600bp
产出 30G 600G
0.7G 0.0001G
测序时间 10天 14天
7小时 2小时
15
Illumina workflow
❖Sample preparation
▪ Shearing, ligate adapter
Single molecules bound to flow cell in a random pattern
CONFIDENTIAL – DO NOT DISTRIBUTE
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
CONFIDENTIAL – DO NOT DISTRIBUTE
Surface bound adapter 2
Flow cell
Surface of floห้องสมุดไป่ตู้ cell coated
with a lawn of oligo pairs
Cluster generation: Hybridize fragment & extend
> 50 M single molecules hybridize to the lawn of primers
Bound molecules are then extended by polymerases
Adapter sequence
3’ extension
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Denature double-stranded DNA
Double-stranded molecule is denatured.
Original template is washed away.
Double-stranded bridge is denatured. Result: Two copies of covalently bound singlestranded templates.
▪ 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)
高通量测序技术及原理介绍
tong.
公司 Roche/454 Illumina ABI/SOLiD Helicos
系统名 FLX System
HiSeq 2000/miSeq 5500xl SOLiD HeliScope
测序长度 200_700 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
14
测序平台 SOLiD Solexa Hiseq2000 454 3730
测序长度 30bp 150bp X 2
750bp 1000bp X 96
进化过程
❖Cluster generation
▪ Bridge PCR
❖Sequencing on Genome Analyzer IIx
▪ RTA (Run Time Analysis)
❖ Analysis pipeline
▪ Offline analysis, alignment, SNPs calling, reads counting
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
CONFIDENTIAL – DO NOT DISTRIBUTE
Sample Prep - Resequencing
8 channels
Key to the simplified workflow • Clonal clusters are
generated in a contained environment (need no clean rooms) • Sequencing also performed in the flow cell on the generated clusters
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Bridge amplification
double-stranded bridge is formed.
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Bridge amplification
Newly synthesized covalently attached to the flow cell surface.
Original template
discard
Newly synthesized
strand
CONFIDENTIAL – DO NOT DISTRIBUTE
Cluster generation: Covalently bound spatially separated single molecules
15bp 30bp，50bp， 75bp，100bp 100bp，400bp 300bp，600bp
产出 30G 600G
0.7G 0.0001G
测序时间 10天 14天
7小时 2小时
15
Illumina workflow
❖Sample preparation
▪ Shearing, ligate adapter
Single molecules bound to flow cell in a random pattern
CONFIDENTIAL – DO NOT DISTRIBUTE
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
CONFIDENTIAL – DO NOT DISTRIBUTE
Surface bound adapter 2
Flow cell
Surface of floห้องสมุดไป่ตู้ cell coated
with a lawn of oligo pairs