Illumina 测序原理.pptx

Illumina DNA 测序原理
David Chen
北京源泉宜科生物科技有限公司

010-56107385
目录
概览
制备文库(Library Preparation)
在测序芯片上生成DNA簇(Cluster Generation)
边合成边测序(Sequencing by Synthesis)
双侧测序,配对测序(Paired-End, Mate Pair & Multiplexing)
数据分析(Data Analysis)
Library Preparation
制备文库
Sequencing
测序
Data Analysis
数据分析
4
3
Cluster Generation
生成DNA簇
2
测序工作流程概览
1
DNA(- ug)
Library preparation
Cluster generation
5’
5’
3’
G
T
C
A
G
T
C
A
G
T
C
A
C
A
G
T
C
A
T
C
A
C
C
T
A
G
C
G
T
A
G
T
1
2
3
7
8
9
4
5
6
Image acquisition
Sequencing
测序过程概览 Sequencing Overview
Base calling
T G C T A C G A T …
制备文库Library Preparation
Library Prep Overview (gDNA)
DNA Template
1-5 µg,50 µl purified DNA
OD260/280 = -
As intact as possible
Library
Yield 500-1000 ng
OD260/280 =~
SR: 150-250 bp
PE: 300-650 bp
MW = size in bp x 650
DN***段化(Fragmentation)
我们用的片段化方法
Covaris
Bioruptor
别的可选的片段化方法
Nebulizer
Sonication
HydroShear®
Enzymatic
Others
打碎
Covaris
修补末端
T4, Klenow, T4 PNK
加A 碱基
A
A
A
A
A
A
Klenow exo-
P
P
A
A
5’
5’
5’
3’
5’
T
3’
T
A
A
P
3’
5’
T
5’
3’
T
T
3’
5’
T
A
3’
5’
2% Agarose Gel 电泳
切胶选取DNA ,对于单侧测序选取150-250 bp ,对于双侧测序选取600bp
磁珠或Qiagen等回收试剂盒回收DN***段
Adaptors Ligation & Excision
PCR 扩增
5’
T
3’
A
5’
A
T
3’
富集
5’
T
3’
A
5’
A
T
3’
退火
5’
T
3’
A
5’
A
T
3’
3’
A
5’
T
3’
T
A
5’
延伸
5’
3’
5’
T
3’
T
A
A
10 - 12 Cycles
Library QC
双侧测序: 500 bp + 100 bp
单侧测序: 250 bp + 100 bp