高通量测序技术简介

Solid line： at least one read Dashed line：at least 20 reads
Analysis of percentage of reads containing known coding region SNVs in the six tissue samples.
高通量测序的应用
• • • • • • De novo 测序 基因深度测序（genome re-sequencing） 转录组深度测序（transcriptome re-sequencing） Digital expression profiling ChIP-seq Methy-seq
Transcriptome resequencing：
Digital expression profiling & microRNA re-sequencing：
hESC： human embryonic stem cells EB： embryoid bodies
ChIP-seq（1）： 人一号染色体DNA-蛋白相互作用
ChIP-seq（2）：
2、每轮反应只能整合一个核苷酸，仪器读取相应的荧光信号 3、信号读取结束，用化学方法去除阻断基团，进行下一轮测序 反应
T C
G A T
5’
Base calling from the raw data 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 根据每个点每轮反应读取的荧光信号序列，转换成相 应的DNA序列
Gene Description
Ribosomal protein L29 Peroxiredoxin 2 Profilin 2 Stathmin-like 4 Cytochrome c oxidase subunit Vb Gamma-aminobutyric acid (GABA) A receptor, alpha 1 Ubiquitin B Ring finger protein 1 DIRAS family, GTP-binding RAS-like 2 PHD finger protein 20 Immunoglobulin J polypeptide Fibronectin 1 Nuclear factor I/X (CCAAT-binding transcription factor) Topoisomerase (DNA) II alpha 170kDa Keratin 8 Integrin, beta 7 Brain abundant, membrane attached signal protein 1 Dopamine receptor D1 interacting protein Glial fibrillary acidic protein Amyloid beta (A4) precursor-like protein 2
454 sequencing： Emulsion PCR (emPCR)
A
+ PCR Reagents + Emulsion Oil
B
Micro-reactors
Adapter carrying library DNA Mix DNA Library & capture beads (limited dilution)
Solexa 测序 Workflow
ABI SOLiD 连接法测序 Sequence by Ligation 基本原理
文库制备：微珠单分子克隆
SOLiD 利用探针的连接反应读取模板的DNA序列
1024种8碱基探针 4色荧光，4种双核苷酸，每色荧光有256个探针(4^6)
连接法测序 （一）
测序引物与adapter退火
SNV： Single Nucleotide Substitution Variant
Digital expression profiling（1）： 人大脑组织与UHR（Universal Human Reference）的表达差异
Tag Sequence GATCAAACCAAGGCCCAGGC GATCACTGTTAATGATTTGC GATCAGTGTCTTTTCAGCAC GATCATCATGACCAATGAAA GATCATGCTGGCTGCAAAGA GATCCAAACCCAAGTCTTGA GATCCAAGATAAAGAAGGCA GATCCCAGACTGGTTCTTGA GATCCCCAATTGACTCAGAG GATCCGGGGCTGCAGGCTTG GATCCTACAGAAGTGGAGCT GATCCTAGTAATTGCCTAGA GATCCTGCGGGAGTCTCCCG GATCCTGTGAAGGCCTGGAA GATCGAGACACGTGATGGGA GATCGAGGACAGTGCAACCA GATCTCAATGCCAATCCTCC GATCTGCACGCCGCTGACCC GATCTGTGCCCAGAGATGGG GATCTGTGGAGAATGTACAC Brain
454 sequencing： Deposition of DNA beads into the PicoTiter™Plate
Load beads into PicoTiter™Plate
Load Enzyme Beads
Centrifuge Step
Illumina Solexa 合成测序 Sequence by Synthesize 基本原理
第五部分：其他类型的芯片
微缩芯片实验室（Lab-on-a-chip)
药物运输芯片
生理功能辅助芯片

纳米芯片
第六部分 高通量测序技术简介 Next Generation Sequencing
• • • • Sample fragmentation Library preparation Sequencing reaction Data analysis
malignant pleural mesotheliomas (MPMs) ：恶性胸膜间皮瘤 pulmonary adenocarcinoma (ADCA)：肺腺癌
Transcriptome characteristics
Expression difference between
MPM and ADCA sample compare to a lung tissue control
Create “Water-in-oil” emulsion
Adapter complement
Enrich Anneal Seq primer “Break micro-reactors” Isolate DNA containing beads Perform emulsion PCR
Generation of millions of clonally amplified templates on each bead No cloning and colony picking
Clonal Single Molecule Arrays 单分子克隆 Attach single molecules to surface
Amplify to form clusters Prepare DNA fragments Ligate adapters
Sequence ~1000 molecules per ~ 1 µ cluster m ~1000 clusters per 100 µ square m ~40 million clusters per experiment
• • • 高度自动化的系统 读取片段多，适合进行大量小片段的测序，如microRNA profiling 基于可逆反应，随反应轮数增加，效率降低，信号衰减，读取序列较短，给de novo sequencing 拼接带来困难
SOLiD sequencing
• • • 每个碱基读取两次非常高的准确性，特别是对于SNP的检测 灵活的系统，完善的磁珠编码系统，可以进行样品的pooling，分割测序区域 读取长度受连接反应的轮数限制，给de novo sequencing 拼接带来困难
Methy-seq（1）： 肿瘤和MCF7细胞系中 BRCA!启动子区域的甲基化差异
Methy-seq（2）：
Roche 454 焦磷酸测序 Pyrophosphate Sequencing
Illumina Solexa 合成测序 Sequence by Synthesize
ABI SOLiD 连接法测序 Sequence by Ligation
Roche 454 焦磷酸测序 Pyrophosphate Sequencing 基本原理
118 162 85 73 91 48 266 217 91 10 0 46 41 0 8 0 240 51 716 132
UHR
861 163 35 0 96 0 271 1538 0 10 113 799 12 42 346 59 81 0 0 69
Symbol
RPL29 PRDX2 PFN2 STMN4 COX5B GABRA1 UBB RING1 DIRAS2 PHF20 IGJ FN1 NFIX TOP2A KRT8 ITGB7 BASP1 DRD1IP GFAP APLP2
探针连接，检测荧光
切除荧光基团
第二轮探针连接，检测荧光
切除荧光基团 每个探针进行检测的两个碱基后面有三个匹配碱基，因此一条测序引物读取的序列是不完整的
连接法测序 （二）
测序引物沿着Adapter移动5次，确保每个位点都被检测
连接法测序 （三）
0位置是Adapter的最后一个碱基，因此只检测一次， 该碱基是进行解码所必须的。
20 microns
Reversible Terminator Chemistry 可逆终止反应
• All 4 labelled nucleotides in 1 reaction
O HN O PPP 3’ O N
cleavage fluor site
O 5’ DNA O HN O O N
X
block
A T G C G C A G A T G C T T A C G A T A C C C G A
Detect signal Cycle 2-n: Add sequencing reagents and repeat
C T
1、每轮测序反应加入四种带有荧光标记的dNTP，末端带有可 以被去除的阻断基团
Sequenced short reads (typically 25–50 bp) from ChIP-Seq experiments are first mapped onto the reference genome. The mapped reads are then used to estimate statistical parameters, which include the estimation of the average length F of sequenced DNA fragments.
Incorporation Detection Deblock; fluor removal
3’ OH free 3’ end
Next cycle
Sequencing-by-Synthesis (SBS)
3’ 5’
Cycle 1: Add sequencing reagents First base incorporated Remove unincorporated bases
Advantage & disadvantage 454 sequencing
• • • 读取长度大，400bp 可以对未知基因组进行从头测序de novo sequencing 当遇到polwenku.baidu.commer时，如AAAAAA等，荧光强度和碱基个数不成线性关系，判定 重复碱基个数有困难
Solexa sequencing