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

454 SEQUENCING： EMULSION PCR (EMPCR)
A
+ PCR Reagents + Emulsion Oil
B
Micro-reactors
Adapter carrying library DNA Mix DNA Library & capture beads (limited dilution)
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
454 SEQUENCING： DEPOSITION OF DNA BEADS INTO THE PICOTITER™PLATE
Load Enzyme Beads
Load beads into PicoTiter™Plate
Centrifuge Step
Illumina Solexa 合成测序 Sequence by Synthesize 基本原理
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
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
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。
Solexa 测序 Workflow
ABI SOLiD 连接法测序 Sequence by Ligation 基本原理
ABI SOLID测序技术
2010年末又发布了最新产品--SOLiD 5500xl测序 平台。 SOLiD 5500xl含有两张微流体芯片（microfluidic FlowChip），每张芯片含有6条相互独立的运行通 道（run lane）。每条lane都能运行相对独立的测 序反应，这样的设计使得SOLiD 5500xl测序平台 极具灵活性。最大测序读长为75nt，同样支持 Fragment、b（使用最新设计 的nanobeads）。测序的系统准确性能达到 99.99%
ILLUMINA SOLEXA测序技术
Illumina公司于2007年初花费6亿美金巨资收 购了Solexa, 2010年初，Illumina将其第二代测 序仪Genome Analyzer IIx升级到HiSeq 2000 HiSeq 2000含有两张Flow cell，可同时运行或 者只运行其中一张。读长为100nt，同时支持 Fragment、量（读长为 2x100nt）。
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
深度(高通量）测序技术简介 NEXT GENERATION SEQUENCING

Sample fragmentation Library preparation Sequencing reaction Data analysis
Roche 454 焦磷酸测序 Pyrophosphate Sequencing
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

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 m cluster ~1000 clusters per 100 µ m square ~40 million clusters per experiment
Solid line： at least one read Dashed line：at least 20 reads
ቤተ መጻሕፍቲ ባይዱ
Analysis of percentage of reads containing known coding region SNVs in the six tissue samples.
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
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