[医学]测序技术原理介绍.ppt

Base decoding Instrument comparison Instrument 454 GS-FLX Titanium Illumina HiSeq 2000 ABI SOLiD 5500xl Amplification emPCR Bridge PCR emPCR Sequencing Pyrosequencing Reversible termination Ligation Paired ends Yes Yes Yes Length/read (bp) 400-600 100 50-100 Throughput/run (bp) 500M 200G ~155G Time/run 10h 8 days 8 days Reagent cost/Mb $12.4 $0.10 ~$0.07 Total direct cost/Mb $40 $0.25 ~$0.11 Purchase cost $500K $690K $595K Journal of Genetics and Genomics 38:95(2011) Molecular Ecology Resources (2011) Pair-end sequencing Read 1 Read 2 Known Distance Repetitive DNA Single read maps to multiple positions Paired read maps uniquely Mate-pair sequencing Read 1 Read 2 Known Distance Molecular Ecology Resources (2011) Science 327:1190 (2010) Nature Methods 8:41 (2011) Jonathan Rothberg Personal Genome Machine $50K/instrument 100bp/read 1Gb/run 2h/run $1.2/Mb Ion Torrent Detect H+ released as a voltage change—fast Common microchip design standards—low-cost manufacturing Sequencing volume is increasing Semiconductor sequencing Helicos BioSciences 1.将待测DNA随机打成200bp左右的小片段，在每个小片段末端加上ploy-dA，最后一个A有荧光标记。 2.与玻璃片上随机固定的多个poly-dT引物结合。 3.激光刺激，标记荧光的A发光，确定位置。 4.洗去荧光物质。 测序技术原理介绍 化学测序 Sanger测序 NGS（二代测序） Form the next to the third（三代测序） 测序方法分类 手工测序 自动测序 Sanger双脱氧链终止法 Maxam-Gilbert化学降解法 3100/3100-a 3130/3130XL 3700 3730/3730XL Solexa 454 焦磷酸法 SOLiD Gilbert化学降解法1. 目的DNA制备2. 单侧末端标记待测DNA片段3. 碱基的特异性修饰及化学降解4. 聚丙烯酰胺凝胶电泳5. 放射自显影6. 阅读测序结果 Sanger测序法1. 单链DNA模板制备2. DNA模板与测序引物退火3. 掺入法标记反应4. 延伸－终止反应5. 变性聚丙烯酰胺凝胶电泳6. 放射自显影7. 阅读测序结果 Chain-terminator reaction deoxynucleotides, dNTP dideoxynucleotides, ddNTP Frederick Sanger Original Sanger sequencing 1980-1990 Radio – gel Read length: ~102bp ~103 bp / day Automated Sanger sequencing 1990- Fluorescent – capillary Read length: ~103bp ~106bp / day ABI 3730 DNA Analyzer $376K/instrument 650 bp/read 0.06 Mb/run 2h/run $10K/Mb Hierarchical shotgun (map-based) method Long fragment BAC library Physical map Shotg