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1、RNA测序方法的进展孟芳2011.11.5 next-generation DNA sequencing(NGS)NGS领域的领头羊 Illumina,Roche 454,Helicos BioSciences and Life Technologies 四个公司Several developments in RNA-seq methods improvements in transcription start site mappingstrand-specific measurementsgene fusion detectionsmall RNA characterizationdetec
2、tion of alternative splicing eventsa comparison of the different approaches that are available for each application and discuss the current limitations and the potential for future improvementsMapping transcription start sites(TSSs)局限：the technology required high quantities of input RNA and generate
3、d only short reads(20 nucleotides)per TSS.Gene fusion detectiondiscussing two new developments in RNA-seq technologies:direct RNA sequencing(DRS)7 and methods for the reliable profiling of minute RNA quantities,which is important for translational research and clinical applications of RNA-seq.a|Sing
4、le-molecule DNA and RNA sequencing technologies could be modified for single-cell applications.Cells can be delivered to flow cells using fluidics systems,followed by cell lysis and capture of mRNA species on the poly(dT)-coated sequencing surfaces by hybridization.Standard sequencing runs could tak
5、e place on channels with a 127.5 mm2 surface area,requiring 2,750 images to be taken per cycle to image the entire channel area.The surface area needed to accommodate 350,000 mRNA molecules contained in a single cell is 0.4 mm2;thus,only eight images per cycle would be needed.Sequence analysis can b
6、e done with direct RNA sequencing(DRS)7or on-surface cDNA synthesis followed by single-molecule DNA sequencing26.b|Counter system workflow.Two probes are used for each target site:the capture probe(shown in red)contains a target-specific sequence and a modification that allows the immobilization of
7、the molecules on a surface;the reporter probe contains a different target-specific sequence(shown in blue)and a fluorescent barcode(shown by a green circle)that is unique to each target being examined.After hybridization of the capture and reporter probe mixture to RNA samples in solution,excess pro
8、bes are removed.The hybridized RNA duplexes are then immobilized on a surface and imaged to identify and count each transcript with the unique fluorescent signals on the capture and reporter probes.19941994：3 3亿美元测定一个人类基因组亿美元测定一个人类基因组(1:10)(1:10)19841984：3030亿美元测定一个人类基因组亿美元测定一个人类基因组未来目标：未来目标：1000/10
9、0 1000/100 美元测定一个人类基因组美元测定一个人类基因组20042004：3 3千万美元测定一个人类基因组千万美元测定一个人类基因组(1:100)(1:100)20062006：1 1百百5050万美元测定一个人类基因组万美元测定一个人类基因组(1:2000)(1:2000)20082008：5050万美元测定一个人类基因组万美元测定一个人类基因组(1:6000)(1:6000)Solexa测序技术的特点边合成边测序（sequencing by synthesis,SBS）大规模平行化（massively parrellel）新的测序技术会对生物科学的研究产生深远的影响，它会新的测序技术会对生物科学的研究产生深远的影响，它会产生海量的数据。产生海量的数据。