第二代RNA测序方法

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1、RNA测序方法的进展孟芳2011.11.5next-generationDNAsequencing(NGS)NGS领域的领头羊Illumina,Roche454,HelicosBioSciencesandLifeTechnologies四个公司SeveraldevelopmentsinRNA-seqmethodsimprovementsintranscriptionstartsitemappingstrand-specificmeasurementsgenefusiondetectionsmallRN

2、AcharacterizationdetectionofalternativesplicingeventsacomparisonofthedifferentapproachesthatareavailableforeachapplicationanddiscussthecurrentlimitationsandthepotentialforfutureimprovementsMappingtranscriptionstartsites(TSSs)局限：thetechnologyrequiredhigh

3、quantitiesofinputRNAandgeneratedonlyshortreads(~20nucleotides)perTSS.GenefusiondetectiondiscussingtwonewdevelopmentsinRNA-seqtechnologies:directRNAsequencing(DRS)7andmethodsforthereliableprofilingofminuteRNAquantities,whichisimportantfortranslationalres

4、earchandclinicalapplicationsofRNA-seq.a

5、Single-moleculeDNAandRNAsequencingtechnologiescouldbemodifiedforsingle-cellapplications.Cellscanbedeliveredtoflowcellsusingfluidicssystems,followedbycelllysisandcaptureofmRNAspeciesonthepoly(dT)-coatedsequencingsu

6、rfacesbyhybridization.Standardsequencingrunscouldtakeplaceonchannelswitha127.5mm2surfacearea,requiring2,750imagestobetakenpercycletoimagetheentirechannelarea.Thesurfaceareaneededtoaccommodate~350,000mRNAmoleculescontainedinasinglecellis~0.4mm2;thus,only

7、eightimagespercyclewouldbeneeded.SequenceanalysiscanbedonewithdirectRNAsequencing(DRS)7oron-surfacecDNAsynthesisfollowedbysingle-moleculeDNAsequencing26.b

8、Countersystemworkflow.Twoprobesareusedforeachtargetsite:thecaptureprobe(showninred)containsatarget

9、-specificsequenceandamodificationthatallowstheimmobilizationofthemoleculesonasurface;thereporterprobecontainsadifferenttarget-specificsequence(showninblue)andafluorescentbarcode(shownbyagreencircle)thatisuniquetoeachtargetbeingexamined.Afterhybridizatio

10、nofthecaptureandreporterprobemixturetoRNAsamplesinsolution,excessprobesareremoved.ThehybridizedRNAduplexesarethenimmobilizedonasurfaceandimagedtoidentifyandcounteachtranscriptwiththeuniquefluorescentsignalsonthecaptureandreporter