JournalofMicrobiologicalMethods86(2011)1–7

ContentslistsavailableatScienceDirect

JournalofMicrobiologicalMethods

journalhomepage:www.wendangwang.com

Review

TheevolutionofPyrosequencing®formicrobiology:Fromgenestogenomes

R.C.Novaisa, ,Y.R.Thorstensonb

ab

UniversityofStateofRiodeJaneiro,RiodeJaneiro,RJ,BrazilLifeTechnologies,FosterCity,CA,USA

articleinfoabstract

PyrosequencingisauniquesequencingmethodthatwasdevelopedasanalternativetoclassicalDNAsequencingforshort-tomedium-readapplications.Comparedtoothermethods,itishighlyquantitative,fastandinexpensive.Additionaladvantagesincludehighaccuracy, exibilityandabilitytoautomatesamplepreparation.Thisarticlepresentsahistoricaloverviewofpyrosequencing,improvementsthathavebeenmadethroughtheyearsanditsevolutionintoaplatformthatvastlyexpandedthescopeofgeneticanalysisthatcouldbeperformedoutsideofabigsequencingcenter.Inaddition,wedescribenumerousapplicationsinmicrobiologythathavebene tedfromthepyrosequencingmethod.

©2011ElsevierB.V.Allrightsreserved.

Articlehistory:

Received14October2010

Receivedinrevisedform4April2011Accepted5April2011

Availableonline14April2011Keywords:

PyrosequencingSequencingMicrobiology

Contents1.

Introduction........................................1.1.Pyrosequencing:ahistoricaloverview.........................1.2.Low-cost,highresolutionsequencing:applicationsoftheQIAGENpyrosequencingplatform1.3.Sequencinggenomes:massivelyparallelsequencingusingRoche454pyrosequencing..2.Conclusion.........................................References............................................

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1.Introduction

1.1.Pyrosequencing:ahistoricaloverview

Sincethedevelopmentofthedideoxychainterminationtechniquewas rstdescribedmorethanthreedecadesago(Sangeretal.,1977)and uorescentlylabelednucleotideswereincorporatedintoSangersequencing(Smithetal.,1986;Leeetal.,1992),advanceshaveledtotheincreasingexpansionanddevelopmentofhigh-quality,high-throughputsequencing.However,thecostwasprohibitiveformostlaboratories.

Theneedforlow-cost,robust,high-throughputmethodstoreplacetheelegantSangermethodledtothedevelopmentofseveralnewtechnologies,includingPyrosequencing®(QIAGEN).PyrosequencingwasoriginallydevelopedbyPålNyréninthe1990s(reviewedinNyrén,2007)andisbasedonthedetectionofreleasedpyrophosphate

Correspondingauthorat:Depto.deCiências,FaculdadedeFormaçãodeProfessores,UniversidadedoEstadodoRiodeJaneiro,RuaDr.FranciscoPortela,1470,Patronato,SãoGonçalo,RJ,CEP24435-000,Brazil.Tel.:+552125715091.

E-mailaddress:rnovais@uerj.br(R.C.

Novais).0167-7012/$–seefrontmatter©2011ElsevierB.V.Allrightsreserved.doi:

10.1016/j.mimet.2011.04.006

(PPi)duringDNAsynthesis.Inacascadeofenzymaticreactions,visiblelightisgeneratedthatisproportionaltothenumberofincorporatednucleotides.ThecascadestartswithanucleicacidpolymerizationreactioninwhichinorganicPPiisreleasedasaresultofnucleotideincorporationbypolymerase.ThereleasedPPiissubsequentlyconvertedtoATPbyATPsulfurylase,whichprovidestheenergytoluciferasetooxidizeluciferinandgeneratelight(Fig.1).Becausetheaddednucleotideisknown,thesequenceofthetemplatecanbedetermined(Ronaghi,2001).

Thepathofpyrosequencingdevelopmentfromtheconcepttothesuccessfulplatformusedtodayhingedontheresolutionofthreemainissues.The rstwasthatdATPfunctionedasaweaksubstrateforluciferasetoproducelightthatcontributedtohighbackgroundduringtheluminescencemeasurement.TheproblemwassolvedbysubstitutionofdATPfordATPαS,anucleosidethiophosphatewhichwasfoundtobeagoodsubstrateforDNApolymeraseandapoorsubstrateforluciferase(Ronaghietal.,1996).

ThesecondissuewasthelargeamountofATPsulfurylaserequiredtoremovespurioussignalsbetweennucleotideadditions.Thisproblemwassolvedbyaddinganucleotide-degradingenzymecalledapyrase.Apyrasecoulddegradeallfourdeoxynucleotidesequallyandtherefore