Jeannette Reinartz isGroupleaderProduction, LynxTherapeutics,GmbH, Germany. Eddy Bruyns isGroupleaderFunctional Genomics,LynxTherapeutics, GmbH,Germany. Jing-Zhong Lin isSeniorScientist,Lynx Therapeutics,Inc.,Hayward, California,USA. Tim Burcham isVicePresident,ITand Bioinformatics,Lynx Therapeutics,Inc.,Hayward, California,USA. Sydney Brenner isScienti®cAdvisorforLynx andProfessor,SalkInstitute,La Jolla,California,USA. Ben Bowen isVicePresident,Discovery Research,LynxTherapeutics, Inc.,Hayward,California,USA. Michael Kramer isManagingDirector,Lynx TherapeuticsGmbH,Germany. Rick Woychik isChiefScienti®cOf®cer,Lynx Therapeutics,Inc.,Hayward, California,USA. Keywords: gene expression pro®ling, massively parallel signature sequencing, MPSS, Megaclone RickWoychik, LynxTherapeutics,Inc.,25861 IndustrialBlvd.,Hayward,CA 94545,USA Tel:+1510)6709488 Fax:+1510)6709303 E-mail:Rwoychik@lynxgen.com Technique review Massively parallel signature sequencing MPSS) as a tool for in-depth quantitative gene expression pro®ling in all organisms Jeannette Reinartz, Eddy Bruyns, Jing-Zhong Lin, Tim Burcham, Sydney Brenner, Ben Bowen, Michael Kramer and Rick Woychik Datereceivedinrevisedform):6thDecember2001 Abstract MassivelyparallelsignaturesequencingMPSS)isoneofthenewesttoolsavailablefor conductingin-depthexpressionpro®ling.MPSSisanopen-endedplatformthatanalysesthe levelofexpressionofvirtuallyallgenesinasamplebycountingthenumberofindividualmRNA moleculesproducedfromeachgene.Thereisnorequirementthatgenesbeidenti®edand characterisedpriortoconductinganexperiment.MPSShasaroutinesensitivityatalevelofa fewmoleculesofmRNApercell,andthedatasetsareinadigitalformatthatsimpli®esthe managementandanalysisofthedata.Therefore,ofthevariousmicroarrayandnon-microarray technologiescurrentlyavailable,MPSSprovidesmanyadvantagesforgeneratingthetypeof completedatasetsthatwillhelptofacilitatehypothesis-drivenexperimentsintheeraofdigital biology. INTRODUCTION Recentdevelopmentsinthesequencingof manyvertebrate,invertebrate,plantand microbialgenomeshavepromptedan increasinginterestinusinggenomics reagentstostudypatternsofgene expression.Buildinglargerelational databases®lledwithcontentthatincludes in-depthgeneexpressionpro®lesfrom multiplecelltypeswillundoubtedlymake anenormouscontributiontoany experimentaleffortindigitalbiology. SeveralDNAmicroarrayplatforms, 1±6 serialanalysesofgeneexpression SAGE), 7,8 cDNAsequencinganda varietyofothertechnologiesareavailable foranalysingtheexpressionofhundredsto thousandsofgenessimultaneously.Eachof theseexistingtechnologieshaslimitations whenitcomestogeneratingcomplete datasetsforbuildingrelationaldatabases.In thispaper,oneofthenewesttoolsfor evaluatinggeneexpressionisreviewed, calledmassivelyparallelsignature sequencingMPSS), 9,10 whichovercomes manyofthelimitationsofthecurrent technologies.MPSSisanovelmicrobead technologythatistotallyunlikeother bead-basedoperationssuchasthose developedbyIllumina 11 orLuminex. 12 MPSS AS A TOOL FOR QUANTITATIVE GENE EXPRESSION PROFILING Unlikemostmicroarraytechnologiesthat capturedatathatareanalogueinnature, MPSSisoneofthefewtechnologiesthat producesdatainadigitalformat.MPSS capturesdatabycountingvirtuallyall mRNAmoleculesinatissueorcell sample.Allgenesareanalysed simultaneously,andbioinformaticstools areusedtosortoutthenumberofmRNAs fromeachgenerelativetothetotal numberofmoleculesinthesample.At leastonemillionmoleculesaretypically & HENRY STEWART PUBLICATIONS 1473-9550. BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS. VOL 1. NO 1. 95±104. FEBRUARY 2002 95