Quantitativegeneexpressionanalysisplaysanimportantroleinidentifyingdifferentiallyexpressedgenesinvariouspathologicalstates,geneexpressionregulationandco-regulation,sheddinglightongenefunctions.Althoughmicroarrayiswidelyusedasapowerfultoolinthisregard,itissuboptimalquantitativelyandunabletodetectunknowngenevariants.Herewedemonstratedeffectivedetectionofdifferentialexpressionandco-regulationofcertaingenesbyexpressedsequencetaganalysisusingaselectedsubsetofcDNAlibraries.Wediscussedtheissuesofsequencingdepthandlibrarypreparation,andproposethatincreasedsequencingdepthandimprovedpreparationproceduresmayallowdetectionofmanyexpressionfeaturesforlessabundantgenevariants.Withthereductionofsequencingcostandtheemergingofnewgenerationsequencingtechnology,in-depthsequencingofcDNApoolsorlibrariesmayrepresentabetterandpowerfultoolingeneexpressionprofilingandcancerbiomarkerdetection.Wealsoproposeusingsequence-specificsubtractiontoremovehundredsofthemostabundanthousekeepinggenestoincreasesequencingdepthwithoutaffectingrelativeexpressionratioofothergenes,astranscriptsfromasfewas300mostabundantlyexpressedgenesconstituteabout20%ofthetotaltranscriptome.In-depthsequencingalsorepresentsauniqueadvantageofdetectingunknownformsoftranscripts,suchasalternativesplicingvariants,fusiongenes,andregulatoryRNAs,aswellasdetectingmutationsandpolymorphismsthatmayplayimportantrolesindiseasepathogenesis.
