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Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes.doc

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Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

Giannoukosetal.GenomeBiology2012,13:r23 /2012/13/3/r23 METHOD OpenAccess EfficientandrobustRNA-seqprocessforcultured bacteriaandcomplexcommunitytranscriptomes GeorgiaGiannoukos1*,DawnMCiulla1,KatherineHuang1,BrianJHaas1,JacquesIzard2,3,JoshuaZLevin1, JonathanLivny1,AshleeMEarl1,DirkGevers1,DoyleVWard1,ChadNusbaum1,BruceWBirren1and AndreasGnirke1 Abstract Wehavedevelopedaprocessfortranscriptomeanalysisofbacterialcommunitiesthataccommodatesbothintact andfragmentedstartingRNAandcombinesefficientrRNAremovalwithstrand-specificRNA-seq.Weappliedthis approachtoanRNAmixturederivedfromthreediverseculturedbacterialspeciesandtoRNAisolatedfromclinical stoolsamples.Theresultingexpressionprofileswerehighlyreproducible,enrichedupto40-foldfornon-rRNA transcripts,andcorrelatedwellwithprofilesrepresentingundepletedtotalRNA. Background the majority of bacterial mRNA is not polyadenylated, Microbial communities are known to play significant asitisineukaryotes,andcan,therefore,notbeisolated roles in humanhealth, development, and disease [1-4], using oligo-dT selection. Thus, specialized approaches andDNAsequencingisaneffectiveapproachtocharac- are needed to enrich the desired transcripts for terizethestructureandpotentialfunctionofthesecom- sequence-basedcharacterization. munities. Whilesequencing ofthe 16Sribosomal RNA NumerousrRNA-depletionmethodshavebeendevel- (rRNA)genehasbeeninvaluableforidentifyingbacter- oped.Theseincludecommerciallyavailablekitssuchas ial species and taxa in complex communities [5], shot- MICROBExpress(Ambion),whichusescaptureoligonu- gunsequencingofmetagenomesprovidesamuchricher cleotides targeting specific regions of the 16S and 23S view of the community by more fully describing the rRNAs,andmRNA-ONLY(Epicentre), whichutilizes a gene content of the community [6,7]. To understand 5′-monophosphate-dependent exonuclease to degrade which genes and gene pathways are actually expressed processed 5’-phosphorylated RNA molecules such as an