Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing 英文参考文献.docVIP

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Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing 英文参考文献.doc

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Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing 英文参考文献

SimultaneousIdentificationofDNAandRNAViruses PresentinPigFaecesUsingProcess-ControlledDeep Sequencing JanaSachsenro¨der1,SvenTwardziok2,JensA.Hammerl1,PawelJanczyk1,PaulWrede2,StefanHertwig1, ReimarJohne1* 1FederalInstituteforRiskAssessment,Berlin,Germany,2InstituteforMolecularBiologyandBioinformatic,Charite,Berlin,Germany Abstract Background:Animalfaecescompriseacommunityofmanydifferentmicroorganismsincludingbacteriaandviruses.Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, whichwasoptimizedforthepurificationofthetotalfractionofviralparticlesfrompigfaeces.Thegenomesofthepurified DNAandRNAvirusesweresimultaneouslyamplifiedbyPCRandsubjectedtodeepsequencingfollowedbybioinformatic analyses.Theefficiencyofthemethodwasmonitoredusingaprocesscontrolconsistingofthreebacteriophages(T4,M13 andMS2)withdifferentmorphologyandgenometypes.Definedamountsofthebacteriophageswereaddedtothesample andtheirabundancewasassessedbyquantitativePCRduringthepreparationprocedure. Results: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads weregenerated.Alloftheaddedbacteriophageswereidentifiedbysequenceanalysisofthereads.Intotal,7.7%ofthe readsshowedsignificantsequenceidentitieswithpublishedviralsequences.Theymainlyoriginatedfrombacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequenceswithidentitiestothechimpanzeestool-associatedcircularssDNAviruswereidentified.Wholegenomeanalysis indicatesthatthisvirus,tentativelydesignatedaspigstool-associatedcircularssDNAvirus(PigSCV),representsanovelpig virus. Conclusion:TheestablishedprotocolenablesthesimultaneousdetectionofDNAandRNAvirusesinpigfaecesincluding theidentificationofsofarunknownviruses.Itmaybeappliedinstudi