冠状病毒Coronavirusl论文-2016 [Springer Protocols Handbooks] Animal Coronaviruses __ Whole-Genome Sequencing of Porcine Epidemic Diarrhea Virus b.pdf

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1、Chapter18Whole-GenomeSequencingofPorcineEpidemicDiarrheaVirusbyIlluminaMiSeqPlatformLeyiWang,TodStuber,PatrickCamp,SueleeRobbe-Austerman,andYanZhangAbstractPorcineepidemicdiarrheavirus(PEDV)belongstothegenusAlphacoronavirusofthefamilyCoronaviridae.PEDVwasidentiÞedasanemergingpathogeninUSpig

2、populationsin2013.Sincethen,thisvirushasbeendetectedinatleast31statesintheUSAandhascausedsigniÞcanteconomiclosstotheswineindus-try.ActivesurveillanceandcharacterizationofPEDVareessentialformonitoringthevirus.ObtainingcomprehensiveinformationaboutthePEDVgenomecanimproveourunderstandingofthee

3、volutionofPEDVviruses,andtheemergenceofnewstrains,andcanenhancevaccinedesigns.Inthischapter,bothatargetedampliÞcationmethodandarandom-primingmethodaredescribedtoamplifythecompletegenomeofPEDVforsequencingusingtheMiSeqplatform.Overall,thisprotocolprovidesausefultwo-prongedapproachtocompletew

4、hole-genomesequencesofPEDVdependingontheamountofvirusintheclinicalsamples.KeywordsPorcineepidemicdiarrheavirus,TargetedampliÞcation,SISPA,Whole-genomesequenc-ing,MiSeq1IntroductionCompletegenomesequencingandgeneticanalysissigniÞcantlyimprovedourunderstandingoftheevolutionandrelationshipofpo

5、rcineepidemicdiarrheavirus(PEDV)strainsworldwide.TheÞrstPEDVwhole-genomesequencewascompletedfortheproto-typestrainCV777in2001[1].Sincethen,severalPEDVstrainshavebeensequencedandnowover170whole-genomesequenceshavebeendepositedinGenBank.Basedonthephylogeneticanaly-sisofthewhole-genomesequence

6、,PEDVhasbeenclassiÞedintotwoGenogroupsÑ1and2ÑwhichthevariantandclassicalstrainsofUSPEDVbelongto,respectively[2].SincetheÞrst454FLXpyrosequencingplatformwasintro-ducedtothemarketin2005,next-generationsequencing(NGS)hassigniÞcantlyadvancedresearchindiverseÞelds.NGShastheLeyiWang(ed.),AnimalCo

7、ronaviruses,SpringerProtocolsHandbooks,DOI10.1007/978-1-4939-3414-0_18,©SpringerScience+BusinessMediaNewYork2016201202LeyiWangetal.advantagesofhigh-throughputandcost-effectiveness.Currently,thereareseveralplatformsavailable,includingtheGenomeAnalyserdeve