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Choosing a Benchtop Sequencing Machine to Characterise Helicobacter pylori Genomes.doc

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Choosing a Benchtop Sequencing Machine to Characterise Helicobacter pylori Genomes

ChoosingaBenchtopSequencingMachineto CharacteriseHelicobacterpyloriGenomes TimothyT.Perkins*,ChinYenTay,FannyThirriot,BarryMarshall PathologyandLaboratoryMedicine,TheUniversityofWesternAustralia,Perth,WesternAustralia,Australia Abstract ThefullyannotatedgenomesequenceoftheEuropeanstrain,26695wasfirstpublishedin1997and,in1999,itwasdirectly comparedtotheUSAisolateJ99,promotingtwostandardlaboratoryisolatesforHelicobacterpylori(H.pylori)research.With thegenomicscaffoldsavailablefromtheseimportantgenomesandtheadventofbenchtophigh-throughputsequencing technology, a bacterial genome can now be sequenced within a few days. We sequenced and analysed strains J99 and 26695 using the benchtop-sequencing machines Ion Torrent PGM and the Illumina MiSeq Nextera and Nextera XT methodologies. Using publically available algorithms, we analysed the raw data and interrogated both genomes by mappingthedataandbydenovoassembly.Wecomparedtheaccuracyofthecodingsequenceassembliestotheoriginally publishedsequences.WiththeIonTorrentPGM,wefoundaninherentlyhigh-errorrateintherawsequencedata.Usingthe IlluminaMiSeq,wefoundsignificantlymorenon-coverednucleotideswhenusingthelessexpensiveIlluminaNexteraXT comparedwiththeIlluminaNexteralibrarycreationmethod.Wefoundthemostaccurate denovoassembliesusingthe Nextera technology, however, extracting an accurate multi-locus sequence type was inconsistent compared to the Ion TorrentPGM.WefoundthecagPAIfailedtoassembleontoasinglecontiginalltechnologiesbutwasmoreaccurateusing the Nextera. Our results indicate the Illumina MiSeq Nextera method is the most accurate for de novo whole genome sequencingofH.pylori. Citation:PerkinsTT,TayCY,ThirriotF,MarshallB(2013)ChoosingaBenchtopSequencingMachinetoCharacteriseHelicobacterpyloriGenomes.PLoSONE8(6): e67539.doi:10.1371/journal.pone.0067539 Editor:JonathanH.Badger,J.CraigVenterInstitute,UnitedStatesofAmerica ReceivedJanuary31,2013;AcceptedMay20,2013;PublishedJune28,2013 Copyright: ? 2013 Perkins etal. This is