Human mtDNA Genome Guide (15037958)

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1、HumanmtDNAGenomeFortheIlluminaSequencingPlatformIntroduction2Workflow3SampleSheet4FirstPCRAmplification6Quantify9TagmentAmplifiedDNA11Limited‐CyclePCRAmplification13Limited‐CyclePCRClean‐Up17Bead‐basedNormalization19PoolandLoadfromBead‐basedNormalization23Bioanalyzer‐basedNormalization25Poola

2、ndLoadfromBioanalyzer‐basedNormalization27Analysis29SupportingInformation36IMPORTANTThisdocumentprovidesinformationforanapplicationforIlluminatechnologythathasbeendemonstratedinternallyandmaybeofinteresttocustomers.ThisinformationisNOTICEprovidedas‐isandisnotanIlluminaproductandisnotaccompani

3、edbyanyrightsorwarranties.Customersusingoradaptingthisinformationshouldobtainanylicensesrequiredandmaterialsfromauthorizedvendors.Illuminaproductsmentionedhereinareforresearchuseonlyunlessmarkedotherwise.Whilecustomerfeedbackiswelcomed,thisapplicationisnotsupportedbyIlluminaTechnicalSupportan

4、dFieldApplicationScientists.Part#15037958Rev.BPage1IntroductionPage2IntroductionThisprotocolexplainshowtoprepare,sequence,andanalyzetheentirehumanmitochondrialDNA(mtDNA)genomefromclean,intactDNAsamples.Duringsamplepreparation,themtDNAgenomeisamplifiedintwoPCRstogeneratetwolongfragmentsspannin

5、gtheentirehumanmitochondrialgenome(16,569bp).Theampliconsarequantifiedandpooledbeforelibrarypreparation.SubsequentlibrarysequencingontheMiSeqisfollowedbydataanalysiswithMiSeqReporterandvariantcallingwiththemtDNAAnalysisTool.NOTETheprotocolsdescribedinthisguideassumethatyouhavereviewedtheSuppo

6、rtingInformation,onpage36andobtainedalloftherequisiteconsumablesandequipment.What’sNewThefollowingchangesweremadeinthisrevision:•AsafestoppingpointwasaddedattheendoftheLimited‐CyclePCRClean‐Upprocedure.•TheNexteraXTDNASamplePreparationKit(96samples)andNexteraXTDNASamplePreparationIndexKitswer

7、eaddedtotheconsumableslist.WorkflowPage3WorkflowThefollowingfigureillustratestheHumanmtDNAGenomeworkflow.Figure1HumanmtDNAGenomeWorkflowSampleSheetPage4SampleSheetAftercreatingyoursamplesheetusingtheIlluminaExperimentManager(IEM),openthesampl