1709-POD-Performance characteristics BRCA MASTR Dx · Performance characteristics BRCA MASTR Dx and...
Transcript of 1709-POD-Performance characteristics BRCA MASTR Dx · Performance characteristics BRCA MASTR Dx and...
PerformanceCharacteristicsBRCAMASTRDx
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PerformanceCharacteristicsBRCAMASTRDx
withdrMIDDxforIlluminaNGSsystems
ManufacturerMultiplicomN.V.Galileïlaan182845NielBelgium
PerformanceCharacteristicsBRCAMASTRDx
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Figure1:graphicrepresentationoftheMASTRworkflow..................................................................................................3
ListofTablesTable1.PerformancecharacteristicsBRCAMASTRDxandMIDDxforIlluminaMiSeq®ongermlineDNAextractedfrom
blood..................................................................................................................................................................5
Table2.PerformancecharacteristicsBRCAMASTRDxandMIDDxforIlluminaMiSeq®onFFTDNA...............................7
TableofContents
1 WORKFLOW.........................................................................................................................................................3
2 PERFORMANCECHARACTERISTICSOFBRCAMASTRDXANDMIDDXFORILLUMINAMISEQONGERMLINEDNAEXTRACTEDFROMBLOOD............................................................................................................................................4
2.1 STUDYSETUP.......................................................................................................................................................4
2.2 ONTARGETREADCOUNTS..................................................................................................................................4
2.3 UNIFORMITYOFAMPLIFICATION.........................................................................................................................4
2.4 ACCURACY,SENSITIVITY&SPECIFICITY................................................................................................................4
2.5 PRECISION...........................................................................................................................................................5
3 PERFORMANCECHARACTERISTICSOFBRCAMASTRDXANDMIDDXFORILLUMINAMISEQONFRESHFROZENTISSUEDNA..................................................................................................................................................................6
3.1 STUDYSETUP.......................................................................................................................................................6
3.2 ONTARGETREADCOUNTS..................................................................................................................................63.3 UNIFORMITYOFAMPLIFICATION.........................................................................................................................6
3.4 ACCURACY,SENSITIVITY&SPECIFICITY................................................................................................................6
4 LISTOFABBREVIATIONS......................................................................................................................................7
5 DISCLAIMER.........................................................................................................................................................8
ListofFigures
PerformanceCharacteristicsBRCAMASTRDx
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1 WORKFLOW
Figure1:graphicrepresentationoftheMASTRworkflow
PerformanceCharacteristicsBRCAMASTRDx
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2 PERFORMANCECHARACTERISTICSOFBRCAMASTRDXANDMIDDXFORILLUMINAMISEQONGERMLINEDNAEXTRACTEDFROMBLOOD
2.1 Studysetup
ThecombinedperformancecharacteristicsoftheBRCAMASTRDxandtheMIDDxforIlluminaMiSeq®kitswereassessedinthreeindependentmedicalgeneticlaboratories.Intotal146independentDNAsampleswereamplified,sequencedandanalyzed;asdescribedinthecorrespondingIFUs.Eachlaboratoryextracted,amplifiedandsequenced40-51DNAsamplesselectedamongstclinicalsamples.DNAwasextractedbyeitherPhenol/chloroformextractionmethod,NaClextractionmethod,Maxwell® 16 InstrumentMX3030 (Promega), QuickGene DNAWhole blood kit L (FUJIFILM LifeScience),QiagenAutopureLSorchemagicSTARDNABloodKits (PerkinElmer).A referencemethod, suchasSangersequencing and/or 454 sequencing using BRCAMASTR Dx, was performed for these samples. Each laboratory alsoprocessedandsequenced4proficiencyDNAsamples,providedbyMultiplicom.
Thermalcyclersusedbythelaboratories:Biorad,Biometra(Westburg),AppliedBiosystems9700.Fragmentanalyzersused:Qiaxcell(Qiagen)andAppliedBiosystems.
Atotaloffive IlluminaMiSeq®runswasperformed;threerunswiththeIlluminaMiSeq®ReagentKitv2(44sampleseach)andtworunswiththeIlluminaMiSeq®ReagentNanoKitv2(15sampleseach).Thisresultedinthegenerationof3,573,788sequencedbases,ofwhich2,924,088baseshadbeendeterminedbyoneofthereferencemethods.SoftwarefordataanalysisincludedtheSeqNextmodulev4.1.2oftheSequencePilotsoftware(JSImedicalsystems,Kippenheim,Germany), the Sophia DDM® application with pipeline ID ILL1MR1G3 (Sophia Genetics, Ecublens, Switzerland) andinternallywiththeMASTRReporterbyuploadingtheexistingFASTQfilestoMASTRReporterv1.0.
2.2 Ontargetreadcounts
Thetotalnumberoffilterpassedreadspersamplewasmappedtothehumangenomeandtheontargetreadscounted.Thepercentageofon-target,oramplicon-derived,readsarerepresentedinTable1.
2.3 Uniformityofamplification
Foreachsamplethenumberofreadpairsperampliconwerecountedandnormalizedtotheaveragereadcountsperamplicon.Combiningallnormalizeddatashowedthat99.7%ofthetotalreadpairsfallwithin20%ofthemeannumberofreadpaircounts(Table1).
2.4 Accuracy,sensitivity&specificity
Theseparametersarecalculatedusingthefollowingdefinitions:
• Truepositive(TP):variantbasespresentbothintheNGSandthereferencedataset• Truenegative(TN):nonvariantbasespresentbothintheNGSandthereferencedataset• Falsepositive(FP):variantbasespresentintheNGSdataset,absentinthereferencedataset• Falsenegative(FN):variantbasespresentinthereferencedataset,absentintheNGSdataset• Accuracy=(TP+TN)/(TP+TN+FP+FN)• Sensitivity=TP/(TP+FP)• Specificity=TN/(TN+FN)
Onlythoseregionswithsufficientcoverageweretakenintoaccountforthecalculation,thus2,918,750basesinsteadof2,924,088bases.Theobservedvaluesandtheir95%confidenceintervalsforthese3parametersanalyzedwithSequencePilot,SophiaDDM®andMASTRReporterarerepresentedinTable1.
PerformanceCharacteristicsBRCAMASTRDx
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2.5 PrecisionBasedontheNGSdatasetfromthetwoDNAsamplestestedtwiceineachlaboratorythatparticipatedinthevalidationstudies,theinter-runaccuracywascalculatedas100%(95%CI[≥99.996%])andtheinter-laboratoryaccuracywasdetermined100%(95%CI[≥99.996%])withSequencePilot,SophiaDDM®(Table1)andMASTRReporter.
Table1.PerformancecharacteristicsBRCAMASTRDxandMIDDxforIlluminaMiSeq®ongermlineDNAextractedfromblood
*Excludingpurechangesinlengthofhomopolymerstretchesof10bporlonger.**MASTRRep:excludinghomopolymerstretchesof10bporlonger.
Asensitivityof100%(95%CI[≥99.998%])wasachievedintheperformanceevaluationstudydescribedintable1.However,onecouldalsoapplyfollowingadditionalproceduresforattaininghighsensitivityforBRCAMASTRDxwithMIDDxforIlluminaMiSeq®:
• Detailedanalysisofampliconsizes:o Perform,foreachsetofBRCAMASTRDx-derivedamplicons(“Plex”),theproceduredescribedinsection10.5
“QualitycontrolofUniversalPCRbyfluorescentlabelingandfragmentanalysis”oftheIFU.
o PCRproductsfromsamplesthatcontaininsertionsordeletionsofoneormorebaseswillresultinasizechangeoftheamplicon(s)harboringsuchvariants.
o Detailedcomparisonoftheampliconprofilestoareferenceprofilewithoutinsertion/deletionvariants,willallowidentifyingthepresenceofinsertion/deletionvariantsinspecificampliconsofthesampleanalyzed.
o Detailedanalysisofthesequencereadsofampliconswithmodifiedlengthsmayinalimitednumberofcasesallowconfirmingvariantsthatweremissedbystandardvariantcalling.
• ReducedstringentfiltersettingsforthesequencereaddataanalysisusingtheSeqNextmoduleofSeqPilot(JSImedicalsystems):
o Reviewthevariantsinthe“distinct”andthe“other”tablethatareclosetothethresholdof15%inoneorbothdirections.
Parameter Observed[95%CI](SeqNext)
Observed[95%CI](SophiaDDM®)
Observed[95%CI](MASTRReporter)
Ontargetreadcounts 98.4%(range:94.7%-99.6%)
Uniformity ofamplification 99.71%
Sensitivity* 100%[≥99.8%]
100%[≥99.8%]
100%[≥99.97%]
Specificity* 99.99997%[99.989%,100%]
100%[≥99.9999%]
100%[≥99.99%]
Accuracy* 99.99997%[99.989%,100%]
100%[≥99.9999%]
100%[≥99.99%]
Reproducibility* 100%[≥99.996%]
100%[≥99.996%]
100%[≥99.99%]
PerformanceCharacteristicsBRCAMASTRDx
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o Technicalvalidationofvariantsbyexperiencedcustomersmaybeobtainedbyassessingthesupportiveevidence in the sequence reads as visualized by the SeqNext software. In case of doubt, it is highlyrecommendedtoconfirmvariantswithasecondtest
3 PERFORMANCECHARACTERISTICSOFBRCAMASTRDXANDMIDDXFORILLUMINAMISEQONFRESHFROZENTISSUEDNA
3.1 StudysetupThecombinedperformancecharacteristicsof theBRCAMASTRDxandtheMIDDx for IlluminaMiSeq®kitson freshfrozentissue(FFT)wereassessedinonemedicalgeneticlaboratoryandatMultiplicom.DNAextractedfromintotal10ovarianand20breastcancertissues,andtheirmatchedadjacentnormaltissueswasamplified,sequencedandanalyzed;asdescribedinthecorrespondingIFUs.TheDNAofalltissuesampleswasextractedusingNucleonextractionkit(Gen-Probe).
Sincenoreferencedatawasavailableforthesamples,thesequencingresultsobtainedonthenormaltissueswereusedasreferencedataforthedataobtainedonthematchedcancertissues.
Thermal cyclers usedby the laboratories:AppliedBiosystems9700. Fragment analyzers used:Qiaxcell (Qiagen) andAppliedBiosystems.AtotalofthreeIlluminaMiSeq®runswasperformed;tworunswiththeIlluminaMiSeq®ReagentKitv3andonewithIlluminaMiSeq®ReagentKitv2.
Software fordataanalysis included theSeqNextmodulev4.1.2of theSequencePilot software (JSImedical systems,Kippenheim, Germany) and the Sophia DDM® application with pipeline ID ILL1MR1S4 (Sophia Genetics, Ecublens,Switzerland).
3.2 OntargetreadcountsThetotalnumberoffilterpassedreadspersamplewasmappedtothehumangenomeandtheontargetreadscounted.Thepercentageofon-target,oramplicon-derived,readsarerepresentedinTable2.
3.3 UniformityofamplificationForeachsamplethenumberofreadpairsperampliconwerecountedandnormalized.Combiningallnormalizeddatashowedthat99.988%ofallampliconsarecoveredwithmorethan20%ofthemeannumberofreadpaircounts(Table2).
3.4 Accuracy,sensitivity&specificityTheseparametersarecalculatedusingthefollowingdefinitions:
• Truepositive(TP):variantspresentinbothcancerandnormaltissueNGSdatasets• Truenegative(TN):nonvariantpresentinbothcancerandnormaltissueNGSdatasets• Falsepositive(FP):variantbasespresentinthecancerNGSdataset,absentinthenormaltissueNGSdataset,except
whenSangersequencingconfirmedthisobservation• Falsenegative(FN):variantbasespresentinthenormaltissueNGSdataset,absentinthecancerNGSdataset,except
whenSangersequencingconfirmedthisobservation• Accuracy=(TP+TN)/(TP+TN+FP+FN)• Sensitivity=TP/(TP+FP)• Specificity=TN/(TN+FN)
PerformanceCharacteristicsBRCAMASTRDx
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Noregionswereexcludedduetoinsufficientcoverage.Theobservedvaluesandtheir95%confidenceintervalsforthesethreeparametersanalyzedwithSequencePilotandSophiaDDM®arerepresentedinTable2.
Table2.PerformancecharacteristicsBRCAMASTRDxandMIDDxforIlluminaMiSeq®onFFTDNA
*Excludingpurechangesinlengthofhomopolymerstretchesof10bporlonger.
4 LISTOFABBREVIATIONS
CE: TheCEsymbolcertifiesthataproductcomplieswiththeEuropeanstandards.DNA: DeoxyribonucleicacidFFT: FreshFrozenTissueFN: FalseNegativeFP: FalsePositiveIFU: InstructionsForUseIVD: ForInVitroDiagnosticUseMASTRDx: MultiPlexAmplificationofSpecificTargetforResequencingforDiagnosticsdrMID: dualreadMolecularIdentifierNGS: Next-GenerationSequencingPCR: PolymeraseChainReactionPlex: SetofMASTRDx-derivedampliconsTN: TrueNegativeTP: TruePositive
Parameter Observed[95%CI](SeqNext)
Observed[95%CI](SophiaDDM®)
Ontargetreadcounts 97.8%(range:91.8%-98.7%)
Uniformityofamplification 99.988%
Sensitivity* 100%[≥99.3%]
100%[≥99.3%]
Specificity* 99.996%[≥99.988%]
99.996%[≥99.988%]
Accuracy* 99.996%[≥99.988%]
99.996%[≥99.988%]
PerformanceCharacteristicsBRCAMASTRDx
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5 DISCLAIMER
Thepurchaseofthisproductenablesthepurchasertouse it fortheamplificationofnucleicacidsequencesforhumangenes.Purchaserhastotakeintoaccountthatinformationobtainedfromampliconsgeneratedusingthisproductmaynotbeusedinproceduresthatareprotectedbyvalidclaimsownedand/orcontrolledbyathirdparty,unlesspriorwrittenapprovalofsuchpartyhasbeenobtained.IVDproductperformanceclaimsapplyonlywhencombiningMASTRDxandMIDDxandwhenbothCE-MarkkitsareusedaccordingtothespecificCE-IVDInstructionsForUse.
5991-8424ENE PrintedinBelgium,September2017
PRNUMBER
PR7000-1427