1709-POD-Performance characteristics BRCA MASTR Dx · Performance characteristics BRCA MASTR Dx and...

8
www.agilent.com For In Vitro Diagnostic Use. Revision date: July 27, 2017 Page 1 of 8 Performance Characteristics BRCA MASTR Dx with drMID Dx for Illumina NGS systems Manufacturer Multiplicom N.V. Galileïlaan 18 2845 Niel Belgium

Transcript of 1709-POD-Performance characteristics BRCA MASTR Dx · Performance characteristics BRCA MASTR Dx and...

PerformanceCharacteristicsBRCAMASTRDx

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page1of8

PerformanceCharacteristicsBRCAMASTRDx

withdrMIDDxforIlluminaNGSsystems

ManufacturerMultiplicomN.V.Galileïlaan182845NielBelgium

PerformanceCharacteristicsBRCAMASTRDx

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page2of8

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

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page3of8

1 WORKFLOW

Figure1:graphicrepresentationoftheMASTRworkflow

PerformanceCharacteristicsBRCAMASTRDx

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page4of8

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

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page5of8

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

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page6of8

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

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page7of8

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

www.agilent.com ForInVitroDiagnosticUse.Revisiondate:July27,2017 Page8of8

5 DISCLAIMER

Thepurchaseofthisproductenablesthepurchasertouse it fortheamplificationofnucleicacidsequencesforhumangenes.Purchaserhastotakeintoaccountthatinformationobtainedfromampliconsgeneratedusingthisproductmaynotbeusedinproceduresthatareprotectedbyvalidclaimsownedand/orcontrolledbyathirdparty,unlesspriorwrittenapprovalofsuchpartyhasbeenobtained.IVDproductperformanceclaimsapplyonlywhencombiningMASTRDxandMIDDxandwhenbothCE-MarkkitsareusedaccordingtothespecificCE-IVDInstructionsForUse.

5991-8424ENE PrintedinBelgium,September2017

PRNUMBER

PR7000-1427