11high-throughputsequencing2nextgenerationsequencingNGSmassivelyparallelsequencingMPSNGSDNA/RNANGSInVitroDiagnosisIVDNGSNGSNGSNGS3/DNARNANGS4IVD/BaseCallQualityscoresReads%StandardOperatingProcedureSOPNGS/NGS51.2.Deoxyribonuclease,DNaseRibonucleaseRNase3.NGS/64./4.14.1.1NGS71MSNVDNA/4.1.24.24.3/4.4PCRNoTemplateControlNTCNTCNGS8NGS1.2./deoxy-ribonucleotidetriphosphatedNTP3.//9NGS1.NGSNGS/1.1NGS//101.2DNAcDNAPCRDNAPCR1.3DNA11//reads1.4121.4.1FASTQGCBaseCallQualityQ1.4.2BAMSequenceAlignment/Map1.4.31.4.41.5131.6NGS1.7////2.2.1142.22.2.1NGS2.115PositivePercentAgreementPPANegativePercentAgreementNPAPositivePredictiveValuePPVSingleNucleotideVariantSNVPPANPA1ABA+BCDC+DA+CB+DA+B+C+D1PPAAA+CNPADB+D2PPANPA2ABA+BCDC+DEFE+FA+C+EB+D+FN162E+F/N95E/A+C+EF/B+D+FN=A+B+C+D+E+F2.2.22.3LimitofDetectionLoDLoDLoDLoD95%LoDLoDNGS172.42.5DNA2.6182.72.8NGS2.9SNV192.10High-throughputsequencingNext-generationsequencing20DNADNAdenovosequencingDNAreadsreadsPCR%GCGCcontentG21CbasecallqualityscoresbasecallqualityQindexbarcode1..2018.122..2018.23.StuartM.Brown.2017.14..NGS1005.226.EVALUATIONOFAUTOMATICCLASSIIIDESIGNATIONFORMSK-IMPACTIntegratedMutationProfilingofActionableCancerTargets7.TheAssociationforMolecularPathologyAmericanSocietyofClinicalOncology,andCollegeofAmericanPathologistsStandardsandGuidelinesfortheInterpretationandReportingofSequenceVariantsinCancerTheJournalofMolecularDiagnosticsVol.19No.1January20178.VerificationandValidationofMultiplexNucleicAcidAssaysApprovedGuideline.ClinicalandLaboratoryStandardsInstitute.NCCLSMM17-AVol.28No.9ISBN1-56238-661-19.GuidelinesforDiagnosticNext-GenerationSequencing.EuropeanJournalofHumanGenetics20152411584-158910.ACMGClinicalLaboratoryStandardsforNext-GenerationSequencing.GeneticsinMedicine201315911.EwingADHoulahanKEHuYEllrottKCaloianCYamaguchiTNBareJCP'ngCWaggottDSabelnykovaVYetalCombiningtumorgenomesimulationwithcrowdsourcingtobenchmarksomaticsingle-nucleotide-variantdetection.Naturemethods2015127623-630.12.FDA.ConsiderationsforDesign,DevelopmentandAnalyticalValidationofNextGenerationSequencingNGSBasedInVitroDiagnosticsIVDsIntendedtoAidintheDiagnosis23ofSuspectedGermlineDiseases13.FDA.Useofpublichumangeneticvariantdatabasestosupportclinicalvalidityforgeneticandgenomic-basedinvitrodiagnosticsDocumentissuedonApril132018.14.FoundationFocus?CDxBRCATechnicalInformationSummary15.CAGCCSCONGS2016.04.2316.98262018.7.10