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MetagenomicNGSfor
PathogenDetection:
QualityControlIssuesand
Strategies
SteveMillerMDPhD
DepartmentofLaboratoryMedicine
UniversityofCaliforniaSanFrancisco
Disclosures
?Majorfundingprovidedby:
–CAInitiativetoAdvancePrecisionMedicine
–SandlerandBowesFoundations
–SchwabFoundation
–MarcusFoundation
RNAVirusDNAVirus
RNAVirus
DNAVirus
Bacteria
Fungi
BioinformaticsAnalysis
mNGSPathogenDetection
ClinicalSample
NucleicAcid
LibraryPreparation
Seq
Sequencing
Report
Pa
Parasite
600μlsampleinputTotalNucleicAcidExtractionSerum,WB,Respiratory,CSFStool,Tissue,
600μl
sample
input
TotalNucleic
Acid
Extraction
Serum,WB,Respiratory,CSFStool,Tissue,BoneMarrow
1hour
1hour
SequenceGeneration
12-40hours
cDNASynthesis1)RNA(DNase)
2)DNA(methyldepletion)
2-4hours
2-3hours
TOTALTURNAROUND
TIME:48-72hours
P
Pre-Treatment
(Beadbeating)
Li
LibraryPreparationandQualityControl
Analy
Analysis
Pipelineand
Report
Generation
10min-6hours
CLIAImplementationofmNGS
?Requiredforpatienttesting
–LaboratoryDevelopedTest(LDT)
?ProtocolDevelopment
?AssayValidation
?QualityControl/QualityAssurance
?StaffTraining/Competency
?ProficiencyTesting
QualityControlApproach
?AdaptedfromCLIARegulations
–QA/QCmonitoring
?FailureModeAnalysis
–Likelypointsofassayfailure
–Developmethodtodetectfailure
?AssayMonitoring
–Defineexpectedperformance
–Monitorchangesovertime
QualityControlMethods
?ReagentQualityControl
?ExternalControls
?InternalControls
?ProcessControls/Checkpoints
?ContaminationControl
ReagentQualityControl
?WhatreagentsneedQC?
?Howtoperform?
?Whatareacceptiblecriteria?
ReagentQC
?Define“Critical”Reagents
–Enzymes
–Majorassaysteps
–Confirmperformancebeforeputtingintouse
?ExternalcontrolmNGSorsurrogateassaysacceptable
?Non-criticalreagents
–ManufacturerQCacceptable
–Confirmacceptabilityusingexter
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