宏基因组NGS病原体检测：质量控制.pptx

MetagenomicNGSfor

PathogenDetection:

QualityControlIssuesand

Strategies

SteveMillerMDPhD

DepartmentofLaboratoryMedicine

UniversityofCaliforniaSanFrancisco

Disclosures

•Majorfundingprovidedby:

–CAInitiativetoAdvancePrecisionMedicine

–SandlerandBowesFoundations

–SchwabFoundation

–MarcusFoundation

mNGSPathogenDetection

RNAVirus

DNAVirus

Bacteria

Fungi

ClinicalSample

NucleicAcid

Report

BioinformaticsAnalysis

LibraryPreparation

Sequencing

Parasite

mNGSAssayWorkflow

TotalNucleic

Acid

Extraction

SequenceGeneration

600μl

sample

input

Pre-Treatment

(Beadbeating)

cDNASynthesis1)RNA(DNase)

2)DNA(methyldepletion)

Analysis

Pipelineand

Report

Generation

Serum,WB,Respiratory,CSFStool,Tissue,BoneMarrow

10min-6hours

12-40hours

2-3hours

2-4hours

1hour

1hour

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

–Confirmacceptabilityusingexternalcontrol

results

BaselineDNase

Epicentre-Illumina

Cneoformans

AmericanTypeCultureCollection

DNACleanConcentratorKit-5CappedColumns

ZymoResearch

dNTPs

Ther