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Developing a rapid clinical sequencing system to classify meningiomaWebinars @ QIAGEN 19/02/2016Hiroshi Nishihara, MD, PhD
Department of Translational Pathology,Hokkaido University, Graduate School of MedicineTranslational Research Laboratory,Hokkaido University HospitalSapporo, Japan
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1Hokkaido University
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Hokkaido Univ. TokyoKyotoLocated in Sapporo cityInitially, Sapporo Agricultural Collage was built by Dr. ClarkPopulation Sapporo city; 2,000,000 Hokkaido state; 5,000,000
Hokkaido UniversitySapporo
Beautiful Campus Conveniently Located
Our location is very convenient and we have wide and beautiful campus including 200ha of primeval foreestOver ten thousands of people visit our campus every year just for sightseeing. 5
Self Introduction
Individualized medicine based on gene profile
Hiroshi Nishihara MD, PhD.Professor, Dept of Translational Pathology, Sch. of MedDirector, Translation Research Laboratory, Hokkaido Univ. Hosp.
Development of Clinical Science
Clinical ResearchARO)
Development of genomic medicine promotes Individualized Medicine.Integrate multiple patients genomic data
High qualityClinical InformationBiostatistics
SNVsCNVs
Clinical SequencingReal-time genomic analysisAnalysis of individual samplesClinically relevant outputsIndividualized Medicine
Clinical BioBank (Translational Research Laboratory)8
Biobank JapanNational Center.
Clinical Biobank in Hospital
Sampling
Rapid and adequate sample processingClinical sequencing in TR Laboratory
Rapid feedback to physicians
High quality sample linked with clinical information
Promote specific clinical studiesUniversitiesInstitutes
High quality biospecimenMass AnalysisSpecific institutes or universities
Research paper
Drug discovery
Government oriented clinical studiesPharmaceutical companies
9For use, not for storage of biospecimenOn-Demand type banking and advanced analysis of biospecimen
Valued biospecimen, especially for clinical studiesAvailability for genomic and molecular analyses
Physical support for clinical studies Core facility for clinical study
Sampling and storage based on specific protocolsProject-based repository systemA novel system of biospecimen repository which vigorously promotes variable clinical studies and clinical sequence
Clinical BioBankClinical BioBank (Translational Research Laboratory)
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Tissue sampling
PFPE tissue blocksPMBC for controlFFPE blocks from pathological materialsFrozen materials
Pathological evaluationTumor content rate, viabilityExtraction of DNA, RNA
dsDNAQubit
CTRT-PCR
RINBioanalyzer
QIA symphony
Targeted Amplicon Sequence with GeneReadMiSeqIllumina
Original pipeline for SNV, CNV analysis
Team conference for final diagnosis with physician, bioinformatican
Individualized MedicineClinical Sequence System in Hokkaido University Hospital
Clinical Sequence System for Brain Tumor (Meningioma)11Meningioma, arising from meningothelial cells, is the most common primary brain tumor, and accounts for about 25% of all intracranial tumors.Loss of neurofibromin 2 (NF2) has been found in about half of sporadic meningiomas.Mutations in TRAF7, KLF4, AKT1 and SMO were recently reported in non-NF2 meningiomas by NGS analysis.We established a clinical sequence system for meningioma to determine the genotype as a routine laboratory examination in addition to concurrent pathological diagnosis.
PFPE Tissue Specimen (Brain Tumor biobank)
Extraction of DNA
Targeted Amplicon Sequence; GeneRead(Whole exon for NF2, TRAF7, KLF4, AKT, SMO)
NF2; Mutation and LOHTRAF7, KLF4, AKT, SMO: Mutation
GeneRead DNAseq Targeted Panel V2Multiplex PCR-enabled enrichment of any region, gene, or set of genes in the human genome average amplicon size 150 bpNeed just 10 ng of DNA/poolTakes only 3 hours for target enrichment Integrated data analysis and biological interpretation
Vikram Devgan
Sample to InsightClinically Relevant Panels
13TypePanel nameSolid tumorClinically Relevant TumorTumor Actionable MutationsHematologic malignanciesMyeloid NeoplasmsTissue-specificBreast CancerColorectal CancerLiver CancerLung CancerOvarian CancerProstate CancerGastric CancerCardiomyopathyComprehensiveCancer PredispositionComprehensive CancerCarrier TestingGene specificBRCA1/2 Panel
Reference databases:
The Cancer Genome AtlasNational Comprehensive Cancer NetworkCOSMIC Cancer Genome CensusOMIMClinVar (NCBI)
Largest collection of wet-bench verified catalog gene panelsVikram Devgan
Sample to Insight
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GeneRead DNAseq Custom Panel V214Online custom panel builder:
Increase breadth of coverageAdjust amplicon length Allow denser tilingGene(s) or genomic region(s) of interest Fully customized panel
Turnaround time: 3 weeks
Vikram DevganWhole exon for NF2, TRAF7, KLF4, AKT, SMO
Sample to InsightTitle, Location, Date15Simple Protocol
GeneRead DNAseq Targeted Panel V2PCR primer mixAdd genomic DNA (10 ng/reaction) andGeneRead DNAseq Panel PCR Kit V2Pool reactions for each sample and purify (AMPure bead purification)
PCR amplification3 hours
NGS library preparation
Sample to InsightStandardized and rapid workflow16
AMPure bead purification
GeneRead amplification
GeneRead Library Prep
GeneRead DNAseq Panel
GeneRead Size Selection
QIAquick PCR Purification RUO Hybrid Workflow
GeneRead Library Quant Kit
GeneRead QuantiMIZE Kit
FFPE DNA isolation
Sequencing
CLC Cancer Work bench(hh:mm)
3:45
2:00
3:00
1:00
2:00
1:15
1:00
0:45
0:30
3:00
24:00
5:00
Day 1Day 2Day 3For 12 samples
Turnaround time:4 days
AMPure bead purificationDay 4
Sample to Insight
PAXgene-fixed formalin-embedded (PFPE) blocks
Confirmation of histology
GeneRead Mix-n-Match Panel - NF2, AKT1, SMO, ERBB2, KIT, METGeneRead Custom Panel - TRAF7, KLF4GeneRead DNAseq Targeted Panel V2GeneRead DNAseq Variant Calling Service (QIAGEN)BioReT System (Amelieff)
Frozen tumor samples(median size in diameter 7 mm, range 2.512)Workflow of NGS
Mix-n-Match Panel (559 genes)17
MRI and HE staining of representative cases
CNA analysis by NGS
The score Q was used to set a threshold (Q 50) to identify clusters with significant copy number changes and socre P correlated with the precisionof the copy number estimate.
Score QScore PCopy number min maxBMC Bioinformatics. 2015 Jan 28;16:17.GeneRead DNAseq Variant Calling Service(http://ngsdataanalysis.sabiosciences.com/NGS2/)
The score P was a useful indicator for the precision of the assigned copy number. Assigned copy numbers with P above 20 generally differ by less than 20 percent from the real copy number. Assigned copy numbers with P greater than 30 roughly show a maximal difference of 10 percent from the true copy number.19
Individualized medicine based on gene profile
StaffDoctor, Technical staffTR LaboratoryDivision of Cancer-Genetic DiagnosisProf. NishiharaSample preparationNGS sequencingData analysis
Data analysisAnnotationDept. of OncologyCancer Chemo. CenterMSSMitsubishi Space SoftwareRaw FastQ dataCancer genetic diagnosis for out patients without health insuranceOncoPrime, MSK-IMPACT (outsourcing)In house targeted sequencing Team conference for final diagnosis
OncoPrimeMSK-IMPACTDivision of Cancer-Genetic DiagnosisOut PatientOutsourcing
Genetic MedicineGene counseling for hereditary cancer (BRCA1, Rinch-syndrome)CollaboResult
Dept. of PathologyCompanion diagnosis for insurance-covered gene (Ras, EGFR, RAF for specific types of cancer)Collabo
PresidentHiroshi Nishihara MD. PhD (Hokkaido University)Vice president Shinichi Toyooka MD, PhD (Okayama University) DirectorManabu Mutoh MD, PhD (Kyoto University)Director Kazuyuki Matsushita MD, PhD (Chiba University)DirectorKazuhiro Okano PhD (QIAGEN)Clinical BioBank Study Group Handing of high quality biospecimenSOP for tissue samplingSOP for sample storage Linkage of sample and clinical information
Analysis of biospecimen for clinical sequenceQuality certification of biospecimenIn house analysis using desktop type NGSAdequate system for supporting clinical research
Networking of clinical biobank in JapanCollaboration with Mega-Bank in JapanSharing the banking system and data base
A couple of months ago, we just started Clinical biobank study group in Japan by collaboration of 4 national universities. We named Clinical Biobank on our banking system designed for supporting clinical research and clinical sequence. In this study group, we discuss about the SOP for handing of high quality biospecimen and establishment of clinical sequence in Japan. We hope that our concept strongly promotes clinical research and clinical sequence.Thank you for your attention. 26
Thank you for your attention!
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