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Department of Genetics,
University Medical Center Groningen
The Netherlands
Next-generation sequencing can replace Sanger sequencing in clinical diagnostics.
Birgit Sikkema-Raddatz
Department of Genetics
University Medical Center Groningen
No. beds: 1,339 No. employees: >11,0000 Department of Genetics
~ 300 employees Many different nationalities Counseling Diagnostics Research Teaching
Section Genome Diagnostics 10 laboratory specialists, 70 technicians >20,000 genetics tests
Karyotyping SNP & Oligo Arrays FISH Sequencing analysis
Department of Genetics
NGS into diagnostics
Genome Diagnostics Richard Sinke Eddy de Boer Krista van Dijk-Bos Jan Jongbloed Yvonne Vos Eva van den Berg-de Ruiter Trijnie Dijkhuizen Annemieke van der Hout Henny Lemmink Renee Niessen Jos Dijkhuis Annelies ten Berge Margaret Burton Martine Meems-Veldhuis Inge Mulder Arjen Scheper Martijn Viel
Clinical Genetics Rolf Sijmons Tom de Koning Conny van Ravenswaaij Peter van Tintelen Joke Verhey Jan Oosterwijk Corien Verschuuren-Bemelmans Irene van Langen Beike Leegte
Cisca Wijmenga Genomics coordination center Morris Swertz Freerk van Dijk Pieter Neerincx Martijn Dijkstra Rowida Almomani Lennart Johansson Patrick Deelen
Department of Genetics Department of Genetics
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Many diseases are heterogeneous
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Department of Genetics
Cardiomyopathies
Department of Genetics
DCM; dilated cardiomyopathy
normal heart
HCM; hypertrofic cardiomyopathy
ACM; aritmogenic cardiomyopathy
Wilde & Behr (2013) Nat. Rev. Cardiol. 10(10):571-83
Department of Genetics
Cardiomyopathies
Paul van der Zwaag, thesis, 2012
Num
ber o
f gen
es re
porte
d
Department of Genetics
Cardiomyopathies
Genetic overlap Up to 50 genes involved
Up to 6 genes in current Diagnostics
Department of Genetics
Arg294Stop CGA-TGA
Data analyses
9
Sanger sequencing
Sanger Sequencing: analysis of 1 gene Growing number of genes need to be tested for a particular disease
Department of Genetics
Novel strategies
Candidate Gene Screening
Next Generation Sequencing
Aim: Apply one comprehensive test. Design and implement various targeted next generation sequencing (NGS) gene-panels.
Department of Genetics
Novel strategies
Candidate Gene Screening
Next Generation Sequencing
What kind of sequencing machine?
Which technique?
Department of Genetics
Exome Sequencing (Agilent)
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Exome Library
Fragments of 120bp labeled with Biotine. Contains coding exons of all genes (+/- 20.000)
Department of Genetics
Exome vs. Targeted
- Coverage performance in exome data varied significantly between exons and (for some exons) insufficient to rely on exome sequencing only
- Insufficient coverage may result in missing clinically relevant mutations
Exome sequencing: High number of variants ! clinical interpretation Insufficient coverage ! missing mutations Targeted resequencing = capturing of exons of certain genes
Department of Genetics
Targeted NGS
What kind of gene panels should we construct to replace
Sanger sequencing?
Based on the indication;
Cardiomyopathies
Hereditary cancer (breast cancer, Lynch syndrome)
Epilepsy
Neurological /movement disorders
Skin disorders
(mental retardation)
Department of Genetics
!
!Custom made library
Custom made Library Contains all coding exons of 48 cardio genes.
Baits: fragments of 120bp labeled with Biotine.
Department of Genetics
Cardio: Genes enriched
ABCC9, ACTC1, ACTN2, ANKRD1, BAG3, CALR3, CAV3, CRYAB, CSRP3/MLP, DES, DMD, DSC2, DSG2, DSP, DTNA, EMD, EYA4, GATAD1, GLA, JPH2, JUP, LAMA4, LAMP2, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYPN, MYOZ1, MYOZ2, NEXN, PKP2, PLN, PRKAG2, PSEN1, PSEN2, RBM20, RYR2, SCN5A, SGCD, TAZ,
TBX20, TCAP, TMEM43, TNNC1, TNNI3, TNNT2, TPM1, TTN, TXNRD2, VCL, ZASP/LDB3
Department of Genetics
Cardio: validation
MiSeq capacity: 1 channel 5 miljoen reads
Readlength 150 bp 5.000.000 x 150 bps = 750.000.000 bp
Paired-end 750.000.000 x2 = 1.500.000.000 bp
Accuracy 75% 75% x 1.500.000.000 = 1.125.000.000 bp
Size Cardio Custom 320.000bp 1.125.000.000 bp /320.000 bp = 3515 bp
12 patients multiplex 3515 /12 = 292
Average coverage 292x
Validationcriteria: Coverage minimal 30 for each nucleotide compared to Sanger: Specificity 100% Sensitivity 98%
Department of Genetics
Results
1. Technical validation: Coverage Specificity/ Sensitivity compared to Sanger Sequencing
2. Clinical validation:
Diagnostic yield
Department of Genetics
48 genes, 1134 targets:
Coverage >30: 99% (<30: 4,398 bps out of 323,651 bps)
Coverage; cardio
Department of Genetics
Coverage; on average 250x (151 bp paired-end reads )
29 targets out of 1134 in at least one sample
15 exons with insufficient coverage
Results - Coverage gaps
Department of Genetics
Conclusions
Resequencing of gene panels on the MiSeq can be
used in routine diagnostics
99% of all bases coverage >30x
No false positives/ negatives
12-16 patients can be multiplexed
Average coverage: 250x
~15 exons per require Sanger sequencing in parallel
Department of Genetics
Interpretation tree Cartagenia
Filtering: - quality - 1000 genomes - GoNL - ESP - SNP database - “managed variant lists”
Department of Genetics
Interpretation tree Cartagenia
Filtering: Quality 1000 genomes GoNL ESP SNP database “managed variant lists”
Per
Per patient 5 – 10 variants
Department of Genetics
Transfer to results form
Previous Classification Present in HGMD Relevante isoforms Allele frequency Population frequency (1000 G, GoNL) Conservation Splicing effects Conclusion Category:
Prediction programas: Alamut (PhyloP score, Mutation Taster, Polyphen,SIFT)
Benign Likely Benign VOUS Likely Pathogenic Pathogenic
Department of Genetics
Cardio-panel v2; 55 genes Clinical yield
Since September 2012 in Routine Diagnostics: >800 patients received >450 finished reports >1 MiSeq run per week
Note: ~15% >1 P/LP
43 pathogenic; 11%
134 likely pathogenic; 34%
213 no mutations/ potentially benign; 55%
390 patients
Diagnostic yield:
Department of Genetics
Challenges
Data sharing!
Existing LOVDs Cartagenia Users Dutch UMCs
Other LSDBs
MMDB
ClinVar
Department of Genetics
Targeted NGS panels
Panel No. of genes Coverage > 30 for each base
No. of patients analyzed
Cardio 55 99,6 >800 Onco 73 99.5 120 Movement 88 99.37 28 Skin 63 in progres not yet Epilepsy 147 99.63 not yet Neuro Construction Liver Construction
Department of Genetics
Onco; genes enriched
BRCA1, BRCA2, PTEN, NF1, CDK4, MUTYH, APC, MSH2, MSH6, MLH1, PMS2, CDH1, STK11, SDHB, RET, SDHD, WT1, SDHC, MEN1, SDHA, FLCN,VHL, NF2, PTCH, FH, BMPR1A, SMAD4, CHEK2, RAD51C, RAD51D, BRIP1, XRCC2, BARD1, HOXB13, KLLN, MITF, ENG, AXIN2, BMP4, TMEM127, CDC73, AIP, CDKN2B, CDKN2C, CDKN1A, CDKN1B, SDHAF2,MAX, PHOX2B, TERT, RUNX1, CEBPA, GATA2, PTCH2, MET, SUFU, TP53, CDKN2A, BAP1, PALB2, DICER1, SMARCB1, SMARCA4, BUB1B, PALLD, EGFR, PDGFRA, KIT, PRKAR1A, ATM, CEP57
Department of Genetics
Targeted NGS; onco
Class 2 (n = 32/70 ) e.g. RAD51C, MAX, ALK, ! Preventive options available for the frequently associated
tumor types No official guidelines yet
3 virtual sub-panels based on preventive options
Class 1 (n= 25/70 ) e.g. BRCA1, MLH1, RET,!
Preventive options available for the frequently associated
tumor types
Following national / international guidelines
Class 3 (n = 13/70) e.g. TP53, KIT, BAP1, !.
No preventive options available for frequently associated
tumor types (e.g. pancreatic cancer, sarcoma)
Department of Genetics
Targeted NGS; onco
Class 2 (n = 32/70 ) e.g. RAD51C, MAX, ALK, ! Preventive options available for the frequently associated
tumor types No official guidelines yet
3 virtual sub-panels based on preventive options
Class 1 (n= 25/70 ) e.g. BRCA1, MLH1, RET,!
Preventive options available for the frequently associated
tumor types
Following national / international guidelines
Class 3 (n = 13/70) e.g. TP53, KIT, BAP1, !.
No preventive options available for frequently associated
tumor types (e.g. pancreatic cancer, sarcoma)
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Department of Genetics
Detection of exon deletions/ duplications
Average coverage per target
Normalisation (first) per sample and (second) per target Calculation of variation coefficient
Targets from X chromosome
One serie of 12 samples Another serie of 12 samples
Department of Genetics
Detection of exon deletions/ duplications
Validation of 120 samples, including 10 known deletions/ duplications
On average 907 of the 930 targets of the onco panel pass the thresholds. No false negative results.
Department of Genetics
Conclusion
Obsolete at a coverage of >30 per nucleotide.
In parallel with NGS at a coverage structural <20x.
Exon deletions/ duplication can be detected.
Targeted NGS can replace Sanger Sequencing
Department of Genetics
Improvements
Reducing Turn-Around-Times
1. Further robotization of sample processing
2. Optimizing the “pipe line”
- Filtering parameters
- Generation of reporting files
3. Automation of interpretation process
How to deal with homologe/ repetitive sequences and
pseudogenes.
Department of Genetics
Targeted NGS, what else !.
(1) Non invasive prenatal diagnostics
(2) Hematology :
• amplicon panels for mutation detection
• TLA panels for structural abnormalities
(3) Neonatology: 72 h Whole Genome
Sequencing