NEXT GENERATION SEQUENCING OF MULTIPLE GENES IN PARALLEL IN GENETICALLY HETEROGENEOUS DISORDERS AFTER

ENRICHMENT

HETEROGENEITY IN MONOGENIC DISORDERS

Disease # (known) Genes

Breast cancer 2

Recessive ataxias >20

Blindness >200

Mental retardation ~1000

From: Cover of Nature human genome issue, published on 15 February 2001

CHALLENGES

• Sequencing of multiple genes in parallel for heterogeneous hereditary disorders

• Multiplexing of samples for sequencing to increase throughput and reduce costs

• Automated data-analysis, including

• Interpretation of potential pathogenicity of identified variants

• Validation (in collaboration with EUGT) )

• Cost effectiveness (Katherine Payne, Manchester)

• Ethical issues (Anne Cambon-Thomsen, Toulouse)

• Dissemination of knowledge (Milan Macek, Prague)

• Management

TECHGENE - OTHER ACTIVITIES

TECHGENE - WHO ARE WE?PARTNER CENTRE COUNTRYScheffer/Veltman Radboud University Nijmegen Medical

CentreThe Netherlands

Matthijs/Cuppens KU Leuven BelgiumMacek Charles University - 2nd School of

Medicine, PragueCzech Republic

Estivill Center for Genomic Regulation (CRG), Barcelona

Spain

Gasparini University of Trieste ItalyRiess University of Tuebingen GermanyBakker/Den Dunnen Leiden University Medical Center The NetherlandsBanfi Telethon Foundation, Naples ItalyNigro Second University of Naples ItalyCambon-Thomsen University Paul Sabatier, INSERM

U558, ToulouseFrance

Payne University of Manchester United KingdomSak Asper Biotech, Tartu Estonia

TECHGENE

• Is a network of ISO15189 accredited clinical molecular genetic laboratories and renowned genome research laboratories

• Has been positioned as a technological innovation platform connected with the EUGT Network of Excellence dealing with Quality Standards in Clinical (Laboratory) Genetics

TECHGENE - SCIENTIFIC ADVISORY BOARD

Name Affiliation Expertise

Prof. Dr. Jean-Jacques Cassiman

Katholieke Universiteit Leuven, Eurogentest, ESHG

Molecular Genetics, Quality Assurance, EU Regulatory Affairs

Prof. Dr. David Barton

National Centre for Medical Genetics, University College Dublin

Clinical Molecular Genetics

Dr. Cor Oosterwijk

European Genetic Alliances Network

Patient Organisations, Commercial contacts

PLATFORMS

COMPANY TECHNOLOGY CURRENT READ LENGTH

CURRENT THROUGHPUT

Roche/454(GS FLX Titanium)

Sequencing by synthesis

300-500 bases 500 Mb/10h

Illumina/Solexa(Illumina Genome Analyzer System)

Sequencing by synthesis

35 bases 4.5-6.0 Gb/2 days

Life Technologies(SOLiD3)

Sequencing by ligation

50 bases 60 Gb/5 days

Validation

WP1

Hemoglobinopathies Breast Cancer

Few Genes with Multiple Mutations

Long PCR Enrichment

WP6

Economic Issues

Read-out & Data Anal.

WP7

Dissemination & Training

WP5

Ethical Issues

WP8 Management of TECHGENE

Array Enrichment

WP4

Mental Retardation

Rare Mutations in Rare Genes

WP2

Sensory Disorders

1 Major Gene & Multiple Minor Genes

WP3

Ataxia & Paraplegia

Multiple Equally Important Genes

� Next Generation Sequencing in Diagnostics

TECHGENE - HOW? Overview of Plan of Investigation

ARRAY-BASED GENE CAPTURE: 7 KNOWN DISEASE GENES (AR ATAXIA)

Target region, i.e. genomic gene sequence +/- 5kb

Unique sequence represented on array

7 disease genes:• 278 exons• 1,110,372 targeted bp • 886,569 tiled bp

APTXSYNE1

TDP1

SACS

SETX

ATM

FXN

SYNE 2: EXONS ONLY

SYNE2:• 116 exons• 58,331 targeted bp • 63,040 tiled bp (represented on array)

NOVEL CANDIDATE LOCUS (HOMOZYGOSITY MAPPING): 1.3MB

Candidate locus:• 8 genes (out of 80)• 118 exons• 1,337,276 targeted bp • 1,012,745 tiled bp (represented on array)

AUTOSOMAL RECESSIVE ATAXIA: ARRAY SUMMARY

• 16 genes • 512 exons• 2,505,979 bases targeted• 1,962,354 bases tiled (represented on array)

KEY QUESTIONS

• Does array-enrichment work?– Coverage of targeted vs. near-targeted regions– How much non-targeted sequence is still present?

• Is mutation detection possible?– Can known mutations be identified?– Identification of known SNPs

AVERAGE MAPPING DATA

* * *

% mapped 98.80%Mapped bases in regions 76.49%

Mapped bases near region (500bp) 3.82%Mapped bases outside region 19.68%

Target bases covered 98.05%Target bases with >5x coverage 91.26%

Target bases with >10x coverage 80.89%Target bases with >30x coverage 33.49%Target region bases not covered 1.95%

ENRICHMENT –FULL GENOMIC SEQUENCES, E.G. SYNE1

Ø 39-fold enrichment for all genes

ZOOM IN SYNE1 - ENRICHMENT AND SEQUENCING IS REPRODUCIBLE

ARE THERE REGIONS NOT COVERED?

~3.6-8.9% of targeted regions are not covered (with the given coverage) All intronic sequences

CAN WE DETECT MUTATIONS?

ARSACS patient:

A 5 bp heterozygous deletion in exon 5 of the SACS gene

Chr13:22810013-22810017CTTTT deletion

Found in 63.0% of 27 reads(11/13 forward, 6/16 reverse)

Leads to frame shift

CAN KNOWN MUTATIONS BE IDENTIFIED?

CAN PATHOGENIC MUTATIONS BE IDENTIFIED AGAINST A BACKGROUND OF APPR. 1,750 HIGH CONFIDENTIALITY (HC) DIFFERENCES ?

ARSACS patient: compound heterozygous for SACS c.5559-5563 delCTTTT / c. 11719C>T (p.Q3907X)

Roche Mapping software (HC differences) output:* exclude variants outside coding regions* exclude known SNPs (dbSNP)* sort for conservation (phastCons & phyloP)���� Top 2 on the list are the known mutations!

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AT patient: c.7875-7576 TG>GC (homozygous mutation ATM gene)

CAN KNOWN MUTATIONS BE IDENTIFIED?

CAN PATHOGENIC MUTATIONS BE IDENTIFIED AGAINST A BACKGROUND OF APPR. 1,750 HIGH CONFIDENTIALITY (HC) DIFFERENCES ?

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AT Patient: c.7875-7576 TG>GC (homozygous mutation ATM gene)

Roche Mapping software (HCdifferences) output:���� Top 1 on the list is the known mutation!

MUTATION DETECTION IN EIGHT SAMPLES

CONCLUSIONS

Nimblegen Sequence Capture & Roche 454 FLX Titanium Sequencing

• Enrichment works• Enrichment is specific• Known mutations can be identified by sequence capture

• Concept is proven• Diagnostic potential for many genetically heterogeneous diseases!

• Next phase is design of seq cap tools for parallel sequencing of appr. 100 genes in hereditary blindness, and hereditary movement disorders, respectively; comparison of array-based seq capture, seq capture in solution, Raindance technology, and others)

• Establishment of a diagnostic NGS knowledge network (academic labs offering diagnostic NGS service, commercial companies involved in diagnostic NGS development)

•Regular meetings, workshops, teleconferences, etc. , open to interested researchers and laboratory scientists

• Facilitating open scientific discussions and collaboration

• TECHGENE Scientific Meeting 18 March 2010

DIAGNOSTIC NGS KNOWLEDGE NETWORK

Alexander Hoischen Sascha Vermeer

Walter van der Vliet Nine van Slobbe-Knoers

Peer Arts Berry Kremer

Christian Gilissen Jorge Sequeiros

Nienke Wieskamp

Rowdy Meijer

Michael Buckley

Joris Veltman

Hans Scheffer www.techgene.eu

www.techgene.org

ACKNOWLEDGEMENTS