Download Affymetrix Resequencing Arrays

Affymetrix Resequencing
Arrays
Matthew Smith
Trainee Presentation
West Midlands Regional Genetics
Laboratory
Introduction
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Autosomal recessive disorders are a major cause of infant morbidity
and mortality
Significantly higher in WM than rest of country (Bundy report, 1990)
Clinical phenotypes can be caused by mutations in one of several
genes or different mutated genes can cause very similar clinical
phenotype
Genes are analysed sequentially until a mutation is identified
– Time consuming
– Expensive
– Medical management in absence of key information
Solution
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Screening all the genes at once
– Next generation sequencing
– Resequencing Arrays
• Offer rapid small scale diagnosis
• Influence clinical management and prognosis
Resequencing Arrays
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300kb sequence in 48hours
Sequences both forward and reverse strands simultaneously
Sequencing for “less than a penny a base”
Overall 1/6 the cost of conventional sequencing
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Amplify target of interest
Fragment DNA
Label DNA
Wash over Array
DNA hybridises to probe with
complementary sequence
Signal amplification
Signal Intensity determines
identity of base
Trainee Project
MitoChip
50kb
Enhanced Genetics Services Project
(Heart of Birmingham PCT/
WMRGL/University of Birmingham’s
Dept of Molecular and Medical
Genetics)
Infant Morbidity and Mortality
Mito genome + 500
common haplotypes
Custom Array (300kb)
112 genes
1450 additional frameshift mutations
Initial Validation
Custom Array Design
Exonic Tiling
Mutation Tiling
1231 Exons
1450 frameshift mutations
Known Frameshift Detection Strategy
CATGGACATTAAGCAGATGAaGAATTTCGTGTCCCAGGAGC
GCAGATGAGAATTTCG
Mutation Tiling
Exon Tiling
Reference
delA
Wildtype
delA
Wildtype
Initial Validation
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Validation of a subset of genes
– SDHB, SDHC, SDHD, VHL
partial coverage of RET
– PLA2G6
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Developed LPCRs
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Single exons from 26 patients
comprising 27 nonsynonymous
pathogenic mutations were
interrogated on the array
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27/27 mutations were detected.
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Sequenced 24988 bases
Call rate over 90% and a 99.8%
concordance with capillary
sequencing
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No cross hybridisation
Capillary Sequencing
GeneArray Sequencing
Current Technical Limitations
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Reduced ability to detect insertions and deletion mutations
– Inclusion of probes complementary to known insertions or
deletions
– Possible to design array to detect these sorts of mutations
– 10kb target
• 1-5bp deletions – 100,000 probes
• 1-5bp insertions – 27,000,000 probes
– Advances in software and improvements in the GSeq algorithm
– SeqC from JSI medical claims to detect insertions and deletions
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No Calls
– GSeq analysis is based on a learning algorithm
– When it can not assign a genotype it assigns a no call
– Majority of No calls are due to strings of C’s (60%) and can be
called uni-directionally.
– Comparison of unique no calls could be indication of frameshift
mutation
Project outcomes
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Gained valuable expertise in the design and development of
resequencing array technology
Highlighted areas of development to make it suitable for diagnostics
Continuation of project
– Enhanced Genetics Service Project (Heart of Birmingham/WMRGL)
– Major initiative to reduce childhood morbidity and mortality
• Carrier testing
• Prenatal diagnosis
– 20 highest priority autosomal recessive conditions (clinical study)
– Continue evaluation of resequencing methodology for diagnostic
use
– Development of methods for unknown frameshift detection
(bioinformatics)
– Evaluation of Array design
Conclusions
Next Generation Sequencing
Huge Impact in Research Labs
Impact on diagnostic Service
Full use of
capacity
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IT issues
Gene
Targeting
Bridges the gap between current sequencing technology and next
generation technology
Potentially a powerful method for complex disease screening
Resequencing offers a targeted approach to mutation detection
– Rapid
– Acute medical management
Future Array designs
• Smaller capacity array with a smaller number of genes
• Modifier genes
Acknowledgments
University Dept of Medical and Molecular Genetics
• Paul Gissen
• Chris Bruce
• Fatimah Rahman
WMRGL
• Fiona MacDonald
• Jennie Bell
• Dominic McMullan