1. Affymetrix Resequencing Arrays Matthew Smith Trainee Presentation West Midlands Regional Genetics Laboratory

2. Introduction 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 Screening all the genes at once –Next generation sequencing –Resequencing Arrays Offer rapid small scale diagnosis Influence clinical management and prognosis

3. Resequencing Arrays 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 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

4. Trainee Project MitoChip Enhanced Genetics Services Project (Heart of Birmingham PCT/ WMRGL/University of Birmingham’s Dept of Molecular and Medical Genetics) Infant Morbidity and Mortality 50kb Mito genome + 500 common haplotypes Custom Array (300kb) 112 genes 1450 additional frameshift mutations Initial Validation

5. Custom Array Design Exonic Tiling Mutation Tiling 1231 Exons 1450 frameshift mutations

6. CATGGACATTAAGCAGATGAaGAATTTCGTGTCCCAGGAGC GCAGATGAGAATTTCG delA Wildtype Exon Tiling Mutation Tiling delA Wildtype Reference Known Frameshift Detection Strategy

7. Initial Validation Validation of a subset of genes –SDHB, SDHC, SDHD, VHL partial coverage of RET –PLA2G6 Developed LPCRs Single exons from 26 patients comprising 27 nonsynonymous pathogenic mutations were interrogated on the array 27/27 mutations were detected. Sequenced 24988 bases Call rate over 90% and a 99.8% concordance with capillary sequencing No cross hybridisation GeneArray Sequencing Capillary Sequencing

8. Current Technical Limitations 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 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

9. Project outcomes 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

10. Conclusions 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 Next Generation Sequencing Huge Impact in Research LabsImpact on diagnostic Service Full use of capacity IT issues Gene Targeting

11. Acknowledgments University Dept of Medical and Molecular Genetics Paul Gissen Chris Bruce Fatimah Rahman WMRGL Fiona MacDonald Jennie Bell Dominic McMullan