Affymetrix Resequencing Arrays

Matthew SmithTrainee Presentation

West Midlands Regional Genetics Laboratory

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

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

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

Custom Array Design Exonic Tiling

Mutation Tiling

1231 Exons

1450 frameshift mutations

CATGGACATTAAGCAGATGAaGAATTTCGTGTCCCAGGAGC

GCAGATGAGAATTTCG

delA

Wildtype

Exon TilingMutation Tiling

delA

Wildtype

Reference

Known Frameshift Detection Strategy

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

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

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

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 Labs Impact on diagnostic Service

Full use of capacity

IT issues Gene Targeting

Acknowledgments

University Dept of Medical and Molecular Genetics

• Paul Gissen

• Chris Bruce

• Fatimah Rahman

WMRGL

• Fiona MacDonald

• Jennie Bell

• Dominic McMullan