Mutation detection by massively parallel sequencing of solution captured human genomic loci

1. Mutation detection by massively parallel sequencing of solution captured human genomic loci Frances Smith DNA Laboratory Guy’s Hospital CMGS 12th April 2010

2. Aims of the project • Comprehensive diagnostic service to sequence all genes involved in Glycogen Storage Disease (GSD) • New technologies • Agilent SureSelect • Illumina sequencing

3. Clinical Need • GSD • Defects in glycogen synthesis or breakdown in liver or muscle • Broad overlapping clinical phenotype • 18 genes • No comprehensive test • Reduce cost • Speed up diagnosis • Reduce invasive tests

4. Solution capture • Submit genomic intervals to eArray • 18 GSD genes • 29 NMD genes • Total of 4 Mbp and 1200 exons

5. Probe Design Parameters • 120 bp RNA probes • 55 thousand probes per library • 5 x probe tiling (85 bp overlap) • Repeat masking Probes Exon

6. Solution capture B B B Pool B B B Biotinylated RNA ‘probes’ Prepped library Hybridise 24h at 65°C B DNA:RNA hybrids B B Select hybrids -streptavidin PCR Target enriched sequencing library B Wash

7. Results • 8 lanes of sequencing • 17 Gbp • 80% (13 Gbp) maps to human genome • 1.6 Gbp per lane • Equivalent to 66 whole DMD genes • Sensitivity (% target bases giving reads) = 99.5% @ >30x coverage • Specificity (% reads mapping to targets) = 63%

8. Uniformity of captureSensitivities at different levels of coverage Coverage

9. Why do some probes not capture well? Good probe: Poor probe: • GC content • Extremes of GC% not captured well • Secondary structure • Self complimentarity • Sequence context • Close to repeats

10. Probe coverage reproducibility Coverage

11. What do we do about it? • Re-design the library • Increase sequencing output • Sanger sequence persistent gaps

12. Validation • Known GSD mutations captured and sequenced blind • 2 compound heterozygote substitutions • Homozygous frameshift • Compound heterozygote substitution and nonsense mutation • Deletions captured and sequenced • 5bp • 7bp • 13bp • 38bp ….. Testing more

13. Point mutations Heterozygous c.247C>T; p.Gln83X c.925C>T; p.Arg309Trp

14. Deletions Heterozygous 13bp del DMD

15. Heterozygous 38bp del SEPN1 A bit more difficult to find…

16. Problems and Challenges • Bioinformatics • Huge amounts of data • Storage and analysis issues • Cost • Set up and run costs high • Time • Technically challenging • Variation • Large number of genes therefore large number of UV’s • How do we investigate/report these?

17. Summary • GSDv1 probe library designed and validated • Solution capture and illumina sequencing carried out for point mutations and deletions up to 38bp • Alignment software • Ongoing • New versions of GSD library designed • Multiplexing • Other heterogeneous disorders

18. Acknowledgments Biomedical Research Centre Guy’s & St Thomas’ NHS Foundation Trust and KCL • Pete Green • Effie Papouli • Muddassar Mirza Clinical colleagues • Mike Champion • Charu Deshpande Guy’s DNA Lab • Steve Abbs • Michael Yau • Tom Cullup Sanger Institute • Dan Turner • Alison Coffey • Eleanor Howard

19. Lily Foundation The Lily Foundation