1. Mutation detection by massively parallel sequencing of solution captured human genomic loci Frances Smith DNA Laboratory Guy’s Hospital CMGS 12 th 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. 120 bp RNA probes 55 thousand probes per library 5 x probe tiling (85 bp overlap) Repeat masking Probe Design Parameters Probes Exon

6. Solution capture Prepped library Biotinylated RNA ‘probes’ B B B B BB Pool Hybridise24h at 65°C B B B DNA:RNA hybrids Select hybrids - streptavidin B Wash PCR Target enriched sequencing library

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 capture Sensitivities at different levels of coverage Coverage

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

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. Heterozygous 13bp del DMD Deletions

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

19. Lily Foundation The Lily Foundation

20. GSD genes GYS2 G6PC G6PT (SLC37A4) GDE (AGL) GBE1 PYGL PHKA2 PHKB PHKG2 PYGM PFKM PGAMM (PGAM2) ALDOA ENO3 GAA LAMP2 GYS1 LDHM (LDHA) Total of 47 genes and 4 Mbp + 29 NMD genes

21. Library preparation: Genomic DNA Shear DNA ~ 200bp End-repair Blunt ended phosphorylated fragments DNA Polymerase Klenow +P Kinase A- tailing A A Klenow exo 3’ A overhanging fragments Ligate adapters T TA A Ligase PCR Partially double stranded adapters with T overhangs Size select Sequencing library Shear DNA ~ 200bp ShearEnd-repair DNA Polymerase End-repair DNA Polymerase End-repair DNA Polymerase End-repair DNA Polymerase Klenow +P Kinase DNA Polymerase End-repair DNA Polymerase Klenow +P Kinase DNA Polymerase End-repair Blunt ended phosphorylated fragments DNA Polymerase Klenow +P Kinase DNA Polymerase End-repair A A Klenow exo A- tailing A A Klenow exo Ligate adapters T TA A Ligase Partially double stranded adapters with T overhangs Ligate adapters T TA A Ligase Partially double stranded adapters with T overhangs PCR Sequencing library PCR Sequencing library

22. Cluster generation: Target enriched sequencing library Quantify by qPCR Hybridise to flowcell Single stranded adapters bound to surface of flowcell Single stranded library molecule hybridises to complimentary adapter Synthesise complimentary strand DNA polymerase Make single stranded Bridge amplification DNA polymerase Fragment bends over to another adapter Repeated cycles bridge amplification Clonal colonies attached to the flowcell - ‘clusters’

23. Sequencing by synthesis: Sequencing primer Linearise template and block unused adapters 1 st base incorporation A A DNA polymerase Nucleotide Reversible terminator A Excite fluorophoreImage clusters Remove terminator Repeated cycles of base incorporation and imaging Laser A G C T Build reads by stacking images and base calling Cycle 1 = A Cycle 2 = G Cycle 3 = C Cycle 4 = T