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

Frances SmithDNA LaboratoryGuy’s Hospital

CMGS 12th April 2010

Aims of the project

• Comprehensive diagnostic service to sequence all genes involved in Glycogen Storage Disease (GSD)

• New technologies– Agilent SureSelect– Illumina sequencing

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

Solution capture

• Submit genomic intervals to eArray

– 18 GSD genes

– 29 NMD genes

– Total of 4 Mbp and 1200 exons

• 120 bp RNA probes• 55 thousand probes per library• 5 x probe tiling (85 bp overlap)• Repeat masking

Probe Design Parameters

Probes

Exon

Solution capture

Prepped library

Biotinylated RNA ‘probes’

B

B

BB

B BPool

Hybridise 24h at 65°C

B

B

BDNA:RNA hybrids

Select hybrids -streptavidin

B

Wash

PCR

Target enriched sequencing library

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%

Uniformity of capture

Sensitivities at different levels of coverage6%

11%

21%

62%

Coverage

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:

Probe coverage reproducibility

Coverage

What do we do about it?

• Re-design the library

•Increase sequencing output

•Sanger sequence persistent gaps

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

Point mutationsHeterozygous

c.247C>T; p.Gln83X

c.925C>T; p.Arg309Trp

Heterozygous 13bp del DMD

Deletions

Heterozygous 38bp del SEPN1

A bit more difficult to find…

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?

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

Acknowledgments

Guy’s DNA Lab– Steve Abbs– Michael Yau– Tom Cullup

Sanger Institute– Dan Turner– Alison Coffey– Eleanor Howard

Biomedical Research CentreGuy’s & St Thomas’ NHSFoundation Trust and KCL

– Pete Green– Effie Papouli– Muddassar Mirza

Clinical colleagues– Mike Champion– Charu Deshpande

Lily Foundation

The Lily Foundation