Custom Panels via Clinical Exomes

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1 Custom Panels via Clinical Exomes Andrew Wallace Feb 2015 Genomic Diagnostics Laboratory St. Mary s Hospital, Manchester

2 Custom Panel via Exome Approach Pros Reduced validation overhead Single validation Full exome gives flexibility New clinically relevant genes can be covered Standard panels for a clinical presentation evolve over time Minimises potential for coincidental findings Later research analysis of data possible Dependant on consent Standard workflow Enrichment & seq chemistry aligned with diagnostic panels Cons Large off target volume of sequence Lowers overall sequencing capacity Lower overall coverage Coverage gaps

3 Exome Sequencing Agilent SureSelect All Exon v5 (50Mb enrichment) 21,522 genes, >357,000 exons 2 x 100bp paired end HiSeq rapid run 5/6 samples sequenced per HiSeq FlowCell (~60Gb sequence) Lab workflow process batching of 15 samples Target of 90% of exome design coverage at >30x depth ~10-12Gb standard sequence

4 Sample & Custom Panel Process Cases co-ordinated by a single exome lead clinician Patients consented at dedicated exome clinic Spreadsheet of samples for testing +/- candidate genes Clinical Scientist agrees and locks final gene list with lead clinician Range from 2 40 genes to date Clinical Scientist identifies clinically relevant transcript(s) for each gene Clinical Bioinformatician generates bed file for reference transcripts Reference transcript(s) +/-15bp intron Clinical Bioinformatician calculates design coverage of gene list Lab process NGS Data Bioinformatic Analysis

5 Number of Genes Variable number of genes in custom panel Ranges from 2-40 to date

6 Example Clinical Indications Primary ovarian failure Systemic candidiasis Macrocephaly Reproductive Immune system Developmental Pulmonary fibrosis Cutis laxa Spastic parapesis Respiratory Connective tissue disorder Neurological

7 Bioinformatic Analysis Primary analysis De-multiplexing BWA alignment whole genome Hg19 Duplicate removal Coverage reporting Custom panel Variant calling limited to custom panel samtools Pindel Filtering Ensembl v75 OMIM HGMD free EVS In house frequencies Coverage reporting Exome design Secondary analysis In house frequency is based on ~750 research exomes Comprising ~1.4 million variants called. Mean coverage of research is 85.6%. Output summary in MS Excel spreadsheets

8 Coverage Metrics Experimental data (D) Custom panel (C) Agilent v5 design (B) Analytical performance = B/A Clinical performance = D C/C Design coverage = B C/C Exome (A)

9 Test Performance Analytical and clinical performance differs Depends on the panel Analytical Performance Clinical Performance Target %age coverage 30x

10 Custom Panel Interpretation Complex but! Due to limited experience with selected genes Simpler than a full exome Generally 5-15 variants identified & interpreted Refer to OMIM & literature for mutation spectrum, patterns of inheritance etc for each gene Allele freq information EVS, ExAC (Broad), dbsnp In house research exome data extremely useful (~750 exomes) VUS analysis as per ACGS guidelines HGMD, LSDBs, Google etc In silico splice site & missense analysis Confirm by Sanger sequencing

11 Reporting Report to Exome lead clinician only Gene names (HGNC) & reference transcripts used Quality metrics Analytical performance Clinical performance for selected genes coding region +/-15bp) Variants categorised as per VUS guidelines into 5 classes Class 3 to 5 reported and interpreted Class 2 technical report Full interpretation inc supporting publications if relevant

12 Example Case 26 year old patient with pain and loss of vision in her left eye. Investigation revealed a candida infection. Despite treatment with antifungal agents she lost the sight in her eye. She remained on antifungal treatment for 6 months. A further six months later she presented with pain and destruction of her right hip. This resulted in a total hip replacement. Analysis of the removed bone revealed a deep seated candida infection. Diagnosis?systemic candidiasis Referred to Exome Clinic & selected for Exome Sequencing Clinician requested 6 custom genes IL17F Autosomal Dominant Missense CARD9 Autosomal Recessive Nonsense & Missense STAT1 Autosomal Dominant & Recessive Missense (AD/AR) & Frameshift (AR) CLEC6A Unknown Animal disease model TRAF3IP2 Autosomal Recessive Missense Plus Psoriasis association CLEC7A Autosomal Recessive Nonsense polymorphism MAF 0.069

13 Example Case 5 variants in 3 genes TRAF3IP2 Autosomal Recessive Missense Plus Psoriasis association c c>t Hom 121/586 Hom 302/586 STAT1 Autosomal Dominant & Recessive Missense (AD/AR) & Frameshift (AR) c.969c>a p.(his323gln) Het 196/586 Hom 56/586 CARD9 Autosomal Recessive Nonsense & Missense c.1138g>c p.(ala380pro) c.951g>a p.(arg317arg) c g>t -

14 Example Case Segregation studies CARD9 Variant c g>t c.951g>a p.(arg317arg) c.1138g>c p.(ala380pro) Genotype Normal Normal Normal CARD9 Variant c g>t c.951g>a p.(arg317arg) c.1138g>c p.(ala380pro) Genotype Het Het Normal CARD9 Variant c g>t c.951g>a p.(arg317arg) c.1138g>c p.(ala380pro) Genotype Het Het Het De novo occurrence of c.1138g>c p.(ala380pro) supportive of CARD9 involvement Patient now on lifelong anti-fungal treatment, no recurrence of infection Siblings re-assured for their risk Patient re-assured for recurrence for children

15 Acknowledgements Helene Schlecht Eleanor Baker Sanjeev Bhaskar Simon Williams Laura Dutton