Robust Diagnostic Genetic Testing Using Solution Capture Enrichment and a Novel Variant-Filtering Interface

• Published in: Human mutation Vol. 35; no. 4; pp. 434 - 441
• Main Authors: Watson, Christopher M., Crinnion, Laura A., Morgan, Joanne E., Harrison, Sally M., Diggle, Christine P., Adlard, Julian, Lindsay, Helen A., Camm, Nick, Charlton, Ruth, Sheridan, Eamonn, Bonthron, David T., Taylor, Graham R., Carr, Ian M.
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.04.2014; Hindawi Limited; BlackWell Publishing Ltd
• Subjects: Axonemal Dyneins - genetics; Codon, Nonsense; Dyneins - genetics; exome sequencing; Genes, Neoplasm; Genetic Predisposition to Disease; Genetic Testing - methods; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Informatics; Kartagener Syndrome - diagnosis; Kartagener Syndrome - genetics; Laboratories; mutation detection; Neoplasms - diagnosis; Neoplasms - genetics; Polymorphism, Single Nucleotide; Reproducibility of Results; sequence analysis; Software; User-Computer Interface; exome sequencing; sequence analysis; mutation detection; software
• ISSN: 1059-7794; 1098-1004
• DOI: 10.1002/humu.22490

ABSTRACT Targeted hybridization enrichment prior to next‐generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer‐predisposing genes or (2) whole‐exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome‐based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques. We present two programs, AgileExomeFilter and AgilePindelFilter, which enable clinical scientists to rapidly and efficiently filter variants from next generation sequencing data. Using these tools a high diagnostic yield (42%) was obtained in a cohort of 24 primary ciliary dyskinesia (PCD) patients that underwent targeted exome analysis. Autozygosity mapping in the mutation negative cases revealed a homozygous truncating mutation in DNAH8, a presumptive new PCD gene.