title: Robust diagnostic genetic testing using solution capture enrichment and a novel variant-filtering interface.
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.
authors.affiliation: Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds, LS9 7TF, United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Watson
authors.forename: Laura A
authors.initials: LA
authors.lastname: Crinnion
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Morgan
authors.forename: Sally M
authors.initials: SM
authors.lastname: Harrison
authors.forename: Christine P
authors.initials: CP
authors.lastname: Diggle
authors.forename: Julian
authors.initials: J
authors.lastname: Adlard
authors.forename: Helen A
authors.initials: HA
authors.lastname: Lindsay
authors.forename: Nick
authors.initials: N
authors.lastname: Camm
authors.forename: Ruth
authors.initials: R
authors.lastname: Charlton
authors.forename: Eamonn
authors.initials: E
authors.lastname: Sheridan
authors.forename: David T
authors.initials: DT
authors.lastname: Bonthron
authors.forename: Graham R
authors.initials: GR
authors.lastname: Taylor
authors.forename: Ian M
authors.initials: IM
authors.lastname: Carr
mesh_terms: D056444:Axonemal Dyneins / Q000235:genetics*; D018389:Codon, Nonsense; D004398:Dyneins / Q000235:genetics*; D052138:Genes, Neoplasm; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods*; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007619:Kartagener Syndrome / Q000175:diagnosis* / Q000235:genetics; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012984:Software; D014584:User-Computer Interface
chemical_list: D018389:Codon, Nonsense; D056444:Axonemal Dyneins; D004398:Dyneins
keywords: exome sequencing; mutation detection; sequence analysis; software