Robust diagnostic genetic testing using solution capture enrichment and a novel variant-filtering interface

Hum Mutat. 2014 Apr;35(4):434-41. 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.

Keywords: exome sequencing; mutation detection; sequence analysis; software.

MeSH terms

  • Axonemal Dyneins / genetics*
  • Codon, Nonsense
  • Dyneins / genetics*
  • Genes, Neoplasm
  • Genetic Predisposition to Disease
  • Genetic Testing / methods*
  • Genetic Variation
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Kartagener Syndrome / diagnosis*
  • Kartagener Syndrome / genetics
  • Neoplasms / diagnosis*
  • Neoplasms / genetics
  • Polymorphism, Single Nucleotide
  • Reproducibility of Results
  • Software
  • User-Computer Interface

Substances

  • Codon, Nonsense
  • Axonemal Dyneins
  • Dyneins