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.


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 (, 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


  • Codon, Nonsense
  • Axonemal Dyneins
  • Dyneins