Dependable and efficient clinical utility of target capture-based deep sequencing in molecular diagnosis of retinitis pigmentosa

Invest Ophthalmol Vis Sci. 2014 Aug 5;55(10):6213-23. doi: 10.1167/iovs.14-14936.


Purpose: The purpose of this study was to establish a fully validated, high-throughput next-generation sequencing (NGS) approach for comprehensive, cost-effective, clinical molecular diagnosis of retinitis pigmentosa (RP).

Methods: Target sequences of a panel of 66 genes known to cause all nonsyndromic and a few syndromic forms of RP were enriched by using custom-designed probe hybridization. A total of 939 coding exons and 20 bp of their flanking intron regions with a total of 202,800 bp of target sequences were captured, followed by massively parallel sequencing (MPS) on the Illumina HiSeq2000 device.

Results: Twelve samples with known mutations were used for test validation. We achieved an average sequence depth of ∼1000× per base. Exons with <20× insufficient coverage were completed by PCR/Sanger sequencing to ensure 100% coverage. We analyzed DNA from 65 unrelated RP patients and detected deleterious mutations in 53 patients with a diagnostic yield of ∼82%.

Conclusions: Clinical validation and consistently deep coverage of individual exons allow for the accurate identification of all types of mutations including point mutations, exonic deletions, and large insertions. Our comprehensive MPS approach greatly improves diagnostic acumen for RP in a cost- and time-efficient manner.

Keywords: massively parallel sequencing; next generation sequencing; retinitis pigmentosa; target gene enrichment.

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Child
  • Child, Preschool
  • DNA / analysis*
  • DNA / genetics
  • Exons
  • Female
  • Follow-Up Studies
  • High-Throughput Nucleotide Sequencing / methods
  • High-Throughput Nucleotide Sequencing / statistics & numerical data
  • Humans
  • Infant
  • Infant, Newborn
  • Introns
  • Male
  • Middle Aged
  • Molecular Diagnostic Techniques / methods
  • Molecular Diagnostic Techniques / statistics & numerical data*
  • Mutation*
  • Pedigree*
  • Polymerase Chain Reaction
  • Reproducibility of Results
  • Retinitis Pigmentosa / diagnosis*
  • Retinitis Pigmentosa / genetics
  • Sequence Analysis, DNA / methods
  • Sequence Analysis, DNA / statistics & numerical data
  • Young Adult


  • DNA