title: Dependable and efficient clinical utility of target capture-based deep sequencing in molecular diagnosis of retinitis pigmentosa.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Victor W
authors.initials: VW
authors.lastname: Zhang
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Yanming
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Xia
authors.initials: X
authors.lastname: Tian
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Fang-Yuan
authors.initials: FY
authors.lastname: Li
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Cavatina
authors.initials: C
authors.lastname: Truong
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Guoli
authors.initials: G
authors.lastname: Wang
authors.affiliation: Casey Eye Institute, Oregon Health and Science University, Portland, Oregon, United States.
authors.forename: Pei-Wen
authors.initials: PW
authors.lastname: Chiang
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Lewis
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States.
authors.forename: Lee-Jun C
authors.initials: LJ
authors.lastname: Wong
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000032:analysis* / Q000235:genetics; D005091:Exons; D005260:Female; D005500:Follow-Up Studies; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007438:Introns; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000706:statistics & numerical data*; D009154:Mutation; D010375:Pedigree; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012174:Retinitis Pigmentosa / Q000175:diagnosis* / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods / Q000706:statistics & numerical data; D055815:Young Adult
chemical_list: D004247:DNA
keywords: massively parallel sequencing; next generation sequencing; retinitis pigmentosa; target gene enrichment