title: Identification of false-negative mutations missed by next-generation sequencing in retinitis pigmentosa patients: a complementary approach to clinical genetic diagnostic testing.
abstract: OBJECTIVE
  Retinitis pigmentosa (RP) is a major cause of heritable human blindness with extreme genetic heterogeneity. A large number of causative genes have been defined by next-generation sequencing (NGS). However, due to technical limitations, determining the existence of uncovered or low-depth regions is a fundamental challenge in analyzing NGS data. Therefore, undetected mutations may exist in genomic regions less effectively covered by NGS.
  
  
  METHODS
  To address this problem, we tested a complementary approach for identifying previously undetected mutations in NGS data sets. The strategy consisted of coverage-based analysis and additional target screening of low-depth regions. Fifty RP patients were analyzed, and none of the mutations found had previously been identified by NGS.
  
  
  RESULTS
  Coverage-based analysis indicated that, because of a highly repetitive sequence, the RPGR open reading frame (ORF)15 was located in an uncovered or low-depth region. Through additional screening of ORF15, we identified pathogenic mutations in 14% (7/50) of patients, including four novel mutations first described herein.
  
  
  CONCLUSIONS
  In brief, we support the need for a complementary approach to identify mutations undetected by NGS, underscoring the power and significance of combining coverage-based analysis with additional target screening of low-depth regions in improving diagnosis of genetic diseases. In addition to its usefulness in RP, this approach is likely applicable to other Mendelian diseases.
authors.affiliation: 1] Division of Ophthalmic Genetics, Laboratory for Stem Cell and Retinal Regeneration, The Eye Hospital of Wenzhou Medical University, Wenzhou, China [2] The State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health, Wenzhou, China.
authors.forename: Xiu-Feng
authors.initials: XF
authors.lastname: Huang
authors.affiliation: 1] Division of Ophthalmic Genetics, Laboratory for Stem Cell and Retinal Regeneration, The Eye Hospital of Wenzhou Medical University, Wenzhou, China [2] The State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health, Wenzhou, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Wu
authors.affiliation: 1] Division of Ophthalmic Genetics, Laboratory for Stem Cell and Retinal Regeneration, The Eye Hospital of Wenzhou Medical University, Wenzhou, China [2] The State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health, Wenzhou, China.
authors.forename: Ji-Neng
authors.initials: JN
authors.lastname: Lv
authors.affiliation: 1] Division of Ophthalmic Genetics, Laboratory for Stem Cell and Retinal Regeneration, The Eye Hospital of Wenzhou Medical University, Wenzhou, China [2] The State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health, Wenzhou, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: 1] Division of Ophthalmic Genetics, Laboratory for Stem Cell and Retinal Regeneration, The Eye Hospital of Wenzhou Medical University, Wenzhou, China [2] The State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health, Wenzhou, China.
authors.forename: Zi-Bing
authors.identifier: 000000030515698X
authors.initials: ZB
authors.lastname: Jin
mesh_terms: D005136:Eye Proteins / Q000235:genetics*; D040181:Genetic Diseases, X-Linked / Q000209:etiology / Q000235:genetics* / Q000473:pathology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation / Q000235:genetics; D010375:Pedigree; D012174:Retinitis Pigmentosa / Q000209:etiology / Q000235:genetics* / Q000473:pathology
chemical_list: D005136:Eye Proteins; C099929:RPGR protein, human