title: Performance evaluation of the next-generation sequencing approach for molecular diagnosis of hereditary hearing loss.
abstract: OBJECTIVE
  To evaluate the performance of a next-generation sequencing (NGS)-based targeted resequencing genetic test, OtoSeq, to identify the sequence variants in the genes causing sensorineural hearing loss (SNHL).
  
  
  METHODS
  Retrospective study.
  
  
  METHODS
  Tertiary children's hospital.
  
  
  METHODS
  A total of 8 individuals presenting with prelingual hearing loss were used in this study. The coding and flanking intronic regions of 24 well-studied SNHL genes were enriched using microdroplet polymerase chain reaction and sequenced on an Illumina HiSeq 2000 sequencer. The filtered high-quality sequence reads were mapped to reference sequence, and variants were detected using NextGENe software.
  
  
  RESULTS
  A total of 1148 sequence variants were detected in 8 samples in 24 genes. Using in-house developed NGS data analysis criteria, we classified 810 (~71%) of these variants as potential true variants that include previously detected pathogenic mutations in 5 patients. To validate our strategy, we Sanger sequenced the target regions of 5 of the 24 genes, accounting for about 29.2% of all target sequence. Our results showed >99.99% concordance between NGS and Sanger sequencing in these 5 genes, resulting in an analytical sensitivity and specificity of 100% and 99.997%, respectively. We were able to successfully detect single base substitutions, small deletions, and insertions of up to 22 nucleotides.
  
  
  CONCLUSIONS
  This study demonstrated that our NGS-based mutation screening strategy is highly sensitive and specific in detecting sequence variants in the SNHL genes. Therefore, we propose that this NGS-based targeted sequencing method would be an alternative to current technologies for identifying the multiple genetic causes of SNHL.
authors.affiliation: Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA. Siva.Theru_Arumugam@cchmc.org
authors.forename: Theru A
authors.initials: TA
authors.lastname: Sivakumaran
authors.forename: Ammar
authors.initials: A
authors.lastname: Husami
authors.forename: Diane
authors.initials: D
authors.lastname: Kissell
authors.forename: Wenying
authors.initials: W
authors.lastname: Zhang
authors.forename: Mehdi
authors.initials: M
authors.lastname: Keddache
authors.forename: Angela P
authors.initials: AP
authors.lastname: Black
authors.forename: Brad T
authors.initials: BT
authors.lastname: Tinkle
authors.forename: John H
authors.initials: JH
authors.lastname: Greinwald
authors.forename: Kejian
authors.initials: K
authors.lastname: Zhang
mesh_terms: D000293:Adolescent; D000367:Age Factors; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D005260:Female; D005500:Follow-Up Studies; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods; D006319:Hearing Loss, Sensorineural / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006776:Hospitals, Pediatric; D006801:Humans; D015994:Incidence; D007223:Infant; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D012189:Retrospective Studies; D018570:Risk Assessment; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012737:Sex Factors; D014481:United States
keywords: clinical diagnosis; genetic etiology; hearing loss; microdroplet PCR; mutation screening; next-generation sequencing