Performance Evaluation of the Next-Generation Sequencing Approach for Molecular Diagnosis of Hereditary Hearing Loss.

Sivakumaran TA, Husami A, Kissell D, Zhang W, Keddache M, Black AP, Tinkle BT, Greinwald JH Jr, Zhang K.

Source

Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

Abstract

ObjectiveTo 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).Study DesignRetrospective study.SettingTertiary children's hospital.Subjects and MethodsA 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.ResultsA 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.ConclusionThis 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.