lunes, 8 de abril de 2013

Performance Evaluation of the Nex... [Otolaryngol Head Neck Surg. 2013] - PubMed - NCBI

Performance Evaluation of the Nex... [Otolaryngol Head Neck Surg. 2013] - PubMed - NCBI

2013 Mar 22. [Epub ahead of print]

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


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


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.

[PubMed - as supplied by publisher]

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