Abstract
Objective: Recently, next-generation sequencing (NGS) technology has emerged as a possible revolutionary force changing the paradigm of molecular diagnosis by offering high-throughput sequencing. In the present study, we have evaluated the performance of NGS to identify the sequence variants in a panel of 24 genes associated with hereditary hearing loss.
Method: The coding and flanking intronic regions covering 177 kb target sequence were enriched using microdroplet PCR from 8 samples and sequenced in a single lane on Illumina HiSeq 2000 instrument. The quality filtered sequence reads were mapped to the human reference sequence. The sequence variants were evaluated using novel pathogenecity filter.
Results: Of the 1148 variants detected in all 8 samples, 810 (71%) were classified as true variants using our filtering criteria. To validate our strategy, we Sanger sequenced regions of 4/24 genes, accounting for about 25% of all target sequences. Of the 373 variants detected in these 4 genes, we classified 265 variants as potentially true using our filtering criteria. We were able to detect 259 variants found on sequencing, including deletions/insertions up to 22 bp. Our NGS-based mutation screening strategy showed 99.99% concordance between NGS and Sanger sequencing, resulting in the analytical sensitivity and specificity of 100% and 99.99%, respectively.
Conclusion: This study demonstrated that our NGS-based mutation screening strategy is highly sensitive in detecting sequence variants in the selected hearing loss genes. Therefore, this approach would be a good alternative to current technologies for identifying the multiple genetic causes of hearing loss.
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