Identification of false-negative mutations missed by next-generation sequencing in retinitis pigmentosa patients: a complementary approach to clinical genetic diagnostic testing |
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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 |
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Abstract: | PurposeRetinitis 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.MethodsTo 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.ResultsCoverage-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.ConclusionIn 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.Genet Med 17 4, 307–311. |
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