Institute of Medical Molecular Genetic, University of Zurich, 8952 Schlieren, Switzerland.
Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8006 Zurich, Switzerland.
Genes (Basel). 2020 Jul 15;11(7):800. doi: 10.3390/genes11070800.
exon ORF15 variants are one of the most frequent causes for inherited retinal disorders (IRDs), in particular retinitis pigmentosa. The low sequence complexity of this mutation hotspot makes it prone to indels and challenging for sequence data analysis. Whole-exome sequencing generally fails to provide adequate coverage in this region. Therefore, complementary methods are needed to avoid false positives as well as negative results. In this study, next-generation sequencing (NGS) was used to sequence long-range PCR amplicons for an IRD cohort of African ancestry. By developing a novel secondary analysis pipeline based on de novo assembly, we were able to avoid the miscalling of variants generated by standard NGS analysis tools. We identified pathogenic variants in 11 patients (13% of the cohort), two of which have not been reported previously. We provide a novel and alternative end-to-end secondary analysis pipeline for targeted NGS of ORF15 that is less prone to false positive and negative variant calls.
外显子 ORF15 变异是遗传性视网膜疾病（IRDs），特别是色素性视网膜炎的最常见原因之一。该突变热点的低序列复杂性使其容易发生插入缺失，这对序列数据分析来说是一个挑战。全外显子组测序通常无法为该区域提供足够的覆盖度。因此，需要互补的方法来避免假阳性和假阴性结果。在这项研究中，我们使用下一代测序（NGS）对非洲裔遗传性视网膜疾病队列的长程 PCR 扩增子进行测序。通过开发基于从头组装的新型二级分析管道，我们能够避免标准 NGS 分析工具生成的变异误报。我们在 11 名患者（队列的 13%）中鉴定出了致病性变异，其中有两个以前没有报道过。我们为 ORF15 的靶向 NGS 提供了一种新颖且替代的端到端二级分析管道，该方法不太容易出现假阳性和假阴性变异呼叫。
