Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland.
Department of Ophthalmology, University Hospital Zurich and University of Zurich, 8091 Zurich, Switzerland.
Int J Mol Sci. 2021 Feb 3;22(4):1508. doi: 10.3390/ijms22041508.
The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel ( = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.
本研究旨在开发一种灵活、经济高效的下一代测序（NGS）遗传测试协议。设计了长达 20kb 的长距离聚合酶链反应（PCR）扩增子，以扩增整个基因组区域，用于一组（=35）遗传性视网膜疾病（IRD）相关基因座。将扩增子混合并通过 NGS 进行测序。该分析应用于 227 名被诊断为 IRD 的先证者：（A）108 名先前通过分子诊断，（B）94 名先前未进行基因检测，（C）25 名在全外显子测序（WES）后仍未确诊。该方法在队列 A 中验证的灵敏度为 100%。基于长距离 PCR 的测序分别在队列 B 和 C 的 51%和 24%的先证者中揭示了可能的致病变异。可以对 3 个拷贝数变异（CNV）的断点进行特征描述。长距离 PCR 文库 Spike-in 扩展了 WES 的覆盖范围。读相位证实了 5 名先证者的复合杂合性。所提出的测序方案提供了整个基因的深度覆盖，包括内含子和启动子区域。我们的方法可以用于（i）作为降低基因检测成本的一级检测，（ii）阐明遗传缺失病例，（iii）以核苷酸分辨率表征 CNV 的断点，（iv）通过 Spike-in 长距离 PCR 文库将 WES 数据扩展到非编码区域，以及（v）帮助候选变异的相位。
