Purpose: To develop a flexible, cost-efficient next-generation sequencing (NGS) protocol for genetic testing. Methods: Long-range polymerase chain reaction (LR PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n=35) of loci associated with retinal diseases (RDs). Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with RD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, (C) 25 undiagnosed after exome sequencing (ES). Results: The method was validated with 100% sensitivity on cohort A. LR PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 CNVs could be characterized. LR PCR libraries spike-in extended coverage data of ES. Read phasing confirmed compound heterozygosity in 5 probands. Conclusion: 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 the nucleotide level, iv) to extend WES data to non-coding regions by spiking-in LR libraries, and v) to help with phasing of candidate variants.

中文翻译：

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基于远程PCR的NGS在孟德尔视网膜疾病诊断中的应用

目的：开发一种灵活，经济高效的下一代测序（NGS）方案用于基因检测。方法：设计最大大小为20 kb的远程聚合酶链反应（LR PCR）扩增子，以扩增与视网膜疾病（RDs）相关的一组基因座（n = 35）的整个基因组区域。汇集扩增子并通过NGS测序。该分析适用于227位经RD诊断的先证者：（A）108位先前被分子诊断的患者，（B）94位未经先前的基因测试的患者，（C）25位在外显子组测序（ES）后未被诊断的患者。结果：该方法对队列A的敏感性为100％。基于LR PCR的测序表明，队列B和队列C的先证者分别有51％和24％可能存在致病性变异。可以表征3个CNV的断点。LR PCR文库增加了ES的扩展覆盖率数据。读阶段确定了5个先证者的化合物杂合性。结论：拟议的测序方案提供了包括内含子和启动子区域在内的整个基因的深入覆盖。我们的方法可用于（i）作为第一级测定以降低基因检测成本，ii）阐明遗漏的遗传情况，iii）在核苷酸水平表征CNV的断点，iv）将WES数据扩展到非编码通过插入LR库来确定区域，以及v）有助于逐步确定候选变体。