利用大型遗传性视网膜疾病队列开发高通量 RPGR ORF15 临床检测方法。
Development of High-Throughput Clinical Testing of RPGR ORF15 Using a Large Inherited Retinal Dystrophy Cohort.
机构信息
Molecular Vision Laboratory, Hillsboro, Oregon, United States.
Australian Inherited Retinal Disease Registry and DNA Bank, Department of Medical Technology and Physics, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia.
出版信息
Invest Ophthalmol Vis Sci. 2018 Sep 4;59(11):4434-4440. doi: 10.1167/iovs.18-24555.
PURPOSE
Mutations in the ORF15 region of RPGR account for approximately half of all X-linked retinitis pigmentosa cases. However, a robust high-throughput method for the detection of ORF15 mutations has yet to be validated. We set out to develop the first clinically validated next-generation sequencing (NGS) method for the detection of mutations in this difficult-to-sequence region, including test accuracy and coverage data.
METHODS
As part of a blind-test, 145 research samples, previously tested by Sanger sequencing, and 81 clinical samples were evaluated using NGS of long-range PCR products fragmented with Illumina's Nextera library preparation kit (method 1), or with Centrillion's OneTube technology, supplemented with duplication analysis using an ORF15-specific in-silico array (method 2). DNA fragments were analyzed using Agilent's DNA 1000 assay, and sequencing was done on Illumina's MiSeq 2×150 or HiSeq2500 2×100. NextGENe by SoftGenetics was used for data analysis and variant calling.
RESULTS
The Nextera library preparation method produced 24 cases of discordance due to (in order of decreasing occurrence) false-negatives, incorrectly called variants, and a false-positive. Subsequent use of a new, OneTube NGS library preparation method, supplemented with duplication analyses, resolved discordance between Sanger and NGS data in all cases. This improvement in variant detection accuracy was largely attributed to improvement in random fragmentation offered by the enzymatic OneTube method, resulting in more complete coverage of the highly repetitive ORF15 region. Minimum coverage was roughly 320 reads for Nextera and 6800 reads for OneTube (normalized for total read counts).
CONCLUSIONS
This paper documents the first clinically validated NGS method for reliable, high-throughput sequencing of RPGR ORF15. Sensitivity and specificity of the new method were 100%, with the caveat of unclear zygosity calling for one large duplication case. These findings demonstrate a reliable and practical implementation for NGS-based diagnosis of RPGR ORF15 mutations. They also provide the foundation for targeted, high-coverage sequencing of any other repetitive regions within the genome.
目的
ORF15 区域的突变约占所有 X 连锁型视色素变性病例的一半。然而，目前还没有经过验证的高通量检测 ORF15 突变的方法。我们着手开发首个经临床验证的用于检测该测序困难区域突变的下一代测序（NGS）方法，包括检测准确性和覆盖范围数据。
方法
作为盲测的一部分，对 145 个研究样本（先前通过 Sanger 测序进行了测试）和 81 个临床样本进行了评估，使用 Illumina 的 Nextera 文库制备试剂盒（方法 1）对长片段 PCR 产物进行 NGS 或 Centrillion 的 OneTube 技术进行 NGS，补充使用 ORF15 专用的原位阵列进行重复分析（方法 2）。使用 Agilent 的 DNA 1000 分析试剂盒对 DNA 片段进行分析，并在 Illumina 的 MiSeq 2×150 或 HiSeq2500 2×100 上进行测序。使用 SoftGenetics 的 NextGENe 进行数据分析和变异调用。
结果
Nextera 文库制备方法产生了 24 个不一致的案例，原因依次为（按发生频率递减的顺序排列）假阴性、错误调用的变异体和假阳性。随后使用一种新的 OneTube NGS 文库制备方法，并补充重复分析，解决了所有情况下 Sanger 和 NGS 数据之间的不一致问题。这种提高变异检测准确性的方法主要归因于酶切 OneTube 方法提供的随机片段化改善，从而使高度重复的 ORF15 区域的覆盖度更加完整。Nextera 的最小覆盖度约为 320 个读取，OneTube 的最小覆盖度约为 6800 个读取（按总读取数归一化）。
结论
本文记录了首个经临床验证的可靠、高通量测序 RPGR ORF15 的 NGS 方法。新方法的灵敏度和特异性均为 100%，但对于一个大的重复案例，其杂合性的检测结果尚不清楚。这些发现为基于 NGS 的 RPGR ORF15 突变诊断提供了可靠且实用的方法。它们还为靶向、高覆盖度测序基因组中的任何其他重复区域奠定了基础。
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