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John (P-W) Chiang, Tina, M Lamey, Jie Duan, Nicholas Wang, Terri L McLaren, Jennifer A Thompson, Jonathan Ruddle, John N De Roach; High Throughput Clinical Testing of RPGR ORF15 Mutations in Patients with Inherited Retinal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2330. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the ORF15 region of RPGR account for around half of all X-linked retinitis pigmentosa (RP) cases and human RPGR gene therapy trials were recently launched, however a robust and reliable, high throughput method for the detection of ORF15 mutations is yet to be validated. Our aim, therefore, was to develop the first clinically validated next-generation sequencing (NGS) method for the detection of mutations in this region. Here, we report test accuracy and coverage data of the newly developed method. With this validated method, a better characterization of mutation in RPGR will be accomplished.
As part of a blind-test, patients previously tested by Sanger sequencing (N=121) were evaluated using NGS on long-range PCR products fragmented with Illumina’s Nextera library preparation kit initially and Centrillion’s OneTube technology as the final validated method. DNA fragments were analyzed using Agilent’s DNA 1000 assay and sequencing was done on Illumina’s MiSeq 2x150. Data analysis and variant calling were done using NextGENe by SoftGenetics.
Using the Nextera library preparation method, ten false negatives, one false positive, and seven incorrectly called mutations were found. Subsequent use of a new, OneTube NGS library preparation method resolved these incorrect calls, and all mutations in this subset (N=28) were correctly identified. Further analysis revealed the OneTube method offers better random fragmentation in the highly repetitive ORF15 region; resulting in better coverage of the region (~320 reads for Nextera while OneTube maintained a minimum coverage of ~6,000).
Here, we developed the first clinically validated robust, NGS method for the reliable, high throughput sequencing of RPGR ORF15. Importantly, our new method is highly accurate for detecting mutations within the hard-to-sequence, highly repetitive, region. Both sensitivity and specificity of the new method is 100%, with the caveat of uncertain zygosity in two cases. These findings demonstrate comprehensive, reliable, and practical implementation of NGS-based diagnosis of RPGR ORF15 mutations and provide the groundwork as the tools for targeted, high-coverage sequencing of any specific region with the genome.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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