TY - JOUR
T1 - Development of high-throughput clinical testing of RPGR ORF15 using a large inherited retinal dystrophy cohort
AU - Chiang, John P.W.
AU - Lamey, Tina M.
AU - Wang, Nicholas K.
AU - Duan, Jie
AU - Zhou, Wei
AU - McLaren, Terri L.
AU - Thompson, Jennifer A.
AU - Ruddle, Jonathan
AU - De Roach, John N.
N1 - Funding Information:
Supported by Retina Australia, the Western Australian DNA Bank, Department of Medical Technology and Physics, Sir Charles Gairdner Hospital, and the National Health & Medical Research Council (NHMRC) of Australia (#1116360). Disclosure: J.P.W. Chiang, Molecular Vision Laboratory (I, E); T.M. Lamey, None; N.K. Wang, None; J. Duan, None; W. Zhou, P; T.L. McLaren, None; J.A. Thompson, None; J. Ruddle, None; J.N. De Roach, None
Funding Information:
Supported by Retina Australia, the Western Australian DNA BankDepartment of Medical Technology and Physics, Sir Charles Gairdner Hospital, and the National Health & Medical Research Council (NHMRC) of Australia (#1116360).
Publisher Copyright:
© 2018 The Authors.
PY - 2018/9
Y1 - 2018/9
N2 - 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.
AB - 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.
KW - Genetic testing
KW - ORF15
KW - RPGR
KW - RPGR ORF15
KW - X-linked retinitis pigmentosa
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UR - http://www.scopus.com/inward/citedby.url?scp=85053075024&partnerID=8YFLogxK
U2 - 10.1167/iovs.18-24555
DO - 10.1167/iovs.18-24555
M3 - Article
C2 - 30193314
AN - SCOPUS:85053075024
SN - 0146-0404
VL - 59
SP - 4434
EP - 4440
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 11
ER -