Development of high-throughput clinical testing of RPGR ORF15 using a large inherited retinal dystrophy cohort

Pei-Wen Chiang, Tina M. Lamey, Nicholas K. Wang, Jie Duan, Wei Zhou, Terri L. McLaren, Jennifer A. Thompson, Jonathan Ruddle, John N. De Roach

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4434-4440
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number11
DOIs
StatePublished - Sep 1 2018
Externally publishedYes

Fingerprint

Retinal Dystrophies
Libraries
Mutation
Nucleic Acid Repetitive Sequences
Retinitis Pigmentosa
DNA
Computer Simulation
Genome
Technology
Sensitivity and Specificity

Keywords

  • Genetic testing
  • ORF15
  • RPGR
  • RPGR ORF15
  • X-linked retinitis pigmentosa

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Development of high-throughput clinical testing of RPGR ORF15 using a large inherited retinal dystrophy cohort. / Chiang, Pei-Wen; Lamey, Tina M.; Wang, Nicholas K.; Duan, Jie; Zhou, Wei; McLaren, Terri L.; Thompson, Jennifer A.; Ruddle, Jonathan; De Roach, John N.

In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 11, 01.09.2018, p. 4434-4440.

Research output: Contribution to journalArticle

Chiang, P-W, Lamey, TM, Wang, NK, Duan, J, Zhou, W, McLaren, TL, Thompson, JA, Ruddle, J & De Roach, JN 2018, 'Development of high-throughput clinical testing of RPGR ORF15 using a large inherited retinal dystrophy cohort', Investigative Ophthalmology and Visual Science, vol. 59, no. 11, pp. 4434-4440. https://doi.org/10.1167/iovs.18-24555
Chiang, Pei-Wen ; Lamey, Tina M. ; Wang, Nicholas K. ; Duan, Jie ; Zhou, Wei ; McLaren, Terri L. ; Thompson, Jennifer A. ; Ruddle, Jonathan ; De Roach, John N. / Development of high-throughput clinical testing of RPGR ORF15 using a large inherited retinal dystrophy cohort. In: Investigative Ophthalmology and Visual Science. 2018 ; Vol. 59, No. 11. pp. 4434-4440.
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abstract = "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.",
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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.

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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.

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