Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients
Abstract Inherited retinal degenerations (IRDs) are a heterogeneous group of predominantly monogenic disorders with over 300 causative genes identified. Short-read exome sequencing is commonly used to genotypically diagnose patients with clinical features of IRDs, however, in up to 30% of patients w...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-05-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-35791-4 |
| _version_ | 1797817970392563712 |
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| author | Kenji Nakamichi Russell N. Van Gelder Jennifer R. Chao Debarshi Mustafi |
| author_facet | Kenji Nakamichi Russell N. Van Gelder Jennifer R. Chao Debarshi Mustafi |
| author_sort | Kenji Nakamichi |
| collection | DOAJ |
| description | Abstract Inherited retinal degenerations (IRDs) are a heterogeneous group of predominantly monogenic disorders with over 300 causative genes identified. Short-read exome sequencing is commonly used to genotypically diagnose patients with clinical features of IRDs, however, in up to 30% of patients with autosomal recessive IRDs, one or no disease-causing variants are identified. Furthermore, chromosomal maps cannot be reconstructed for allelic variant discovery with short-reads. Long-read genome sequencing can provide complete coverage of disease loci and a targeted approach can focus sequencing bandwidth to a genomic region of interest to provide increased depth and haplotype reconstruction to uncover cases of missing heritability. We demonstrate that targeted adaptive long-read sequencing on the Oxford Nanopore Technologies (ONT) platform of the USH2A gene from three probands in a family with the most common cause of the syndromic IRD, Usher Syndrome, resulted in greater than 12-fold target gene sequencing enrichment on average. This focused depth of sequencing allowed for haplotype reconstruction and phased variant identification. We further show that variants obtained from the haplotype-aware genotyping pipeline can be heuristically ranked to focus on potential pathogenic candidates without a priori knowledge of the disease-causing variants. Moreover, consideration of the variants unique to targeted long-read sequencing that are not covered by short-read technology demonstrated higher precision and F1 scores for variant discovery by long-read sequencing. This work establishes that targeted adaptive long-read sequencing can generate targeted, chromosome-phased data sets for identification of coding and non-coding disease-causing alleles in IRDs and can be applicable to other Mendelian diseases. |
| first_indexed | 2024-03-13T09:01:22Z |
| format | Article |
| id | doaj.art-2d58e0200ccd4dd5a2cd7df9cc9d1f13 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-13T09:01:22Z |
| publishDate | 2023-05-01 |
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| series | Scientific Reports |
| spelling | doaj.art-2d58e0200ccd4dd5a2cd7df9cc9d1f132023-05-28T11:16:43ZengNature PortfolioScientific Reports2045-23222023-05-011311910.1038/s41598-023-35791-4Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patientsKenji Nakamichi0Russell N. Van Gelder1Jennifer R. Chao2Debarshi Mustafi3Department of Ophthalmology, Roger and Karalis Johnson Retina Center, University of WashingtonDepartment of Ophthalmology, Roger and Karalis Johnson Retina Center, University of WashingtonDepartment of Ophthalmology, Roger and Karalis Johnson Retina Center, University of WashingtonDepartment of Ophthalmology, Roger and Karalis Johnson Retina Center, University of WashingtonAbstract Inherited retinal degenerations (IRDs) are a heterogeneous group of predominantly monogenic disorders with over 300 causative genes identified. Short-read exome sequencing is commonly used to genotypically diagnose patients with clinical features of IRDs, however, in up to 30% of patients with autosomal recessive IRDs, one or no disease-causing variants are identified. Furthermore, chromosomal maps cannot be reconstructed for allelic variant discovery with short-reads. Long-read genome sequencing can provide complete coverage of disease loci and a targeted approach can focus sequencing bandwidth to a genomic region of interest to provide increased depth and haplotype reconstruction to uncover cases of missing heritability. We demonstrate that targeted adaptive long-read sequencing on the Oxford Nanopore Technologies (ONT) platform of the USH2A gene from three probands in a family with the most common cause of the syndromic IRD, Usher Syndrome, resulted in greater than 12-fold target gene sequencing enrichment on average. This focused depth of sequencing allowed for haplotype reconstruction and phased variant identification. We further show that variants obtained from the haplotype-aware genotyping pipeline can be heuristically ranked to focus on potential pathogenic candidates without a priori knowledge of the disease-causing variants. Moreover, consideration of the variants unique to targeted long-read sequencing that are not covered by short-read technology demonstrated higher precision and F1 scores for variant discovery by long-read sequencing. This work establishes that targeted adaptive long-read sequencing can generate targeted, chromosome-phased data sets for identification of coding and non-coding disease-causing alleles in IRDs and can be applicable to other Mendelian diseases.https://doi.org/10.1038/s41598-023-35791-4 |
| spellingShingle | Kenji Nakamichi Russell N. Van Gelder Jennifer R. Chao Debarshi Mustafi Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients Scientific Reports |
| title | Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients |
| title_full | Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients |
| title_fullStr | Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients |
| title_full_unstemmed | Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients |
| title_short | Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients |
| title_sort | targeted adaptive long read sequencing for discovery of complex phased variants in inherited retinal disease patients |
| url | https://doi.org/10.1038/s41598-023-35791-4 |
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