Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic
Abstract Background Diagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the opportunity to make definitive molecular diagnoses using a single assay capable of...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-06-01
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| Series: | Journal of Translational Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12967-023-04243-y |
| _version_ | 1797806546132926464 |
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| author | Stephanie U. Greer Jacquelin Botello Donna Hongo Brynn Levy Premal Shah Matthew Rabinowitz Danny E. Miller Kate Im Akash Kumar |
| author_facet | Stephanie U. Greer Jacquelin Botello Donna Hongo Brynn Levy Premal Shah Matthew Rabinowitz Danny E. Miller Kate Im Akash Kumar |
| author_sort | Stephanie U. Greer |
| collection | DOAJ |
| description | Abstract Background Diagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the opportunity to make definitive molecular diagnoses using a single assay capable of detecting variants, characterizing methylation patterns, resolving complex rearrangements, and assigning findings to long-range haplotypes. Here, we demonstrate the clinical utility of Nanopore long-read sequencing by validating a confirmatory test for copy number variants (CNVs) in neurodevelopmental disorders and illustrate the broader applications of this platform to assess genomic features with significant clinical implications. Methods We used adaptive sampling on the Oxford Nanopore platform to sequence 25 genomic DNA samples and 5 blood samples collected from patients with known or false-positive copy number changes originally detected using short-read sequencing. Across the 30 samples (a total of 50 with replicates), we assayed 35 known unique CNVs (a total of 55 with replicates) and one false-positive CNV, ranging in size from 40 kb to 155 Mb, and assessed the presence or absence of suspected CNVs using normalized read depth. Results Across 50 samples (including replicates) sequenced on individual MinION flow cells, we achieved an average on-target mean depth of 9.5X and an average on-target read length of 4805 bp. Using a custom read depth-based analysis, we successfully confirmed the presence of all 55 known CNVs (including replicates) and the absence of one false-positive CNV. Using the same CNV-targeted data, we compared genotypes of single nucleotide variant loci to verify that no sample mix-ups occurred between assays. For one case, we also used methylation detection and phasing to investigate the parental origin of a 15q11.2-q13 duplication with implications for clinical prognosis. Conclusions We present an assay that efficiently targets genomic regions to confirm clinically relevant CNVs with a concordance rate of 100%. Furthermore, we demonstrate how integration of genotype, methylation, and phasing data from the Nanopore sequencing platform can potentially simplify and shorten the diagnostic odyssey. |
| first_indexed | 2024-03-13T06:08:54Z |
| format | Article |
| id | doaj.art-e6f7c74520a941a0857be3d702c02e98 |
| institution | Directory Open Access Journal |
| issn | 1479-5876 |
| language | English |
| last_indexed | 2024-03-13T06:08:54Z |
| publishDate | 2023-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Translational Medicine |
| spelling | doaj.art-e6f7c74520a941a0857be3d702c02e982023-06-11T11:24:01ZengBMCJournal of Translational Medicine1479-58762023-06-0121111110.1186/s12967-023-04243-yImplementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinicStephanie U. Greer0Jacquelin Botello1Donna Hongo2Brynn Levy3Premal Shah4Matthew Rabinowitz5Danny E. Miller6Kate Im7Akash Kumar8MyOme Inc.MyOme Inc.MyOme Inc.MyOme Inc.MyOme Inc.MyOme Inc.Department of Pediatrics, Department of Laboratory Medicine and Pathology, University of WashingtonMyOme Inc.MyOme Inc.Abstract Background Diagnosis of rare genetic diseases can be a long, expensive and complex process, involving an array of tests in the hope of obtaining an actionable result. Long-read sequencing platforms offer the opportunity to make definitive molecular diagnoses using a single assay capable of detecting variants, characterizing methylation patterns, resolving complex rearrangements, and assigning findings to long-range haplotypes. Here, we demonstrate the clinical utility of Nanopore long-read sequencing by validating a confirmatory test for copy number variants (CNVs) in neurodevelopmental disorders and illustrate the broader applications of this platform to assess genomic features with significant clinical implications. Methods We used adaptive sampling on the Oxford Nanopore platform to sequence 25 genomic DNA samples and 5 blood samples collected from patients with known or false-positive copy number changes originally detected using short-read sequencing. Across the 30 samples (a total of 50 with replicates), we assayed 35 known unique CNVs (a total of 55 with replicates) and one false-positive CNV, ranging in size from 40 kb to 155 Mb, and assessed the presence or absence of suspected CNVs using normalized read depth. Results Across 50 samples (including replicates) sequenced on individual MinION flow cells, we achieved an average on-target mean depth of 9.5X and an average on-target read length of 4805 bp. Using a custom read depth-based analysis, we successfully confirmed the presence of all 55 known CNVs (including replicates) and the absence of one false-positive CNV. Using the same CNV-targeted data, we compared genotypes of single nucleotide variant loci to verify that no sample mix-ups occurred between assays. For one case, we also used methylation detection and phasing to investigate the parental origin of a 15q11.2-q13 duplication with implications for clinical prognosis. Conclusions We present an assay that efficiently targets genomic regions to confirm clinically relevant CNVs with a concordance rate of 100%. Furthermore, we demonstrate how integration of genotype, methylation, and phasing data from the Nanopore sequencing platform can potentially simplify and shorten the diagnostic odyssey.https://doi.org/10.1186/s12967-023-04243-yLong-read sequencingOxford Nanopore TechnologiesAdaptive samplingCopy number variantsNeurodevelopmental disordersClinical testing |
| spellingShingle | Stephanie U. Greer Jacquelin Botello Donna Hongo Brynn Levy Premal Shah Matthew Rabinowitz Danny E. Miller Kate Im Akash Kumar Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic Journal of Translational Medicine Long-read sequencing Oxford Nanopore Technologies Adaptive sampling Copy number variants Neurodevelopmental disorders Clinical testing |
| title | Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic |
| title_full | Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic |
| title_fullStr | Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic |
| title_full_unstemmed | Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic |
| title_short | Implementation of Nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic |
| title_sort | implementation of nanopore sequencing as a pragmatic workflow for copy number variant confirmation in the clinic |
| topic | Long-read sequencing Oxford Nanopore Technologies Adaptive sampling Copy number variants Neurodevelopmental disorders Clinical testing |
| url | https://doi.org/10.1186/s12967-023-04243-y |
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