Flexible and rapid validation of structural variation using adaptive sampling

Flexible and rapid validation of structural variation using adaptive sampling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Flexible and rapid validation of structural variation using adaptive sampling Lars Feuk, Aida Paivandy, Felix Lenner, Jesper Eisfeldt, Tord Jonson, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7307341/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2026 Read the published version in European Journal of Human Genetics → Version 1 posted 9 You are reading this latest preprint version Abstract Identification of genomic rearrangement by microarrays or short-read sequencing frequently lacks information about the exact architecture and breakpoints of variants due to technical limitations. Independent verification of complex structural variants (SVs) is often performed using custom targeted assays, making confirmation of clinically relevant findings time consuming and laborious. In this study we evaluate Oxford Nanopore long-read adaptive sampling for flexible and rapid confirmation and characterization of complex genomic rearrangements and structural variants. Adaptive sampling is an in silico target enrichment, where continued sequencing or ejection of a fragment is based on whether it matches a defined reference sequence. Using adaptive sampling, we targeted 10 regions of different structural variant types, including deletions, translocations and complex rearrangements. Each sample was analyzed on a MinION flow-cell, and sequencing resulted in between 14.1-18.3 Gb of data per flow cell, with mean autosomal on-target coverage of 28.4x and off-target read depth coverage of 5.3x. We were able to confirm all rearrangements by read depth and identification of breakpoint junction spanning reads. We also show that background reads can be used to detect structural variants in non-targeted regions of the genome. Our results show that adaptive sampling represents a robust, flexible and rapid strategy for confirmation and characterization of clinically relevant complex genomic rearrangements in clinical samples. By providing sequence information, read depth and methylation data, nanopore adaptive sampling has advantages over other assays for variant confirmation used in diagnostic laboratories today. Health sciences/Medical research/Genetics research Health sciences/Medical research/Translational research Long-read sequencing adaptive sampling structural variation chromosomal rearrangement Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Additional Declarations There is no duality of interest Supplementary Files Lennersupplementary.pdf Supplemental material Cite Share Download PDF Status: Published Journal Publication published 23 Feb, 2026 Read the published version in European Journal of Human Genetics → Version 1 posted Editorial decision: revise 25 Nov, 2025 Review # 2 received at journal 21 Nov, 2025 Reviewer # 2 agreed at journal 22 Oct, 2025 Review # 1 received at journal 01 Oct, 2025 Reviewer # 1 agreed at journal 15 Sep, 2025 Reviewers invited by journal 04 Sep, 2025 Submission checks completed at journal 22 Aug, 2025 Editor assigned by journal 06 Aug, 2025 First submitted to journal 06 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7307341","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":510448116,"identity":"d405014a-d0e8-4a70-a09e-40c4b0192c64","order_by":0,"name":"Lars Feuk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIie3RsQqDMBCA4RMhLtKsKeI7CEIXpc+SUIhLB8eOFsGp0FURfBbFwan0FQqCSxfBpVsbxa5Bt0LzDzfl4w4CoFL9YJtp+gDEOEfLCJomF8SsVhNCFx6GjFvbA/VZlrXxM4SaRbgu5cQMdgQoZ7nFEi8dCeHyfQg4Au1Vs0IQ15yI6cgJ7nRx2JsV22om+N7LCeEgDitZTrS4nQgcpUKQDhFKD252YYmeOoGbEC4/DGOu9z3d22nTtEN48uwrrh/yNWN03miB8/2phenDmtcqlUr1P30AQxg/ao+JTMcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2355-2919","institution":"Uppsala University","correspondingAuthor":true,"prefix":"","firstName":"Lars","middleName":"","lastName":"Feuk","suffix":""},{"id":510448117,"identity":"699fbed4-4932-41ce-be1e-3a1ef9d3b854","order_by":1,"name":"Aida Paivandy","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Aida","middleName":"","lastName":"Paivandy","suffix":""},{"id":510448118,"identity":"b114ab5e-29b4-42af-8582-7e6c4f6e504a","order_by":2,"name":"Felix Lenner","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Felix","middleName":"","lastName":"Lenner","suffix":""},{"id":510448119,"identity":"4abd1f6a-3f69-44a6-82ab-27c9bb9404cd","order_by":3,"name":"Jesper Eisfeldt","email":"","orcid":"","institution":"Karolinska institutet","correspondingAuthor":false,"prefix":"","firstName":"Jesper","middleName":"","lastName":"Eisfeldt","suffix":""},{"id":510448120,"identity":"1cd8079c-0b1c-423d-a82b-39e387659222","order_by":4,"name":"Tord Jonson","email":"","orcid":"","institution":"Skåne University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tord","middleName":"","lastName":"Jonson","suffix":""},{"id":510448121,"identity":"4af1f610-4567-469b-8a8f-ff000664c838","order_by":5,"name":"Hans Ehrencrona","email":"","orcid":"https://orcid.org/0000-0002-5589-3622","institution":"Lund University","correspondingAuthor":false,"prefix":"","firstName":"Hans","middleName":"","lastName":"Ehrencrona","suffix":""},{"id":510448122,"identity":"47b35a3f-093c-4302-85ce-d0c4e8494cf1","order_by":6,"name":"Anna Lindstrand","email":"","orcid":"https://orcid.org/0000-0003-0806-5602","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Lindstrand","suffix":""},{"id":510448123,"identity":"2b6ad1af-9569-47e6-9e0f-329dbdc38811","order_by":7,"name":"Stephen Scherer","email":"","orcid":"https://orcid.org/0000-0002-8326-1999","institution":"Hospital for Sick Children","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Scherer","suffix":""}],"badges":[],"createdAt":"2025-08-06 08:16:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7307341/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7307341/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41431-026-02039-4","type":"published","date":"2026-02-23T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91052720,"identity":"4dcb32e0-8586-4a40-a815-7cef68c8ba98","added_by":"auto","created_at":"2025-09-11 07:22:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":240495,"visible":true,"origin":"","legend":"\u003cp\u003eA) Bases that passed quality filters for each sample. B) Depth of target regions from GRCh38 for the samples sequenced on MinION. Regions A - E included in all samples, J in samples S5 - S8, L in samples S6 - 8, and M, N and O in samples S7 and S8. C) Target region depth across four multiplexed samples on a PromethION flow cell. The same regions were targeted in all four samples.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7307341/v1/0988ed89eeb682d47b39835b.png"},{"id":91052725,"identity":"920085f2-1013-48a1-aa26-e0dbe72873a2","added_by":"auto","created_at":"2025-09-11 07:22:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":715481,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of a translocation on chromosome 6, by using sequence from the CHM13-T2T assembly. A. Overview of breakpoint region in hg19 and T2T-CHM13 assemblies. Nearby gene MANEA shown for reference. The breakpoint was previously mapped by FISH to a gap in the hg19 assembly using a BAC probe (RP11-134M2) anchored on the telomeric side of the gap region. The gap region, estimated to 150kb in hg19, corresponds to a \u0026gt;360kb region in T2T-CHM13. Targeting this sequence with adaptive sampling led to identification of the exact chr6 breakpoint. B. Circos plot showing the (t6;22)(q16.1;p13) translocation. C. IGV image showing reads spanning the translocation breakpoints, which can be mapped at nucleotide resolution on chr6, while the breakpoint on the short arm of chr22 maps to a region of identical repeats where no exact breakpoint could be established.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7307341/v1/d7c1140c07efbab3711382fd.png"},{"id":91052721,"identity":"fe69b974-66ab-4d28-a436-1cdca72d2d95","added_by":"auto","created_at":"2025-09-11 07:22:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":278015,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of copy number changes using read depth (log2 ratio) across targeted regions. A. Deletion on chromosome 6 (target region J). B. Complex rearrangement with DUP-NML-DUP (target region A). Dashed lines correspond to the mean copy number within breakpoints regions called with Sniffles2 or identified by IGV. C. Top panel shows read depth across a complex rearrangement on chr16. Copy number can be inferred from the log2 read depth ratio plotted across the region. Colored lines correspond to the mean copy number within breakpoints regions. The lower panel shows the deduced structure of the complex rearrangement, using a combination of read depth and breakpoint reads (breakpoints in IGV shown in Supplementary Figures).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7307341/v1/71e22961e5946161a319f7b9.png"},{"id":91052722,"identity":"87e49ec9-c515-43ca-ae42-dcc0fb47470b","added_by":"auto","created_at":"2025-09-11 07:22:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222943,"visible":true,"origin":"","legend":"\u003cp\u003eA) A copy number loss and gain on chromosome 4 and 9 respectively, called using the rejected reads from adaptive sampling on the P2S. Target regions, common CNV regions and centromeres/telomeres were removed from the analysis. B) Plots of off-target read depth profiles across chromosome 16 from four samples multiplexed on a PromethION flow cell, showing a 1 Mb deletion on chr16 in barcode 4, which can clearly be distinguished as a copy number aberration using read depth data.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7307341/v1/3cc4b129f68148d2c68a265c.png"},{"id":103301444,"identity":"a71941e7-1721-4a94-9027-185703b247e1","added_by":"auto","created_at":"2026-02-24 08:12:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1043904,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Lenneretal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7307341/v1_covered_6f419bc8-ede0-4b13-b867-60c0d3b04fb5.pdf"},{"id":91052726,"identity":"9b4fca57-80eb-48ed-b066-611daceb04f1","added_by":"auto","created_at":"2025-09-11 07:22:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2287320,"visible":true,"origin":"","legend":"Supplemental material","description":"","filename":"Lennersupplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7307341/v1/28c0da4a463e0656b940648e.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Flexible and rapid validation of structural variation using adaptive sampling","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Long-read sequencing, adaptive sampling, structural variation, chromosomal rearrangement","lastPublishedDoi":"10.21203/rs.3.rs-7307341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7307341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Identification of genomic rearrangement by microarrays or short-read sequencing frequently lacks information about the exact architecture and breakpoints of variants due to technical limitations. Independent verification of complex structural variants (SVs) is often performed using custom targeted assays, making confirmation of clinically relevant findings time consuming and laborious. In this study we evaluate Oxford Nanopore long-read adaptive sampling for flexible and rapid confirmation and characterization of complex genomic rearrangements and structural variants. Adaptive sampling is an in silico target enrichment, where continued sequencing or ejection of a fragment is based on whether it matches a defined reference sequence. Using adaptive sampling, we targeted 10 regions of different structural variant types, including deletions, translocations and complex rearrangements. Each sample was analyzed on a MinION flow-cell, and sequencing resulted in between 14.1-18.3 Gb of data per flow cell, with mean autosomal on-target coverage of 28.4x and off-target read depth coverage of 5.3x. We were able to confirm all rearrangements by read depth and identification of breakpoint junction spanning reads. We also show that background reads can be used to detect structural variants in non-targeted regions of the genome. Our results show that adaptive sampling represents a robust, flexible and rapid strategy for confirmation and characterization of clinically relevant complex genomic rearrangements in clinical samples. 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