Nanopore sequencing-based episignature detection

Background: A subset of developmental disorders (DD) is characterized by disease-specific genome-wide methylation changes. These episignatures inform about underlying pathogenic mechanisms and can be used to assess the pathogenicity of genomic variants as well as confirm clinical diagnoses. Currently, episignature detection requires the use of indirect methylation profiling microarrays. We hypothesized that long-read whole genome sequencing would not only enable the detection of single nucleotide variants and structural variants but also episignatures. Methods: Genome-wide nanopore sequencing was performed in forty controls and twenty patients with confirmed or suspected episignature-associated DD, representing thirteen distinct diseases. Following variant and methylome calling, hierarchical clustering and dimensional reduction were used to determine the compatibility with microarray-based episignatures. Subsequently, we developed a support vector machine for each DD. Results: Nanopore sequencing based methylome patterns were concordant with microarray-based episignatures. Our classifier identified episignatures in 17/20 disease samples and none in the control samples. The remaining three patient samples were classified as controls by both our classifier and a commercial microarray assay. In addition, we identified all underlying pathogenic single nucleotide and structural variants and showed haplotype-aware skewed X-inactivation evaluation directs clinical interpretation. Conclusion: This proof-of-concept study demonstrates nanopore sequencing enables concurrent haplotyped genomic and epigenomic analyses. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work has been made possible by FWO-TBM grant T-003819N, FWO grant G0A2622N and KU Leuven grants, C1-C14/18/092 and C14/22/125 to JRV. BH was supported by a Collaborative Doctoral Partnership Agreement of the Joint Research Center F.7/KU LEUVEN B&G 35332. JB is supported by a senior clinical investigator fellowship of the FWO Flanders. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Ethical Committee Research Universitaire Ziekenhuizen KU Leuven/Katholieke Universiteit Leuven (S65304 S65991), and informed consent was obtained from all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data produced are partially available as supplementary with the manuscript and on GitHub.