Both long-read genome sequencing (lrGS) and the recently published Telomere to Telomere (T2T) reference genome provide increased coverage and resolution across repetitive regions promising heightened structural variant detection and improved mapping. Inversions (INV), intrachromosomal segments which are rotated 180 degrees and inserted back into the same chromosome, are a class of structural variants particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage sensitive genes in cis. Here we remapped the genome data from six individuals carrying unsolved cytogenetically detected inversions. An INV6 and INV10 were resolved using GRCh38 and T2T-CHM13. Finally, an INV9 required optical genome mapping, de novo assembly of lrGS data and T2T-CHM13. This inversion disrupted intron 25 of EHMT1, confirming a diagnosis of Kleefstra syndrome 1 (MIM#610253). These three inversions, only mappable in specific references, prompted us to investigate the presence and population frequencies of differential reference regions (DRRs) between T2T-CHM13, GRCh37, GRCh38, the chimpanzee and bonobo, and hundreds of megabases of DRRs were identified. Our results emphasize the significance of the chosen reference genome and the added benefits of lrGS and optical genome mapping in solving rearrangements in challenging regions of the genome. This is particularly important for inversions and may impact clinical diagnostics.

AL has received honoraria from Illumina and PacBio. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics Laboratories. The remaining authors have nothing to declare.

Research reported in this publication was supported by the Swedish Research Council 2019-02078, the Swedish Brain Fund FO2022-0256, the Stockholm Regional Council ALF funding, the Swedish Rare Diseases Research foundation (AL) and the National Institute of General Medical Sciences NIGMS R01 GM132589 (CMBC). Additional support was provided through the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (U01HG007709 and U01HG007942) and the National Institute of Health (NIH S10 1S10OD028587). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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:

Ethics approval for analysis of participant samples was given by the Regional Ethical Review Board in Stockholm, Sweden (ethics permit numbers 2012/222-31/3). This ethics permit allows for use of clinical samples for analysis of scientific importance as part of clinical development. The IRB approval does not require us to get written consent for clinical testing. The research conformed to the principles of the Helsinki Declaration. Patient BH16643-1 was enrolled using research protocol H-47281/Pacific Northwest Research Institute WIRB #20202158 and 15HG0130 with the NIH IRB as part of the Undiagnosed Diseases Network (UDN). Informed consent was obtained from the legal guardians.

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

All data produced in the present study are available upon reasonable request to the authors. The clinical samples are not available due to ethical permissions.