Rapid identification of genomic structural variations with nanopore sequencing enables blood-based cancer monitoring

Somatic genomic structural variations (SVs) are promising personalized biomarkers for sensitive and specific detection of circulating tumor DNA (ctDNA) in liquid biopsies. However, affordable and fast identification of such SV biomarkers is challenging, which hinders routine use in the clinic. Here, we developed a novel approach - termed SHARC - for rapid discovery of somatic SVs as personalized tumor biomarkers. SHARC combines low coverage cancer genome sketching by using Oxford Nanopore sequencing with random forest classification and a dedicated filtering pipeline to enrich for somatic SVs. Our method leverages the real-time and long-read capabilities of nanopore sequencing to identify somatic SV breakpoints at nucleotide resolution from a tumor tissue biopsy within three days. We applied SHARC to tumor samples of high-grade ovarian and prostate cancer and validated on average ten somatic SVs per sample with PCR mini-amplicons. An accompanying method for SV breakpoint detection from liquid biopsies was devised based on digital PCR, enabling detection of cancer in a quantitative manner. Using this method, we retrospectively monitored treatment response in patients with metastatic prostate cancer. Our work demonstrates that SHARC forms a universal framework for rapid development of personalized biomarker assays for blood-based monitoring of any cancer type. ### Competing Interest Statement J.E.V-I, C.S. and W.P.K. have received financial compensation for travel and accommodation expenses to speak at Oxford Nanopore Technologies-organized meetings. The remaining authors declare no competing financial interests. ### Funding Statement This work has been supported by KWF grants UU 2012-5710 and by funding from the Utrecht University to implement a single-molecule sequencing facility. ### Author Declarations All relevant ethical guidelines have been followed; any necessary IRB and/or ethics committee approvals have been obtained and details of the IRB/oversight body are included in the manuscript. Yes All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. 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 and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Nanopore sequencing data is available in ENA and EGA as follows: COLO829 cell line: ENA accession ERX2765498. HGS-3 organoid line: EGA dataset accession EGAD00001005476 Patient material: EGA study accession EGAS00001003963