Structural variations (SV), a hallmark of genomic instability in cancer can either activate oncogenes or inactivate tumor suppressor genes. SVs tend to be recurrent and have been associated with several cancer types. Next-generation sequencing (NGS) is mostly blind to large SVs, lacking sensitivity with false positive rates up to 89% in SV detection. Long-read, single-molecule sequencing platforms can address larger variations as they typically generate read lengths of tens of thousands of bases and have helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. However, the throughput and coverage offered by whole genome long read sequencing makes it infeasible to conduct large-scale genomic studies.Targeted sequencing significantly improves accuracy and coverage by offering the depth necessary to detect rare alleles in a heterogenous population of cells. Using the nanopore Cas9-targeted sequencing (nCATS), a PCR-free enrichment system from Oxford Nanopore Technologies, we targeted 10 prominent cancer genes in MCF 10A and SK-BR-3 breast cell lines. However, we observed that the number of reads generated for targets longer than 30kb were not sufficient to accurately call SVs. To further enhance this approach, we developed an Affinity-based Cas-9-Mediated Enrichment (ACME) step, that uses HisTag DynabeadsTM to pulldown non-target reads. With ACME we achieved ~ 75-fold enrichment of the BRCA2 region and 35-65x coverage of this ~90 kb target on our panel. We observed an increase in enrichment and coverage of other genes on the panel as well, with enrichment as high as 5000-fold for some genes. While ACME is a biochemical approach, recently, adaptive sequencing or “Read Until”, a computational approach that ‘rejects' non-target reads has been used by a couple of groups for target enrichment. Using UNCALLED, a variation of Read Until, we observed an ~10x coverage of our targets. Recognizing the potential and limitations of both, biochemical and computational approaches, our efforts now are directed towards a combined approach where we expect to achieve a minimum enrichment of 200-fold across all targets. With a 200-fold enrichment of a 3.5Mb target, we should get between 200x - 400x coverage of the target, which should be high enough to detect SVs in as low as 10% of the cells in the sample. This would support large scale SV analysis to help define the landscape of such variants in the population and identify regions of therapeutic or diagnostic interest.

Citation Format: Shruti V. Iyer, Melissa Kramer, Sara Goodwin, W. Richard McCombie. Understanding genetic variation in cancer using nanopore targeted sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB201.