Abstract 5045: Cancer multiomics: Whole-genome DNA, methylation, RNA-seq, and direct RNA characterization of cancer genomes using Oxford Nanopore sequencing

Abstract Cancer progression is characterized by the accumulation of both genomic and epigenomic alterations as well as transcription changes, leading to the formation of tumors that are highly heterogeneous. This heterogeneity arises because the tumor consists of a diverse population of cells, each with distinct genomic, epigenomic, and transcriptomic profiles. These differences influence the tumor's behavior, its response to therapy, and its ability to evolve. To better understand the mechanisms driving cancer, it is crucial to adopt a multi-omics approach. Here we describe an approach that leverages Oxford Nanopore Technologies’ sequencing platform to achieve a comprehensive multi-omics analysis, enabling the detection of a wide spectrum of haplotype-specific somatic genomic variants (ranging from single nucleotide changes to large chromosomal rearrangements), epigenetic modifications such as 5mC and 5hmC, chromatin accessibility changes, and detailed transcriptomic profiles. Through both cDNA and direct RNA (dRNA) nanopore sequencing, it captures transcriptional alterations, isoform diversity, and RNA modification landscapes, providing a holistic view of the molecular changes driving cancer progression. We highlight several notable examples of identified somatic genomic mutations in an example tumor vs matching normal sample, including loss of heterozygosity and exon deletion in PTEN gene, as well as apparent promoter 5mC hypermethylation in the tumor as well as genome wide changes in 5hmC levels. We further explore changes in isoform-level transcript expression with both cDNA and dRNA nanopore reads. We demonstrate high concordance between the two approaches with dRNA reads allowing for additional examination of RNA base modifications, including m6A and pseU. We also illustrate how the treatment with artificially induced 6mA in both normal and tumor DNA samples allows for the identification of differences in chromatin accessibility across the whole genome, while preserving the ability to detect somatic variations using a single dataset. Lastly, we highlight the ability of Oxford Nanopore sequencing to produce telomere-2-telomere diploid assemblies of the matching normal sample enabling analyses relative to individualized assembled references, plotting a path towards truly personalized cancer genome analysis. In summary, our approach highlights how Nanopore sequencing facilitates multi-omics analysis, providing a comprehensive characterization of diverse genomic, epigenomic, and transcriptomic alterations in tumor samples using a single platform. Citation Format: Sergey Aganezov, Philipp Rescheneder, Aino Jarvelin, Sean McKenzie, Ella Kennett, George Dodd, Sissel Juul. Cancer multiomics: Whole-genome DNA, methylation, RNA-seq, and direct RNA characterization of cancer genomes using Oxford Nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5045.