Detection and Assembly of Extrachromosomal DNA in Tumors with Nanopore Sequencing

In cancer, parts of linear chromosomes containing coding and regulatory sequences can become excised and form novel circular extrachromosomal DNA (ecDNA) molecules. Because ecDNA lack centromeres, their replication and segregation during cell division is not limited by laws of Mendelian inheritance. This allows ecDNA to drive rapid oncogene amplification and intra-tumor heterogeneity, leading to better tumor adaption to changing microenvironments and increasing the potential for drug treatment resistance. Detection and assembly of ecDNA plays an important role in cancer research and can help inform treatment. We present a computational workflow capable of detecting and assembling ecDNA in tumors by processing long nanopore reads at shallow whole genome coverage levels. Briefly, the workflow first identifies reference genomic regions with sample-specific excess in read-depth coverage (i.e. focal amplifications). Then long nanopore reads overlapping these regions are extracted and used as input for local de-novo assembly. Resulting assemblies are then refined to have a reference-based representation of ecDNA-comprising fragments, highlighting their new relative order, orientation, and structural differences from canonical reference/matching normal sample, followed by reporting of non-reference inserted sequences for further analysis (e.g., viral integration detection). We evaluate the proposed approach on low-pass whole genome nanopore sequencing data from a variety of ecDNA-containing cell lines. We observe that the proposed nanopore-based workflow detects ecDNA presence and assembles its amplicon structure in concordance with previously published results. We further demonstrate the stability of obtained results with ≤1x target WGS coverage levels, suggesting that the output obtained from a MinION nanopore sequencer would be suitable for conducting multi-site tumor multiplex sequencing for analyzing intra-tumor ecDNA abundance and structural heterogeneity. We show that the real-time nature of nanopore sequencing allows for detection and assembly of ecDNA elements within just a few hours of sequencing, paving the way for a rapid turnaround ecDNA analysis. Notably, because of the unique ability of nanopore reads to retain single-molecule methylation signals, the proposed workflow allows for identification of differentially methylated regions both across intra-tumor multi-site samples, as well as in a tumor vs normal comparison, thus shedding light in acquisition/loss of DNA modifications in ecDNA. These results show how nanopore sequencing can be used as cost- and time-effective stand-alone platform to detect ecDNA presence and analyze its structure, while also revealing the dimension of ecDNA methylation in tumor samples. Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition. Citation Format: Sergey Aganezov, John Beaulaurier, Eoghan Harrington, Sissel Juul. Detection and assembly of extrachromosomal DNA in tumors with nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2705.