Circular extrachromosomal DNA (ecDNA) are a genomic lesion occurring in tumors, and represent a foundational, growing frontier in cancer biology. The discovery that focal amplifications exist in multiple topologies and arise by different mechanisms has enabled cancer researchers to study the consequences of different types of focal amplification – revealing that focal amplifications like ecDNA lead to worse patient survival. Consequently, there is an urgent need for bioinformatic methods and tools to study these focal amplifications, particularly ecDNA. This thesis describes novel tools and methods which can be used to study ecDNA, and other focal amplifications. It also demonstrates how those tools and methods can be used to profile focal amplifications across different cancer types, ultimately revealing novel biology about the structure, function and genesis of ecDNA in different contexts.

I first present two methods, AmpliconReconstructor (AR) and FaNDOM, which incorporate optical mapping data to resolve the structures of ecDNA and other focal amplifications. AR incorporates both optical mapping and NGS data and builds upon a prior method for ecDNA detection with NGS data, AmpliconArchitect (AA). FaNDOM utilizes optical mapping solely and enables the rapid characterization of large structural variants using assembled OM contigs or individual OM molecules.

I also describe the landscape of ecDNA in oropharyngeal squamous cell carcinoma, demonstrating that both human and hybrid human-viral ecDNA exist and are associated with distinct patterns of transcriptional splicing. Visualizations of the rearranged ecDNA structures and overlaid transcription-level data reveal the overexpression of genes carried on ecDNA.

Lastly, I describe the genesis of ecDNA in Barrett’s esophagus, the precursor tissue of esophageal adenocarcinoma. We utilized methods for profiling ecDNA, such as AmpliconClassifier, to demonstrate that ecDNA exist in pre-cancerous tissue, are associated with worse histology, that they are subsequently found again in cancer, and that they tend to undergo positive selection during the malignant transformation. These findings solidify ecDNA as a potent driver of cancer, and not an opportunistic passenger.

Optical mapping is a DNA physical mapping technique that can measure large-scale structural variation in genomes and enables more accurate completion of genome assemblies. Particularly, we focus on the case where optical mapping is used to complete genome assembly on pre-identified segments such as may be found in a breakpoint graph. In this work we study methods for aligning optical map contigs with reference genome segments. This work introduces a novel method for optical map alignment that outperforms existing methods for aligning breakpoint graph reference segments to assembled optical map contigs. Also discussed are some additional modifications that can be made to the method.