AmpliconReconstructor: Integrated analysis of NGS and optical mapping resolves the complex structures of focal amplifications in cancer

Oncogene amplification, a major driver of cancer pathogenicity, is often mediated through focal amplification of genomic segments. Recent results implicate extrachromosomal DNA (ecDNA) as the primary mechanism driving focal copy number amplification (fCNA) - enabling gene amplification, rapid tumor evolution, and the rewiring of regulatory circuitry. Resolving an fCNA’s structure is a first step in deciphering the mechanisms of its genesis and the subsequent biological consequences. Here, we introduce a powerful new computational method, AmpliconReconstructor (AR), for integrating optical mapping (OM) of long DNA fragments (>150kb) with next-generation sequencing (NGS) to resolve fCNAs at single-nucleotide resolution. AR uses an NGS-derived breakpoint graph alongside OM scaffolds to produce high-fidelity reconstructions. After validating performance by extensive simulations, we used AR to reconstruct fCNAs in seven cancer cell lines to reveal the complex architecture of ecDNA, breakage-fusion-bridge cycles, and other complex rearrangements. By distinguishing between chromosomal and extrachromosomal origins, and by reconstructing the rearrangement signatures associated with a given fCNA’s generative mechanism, AR enables a more thorough understanding of the origins of fCNAs, and their functional consequences.