AmpliconReconstructor – revealing focally amplified rearrangements in cancer

Oncogene amplification, a driver of cancer pathogenicity, frequently includes focal amplification of the surrounding genomic regions. AmpliconReconstructor utilizes whole-genome optical mapping to reveal these structures.
AmpliconReconstructor – revealing focally amplified rearrangements in cancer

Proto-oncogenes provide the positive signals that cause a cell to grow and divide. In their mutated (oncogenic) form, the genes are unregulated and lead the cell to grow and proliferate into a tumor mass. Often, the mutation is through increased copy number-- there are just more copies of these genes providing pro-growth and proliferative signals. Recent studies indicate that cancers with somatic oncogene amplifications are more aggressive, less amenable to immunotherapy, and result in poor outcomes.

What kinds of genomic rearrangements result in increased copy numbers of oncogenes? Where do all of the extra copies reside in the genome? While the answers to these fundamental biological questions vary from cancer to cancer, recent studies indicate that focal amplification of an oncogene is primarily the result of extrachromosomal DNA (ecDNA) formation [1]. In ecDNA, circular, oncogene carrying elements exist independently of the chromosome and reach higher copy numbers through random segregation and selection [2]. Other mechanisms such as tandem duplications, and breakage fusion bridge cycles, can also result in oncogene amplification. Resolving the architecture--the order and orientation of genomic segments-- of the amplified region is a first step in elucidating the mechanism, but remains challenging with short-read sequencing due to the large sizes and complex rearrangements in the focally amplified regions. The amplicons themselves are often large, frequently greater than 1 Mb, but the next generation sequencing reads from which amplicon structure is inferred, are short, leading to potential challenges in unambiguously deciphering amplicon structure. How do we ensure that we map these large amplicons correctly? 

We developed a computational method called AmpliconReconstructor (AR) to resolve the architecture using Bionano optical mapping technology. AR starts with a prior method, AmpliconArchitect [3], which uses NGS data to build accurate copy number-aware breakpoint graphs of focal amplifications. AR scaffolds together fine-structure resolved chains of rearrangements using assembled optical maps to produce basepair resolved structures of focal amplifications. The short-read sequencing provides accurate identification of copy number and precise breakpoint junction location, while the optical maps provide a long-range scaffold upon which the smaller genomic segments can be ordered and oriented.

We validated the accuracy of AR using extensive simulations using previously detected amplicon structures, and then applied it to resolve the architecture of amplicons in 6 cancer-derived cell-lines. AR reconstructed an unambiguous resolution of ecDNA carrying a mutated EGFR-vIII oncogene in glioblastoma cell-lines, GBM39[4] and HK301. On the lung cancer cell-line, HK460, AR reconstructed a 2.15 Mb amplicon containing Myc, and showed it to be reintegrated into the chromosomes as a homogeneously staining region.

Long-range DNA sequencing technologies are poised to vastly improve our ability to decipher the structure of large amplicons. However, the sheer size of some types of amplifications may well be beyond current long read technologies. Here, our approach may add further value. AR enabled the first sequence-based reconstruction of a 9.5Mbp breakage-fusion-bridge (bfb) amplicon, carrying structural variations different from the classic inverted duplications seen in bfb cycles. It also comprehensively characterized genomic rearrangements accompanying the Philadelphia chromosome in K562, revealing both the (chr22-chr9 BCR-ABL1 fusion) and also segments from chr13 carrying a disrupted form of GPC5.

Together, these results suggest that AR is a powerful computational method to integrate genomic tools for reconstructing the fine structure of focal amplification in cancer, providing insight into the intra and extrachromosomal rearrangement mechanisms that enable the amplification.


  1. Turner, K. M. et al. Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. Nature 543, 122–125 (2017).
  2. Kim, H. et al. Frequent extrachromosomal oncogene amplification drives aggressive tumors. Preprint at (2019).
  3. Deshpande, V. et al. Exploring the landscape of focal amplifications in cancer using AmpliconArchitect. Nat. Commun. 10, 392 (2019).
  4. Wu, S. et al. Circular ecDNA promotes accessible chromatin and high oncogene expression. Nature 575, 699-703 (2019).