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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 driver of 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 fCNA’s 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 its 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.
The diagnostic yield in genetic disease has seen very little improvement over the last few decades, despite the introduction of whole genome sequencing.
The Bionano Genomics platform for genome imaging offers an extremely long-read technology, providing unmatched sensitivity and specificity to detect structural variation, genome-wide, at low cost. Our de novo maps can resolve complex repetitive regions, identify Copy Number Variations, and elucidate genome-wide structural variation like balanced/unbalanced translocations, inversions, and indels with much higher sensitivity and precision than sequencing-based methods.
For mosaic samples, Bionano’s high coverage depth allows for the detection of any type of structural variant with more than 90% sensitivity, present in as little as 10% of the cells, genome wide, and completely unbiased. Examples will be presented of how Bionano’s platform is helping solve genetic mysteries for patients with a variety of genetic disorders by detecting genomic rearrangements and structural variants missed by NGS and cytogenetic methods.
Structural variants (SVs) are an important source of genetic variation in the human genome and they are involved in a multitude of human diseases as well as cancer. SVs are enriched in repeat-rich regions of the human genome, and several remain undetected by conventional short-read sequencing technologies. Here we applied Bionano Genomics’ high-resolution optical mapping to comprehensively identify SVs, leveraging the most recent improvements: a) deep-genome coverage (400x) to enable somatic mutation detection in leukemia samples; b) highest resolution (≥500bp) and no sequencing bias allows detection of SVs refractory to sequencing in rare disease cases.
Deep-genome coverage was used to comprehensively detect somatic SVs on 52 leukemia samples, and allowed the 100% concordance for all aberrations with >10% variant allele fraction that previously required a combination of karyotyping, FISH and/or CNV-microarray. In addition, optical mapping allowed the identification of SVs that remained refractory to detection by classical methods including MLPA, Sanger sequencing, exome and/or genome sequencing. This allowed the identification of likely disease causing SVs in 5/20 research cases. Including a) a partial deletion of the NSF gene located in the distal segmental-duplication in 17q21.31, which likely disrupts NSF in a patients with intellectual disability; this event remained undetected even by long-read SMRT sequencing; b) a retrotransposon insertion in patient with a tumor-predisposition syndrome.
In summary, the full concordance with diagnostic standard assays in leukemia demonstrates the potential to replace classical cytogenetic tests. We furthermore show how the complementary use of mapping rather than sequencing approaches can unmask hidden structural variants.
Whole genome imaging using the Saphyr instrument from Bionano detects structural variants (SVs), such as insertions, deletions, and translocations, not readily evident from standard methods of whole genome analysis. This technology is particularly useful for detecting large (>500bp) and complex SVs that are difficult to detect using traditional short read sequencing alone. We have isolated high-molecular weight DNA (>150,000 bp) from various solid head and neck tumors using a protocol using a nanobind disc, consisting of novel nano structured silica surrounding a thermoplastic paramagnetic disk. This DNA is bar-coded using a direct labeling enzyme at a 6 bp consensus sequence scattered throughout the human genome. Cancer genome maps are assembled de novo based on label overlap and subsequently compared to a labeled human reference genome to identify SVs. We have successfully applied this framework to various head and neck solid tumors, including Human Papillomavirus (HPV)-positive oropharyngeal cancer, tongue cancer, and thyroid cancer. Genome imaging with whole genome sequencing can identify HPV insertion sites into the human genome of oropharyngeal cancers, and we have found viral integration to be associated with high genomic instability and more advanced clinical disease. Anaplastic thyroid cancer is a particularly aggressive form of cancer with strong genetic drivers in cell cycle regulation that can be described using the genome imaging platform. Short read sequencing in the literature has been inconclusive regarding the genetic difference between young and elderly tongue cancer, but genome imaging is able to detect different SVs affecting Ras signaling and the cell cycle between the two cohorts.
Accurate analysis of structural variants begins with isolating ultra-high molecular weight DNA. Obtaining high-quality UHMW DNA can present a challenge since sample collection, preservation, the DNA isolation process and subsequent handling of isolated DNA can significantly affect its quality.
In this webinar, Dr. Ben Clifford, Sr. Application Scientist at Bionano Genomics, discusses tips and tricks for isolating high quality UHMW DNA – right from sample collection, preservation and gentle handling of isolated DNA to minimize shearing and fragmentation.
In addition, we heard from Dr. Sven Bocklandt, Director of Scientific affairs at Bionano Genomics to walk us through genome imaging technology and workflow to demonstrate how it has been used successfully in studying structural variations in cancer and genetic diseases.
“Bionano’s optical mapping technology allowed us to characterize complex structural rearrangements in cancer with unprecedented precision. The results are incredibly robust and easy to interpret with Bionano software, and the team was really helpful for data analysis!”
-Dr. Eric Letouzé
Cyclins A2 and E1 regulate the cell cycle by promoting S phase entry and progression. We recently identified a hepatocellular carcinoma (HCC) subgroup exhibiting cyclin activation through various mechanisms, including HBV and AAV2 viral insertions, gene fusions and enhancer hijacking. Those poor-prognosis HCCs display a unique signature of structural rearrangements, triggered by replicative stress. This signature is strongly enriched in early-replicated active chromatin regions and is characterized by hundreds of tandem duplications and more complex events called Templated Insertion Cycle (T.I.C.).
Structural variation calling from short-read Whole Genome Sequencing provides abnormal junctions by comparing chimeric reads with a reference genome. However, those independent breakpoints are too distant, thus this method is not enough to reconstruct highly complex rearrangements, which may involve up to dozens of regions of the genome linked together. Here we used Bionano data to characterize with certainty large DNA molecules resulting from complex T.I.C.. This analysis allowed us to know which regions of the genome are the acceptor of such complex structural rearrangements. This information is critical in the understanding of how those rearrangements affect genes involved in tumorigenesis by placing oncogenes in different genomic contexts.
Accumulation of structural variations (SVs) across the genome is a known trigger factor for oncogenesis. Identifying these structural genomic alterations – accurately and comprehensively – is crucial for improving research and ultimately therapies for cancer patients, yet one of primary challenges when solely relied on short read sequencing and standard cytogenetic methods (e.g. karyotyping, FISH and chromosomal microarrays).
Optical mapping with genome imaging, enabled by the Bionano Saphyr® System, can accurately assemble and assay relevant regions for complex genomic disorders like cancer, even those involving very large segmental duplications. Genome imaging has to date unraveled a number of genes, never implicated in cancer and shown how they are affected by structural variations, along with deciphering novel structural variants. Listen to this webinar to learn how combining genome imaging with whole genome sequencing offers a strong integrative approach to understand small and large genomic variations in cancers.
This webinar outlines how a team at Radboud University Medical Center is assessing ultra-long read optical mapping on the Bionano Saphyr System to replace classical cytogenetics approaches in routine testing and for the discovery of novel structural variants with potential scientific, prognostic, or therapeutic value that are missed by standard approaches.
