Genome Mapping Research Articles

Optical Genome Mapping as a New Tool to Overcome Conventional Cytogenetics Limitations in Patients with Bone Marrow Failure.

Cytogenetic studies are essential in the diagnosis and follow up of patients with bone marrow failure syndromes (BMFSs), but obtaining good quality results is often challenging due to hypocellularity. Optical Genome Mapping (OGM), a novel technology capable of detecting most types chromosomal structural variants (SVs) at high resolution, is being increasingly used in many settings, including hematologic malignancies. Herein, we compared conventional cytogenetic techniques to OGM in 20 patients with diverse BMFSs. Twenty metaphases for the karyotype were only obtained in three subjects (15%), and no SVs were found in any of the samples. One patient with culture failure showed a gain in chromosome 1q by fluorescence in situ hybridization, which was confirmed by OGM. In contrast, OGM provided good quality results in all subjects, and SVs were detected in 14 of them (70%), mostly corresponding to cryptic submicroscopic alterations not observed by standard techniques. Therefore, OGM emerges as a powerful tool that provides complete and evaluable results in hypocellular BMFSs, reducing multiple tests into a single assay and overcoming some of the main limitations of conventional techniques. Furthermore, in addition to confirming the abnormalities detected by conventional techniques, OGM found new alterations beyond their detection limits.

Genomic Characterization of Partial Tandem Duplication Involving the KMT2A Gene in Adult Acute Myeloid Leukemia.

Gene rearrangements affecting KMT2A are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. KMT2A gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed KMT2A partial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches. We have validated the use of a custom hybrid-capture-based next-generation sequencing (NGS) panel for comprehensive profiling of AML patients seen at our institution. This NGS panel targets the entire consensus coding DNA sequence of KMT2A. To deduce the presence of a KMT2A-PTD, we used the relative ratio of KMT2A exons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM). We analyzed 932 AML cases and identified 41 individuals harboring a KMT2A-PTD. MLPA, NGS, and OGM confirmed the presence of a KMT2A-PTD in 22 of the cases analyzed where orthogonal testing was possible. The two false-positive KMT2A-PTD calls by NGS could be explained by the presence of cryptic structural variants impacting KMT2A and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in KMT2A, while MLPA yielded inconclusive results. MLPA analysis for KMT2A-PTD is limited to exon 4, whereas NGS and OGM resolved KMT2A-PTD sizes and copy number levels. KMT2A-PTDs are complex gene rearrangements that cannot be fully ascertained using a single genomic platform. MLPA, NGS panels, and OGM are complementary technologies applied in standard-of-care testing for AML patients. MLPA and NGS panels are designed for targeted copy number analysis; however, our results showed that integration of concurrent genomic alterations is needed for accurate KMT2A-PTD identification. Unbalanced chromosomal rearrangements overlapping with KMT2A can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.

Two Nested Inversions in the X Chromosome Differentiate the Dominant Malaria Vectors in Europe, Anopheles atroparvus and Anopheles messeae.

The Maculipennis subgroup of malaria mosquitoes includes both dominant malaria vectors and non-vectors in Eurasia. Understanding the genetic factors, particularly chromosomal inversions, that differentiate Anopheles species can provide valuable insights for vector control strategies. Although autosomal inversions between the species in this subgroup have been characterized based on the chromosomal banding patterns, the number and positions of rearrangements in the X chromosome remain unclear due to the divergent banding patterns. Here, we identified two large X chromosomal inversions, approximately 13 Mb and 10 Mb in size, using fluorescence in situ hybridization. The inversion breakpoint regions were mapped by hybridizing 53 gene markers with polytene chromosomes of An. messeae. The DNA probes were designed based on gene sequences from the annotated An. atroparvus genome. The two nested inversions resulted in five syntenic blocks. Only two small syntenic blocks, which encompass 181 annotated genes in the An. atroparvus genome, changed their position and orientation in the X chromosome. The analysis of the An. atroparvus genome revealed an enrichment of gene ontology terms associated with immune system and mating behavior in the rearranged syntenic blocks. Additionally, the enrichment of DNA transposons was found in sequences homologous to three of the four breakpoint regions. This study demonstrates the successful application of the physical genome mapping approach to identify rearrangements that differentiate species in insects with polytene chromosomes.

An R2R3-MYB Transcriptional Factor LuMYB314 Associated with the Loss of Petal Pigmentation in Flax (Linum usitatissimum L.).

The loss of anthocyanin pigments is one of the most common evolutionary transitions in petal color, yet the genetic basis for these changes in flax remains largely unknown. In this study, we used crossing studies, a bulk segregant analysis, genome-wide association studies, a phylogenetic analysis, and transgenic testing to identify genes responsible for the transition from blue to white petals in flax. This study found no correspondence between the petal color and seed color, refuting the conclusion that a locus controlling the seed coat color is associated with the petal color, as reported in previous studies. The locus controlling the petal color was mapped using a BSA-seq analysis based on the F2 population. However, no significantly associated genomic regions were detected. Our genome-wide association study identified a highly significant QTL (BP4.1) on chromosome 4 associated with flax petal color in the natural population. The combination of a local Manhattan plot and an LD heat map identified LuMYB314, an R2R3-MYB transcription factor, as a potential gene responsible for the natural variations in petal color in flax. The overexpression of LuMYB314 in both Arabidopsis thaliana and Nicotiana tabacum resulted in anthocyanin deposition, indicating that LuMYB314 is a credible candidate gene for controlling the petal color in flax. Additionally, our study highlights the limitations of the BSA-seq method in low-linkage genomic regions, while also demonstrating the powerful detection capabilities of GWAS based on high-density genomic variation mapping. This study enhances our genetic insight into petal color variations and has potential breeding value for engineering LuMYB314 to develop colored petals, bast fibers, and seeds for multifunctional use in flax.

Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics

Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5–10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.

Current genetic diagnostics in inborn errors of immunity.

New technologies in genetic diagnostics have revolutionized the understanding and management of rare diseases. This review highlights the significant advances and latest developments in genetic diagnostics in inborn errors of immunity (IEI), which encompass a diverse group of disorders characterized by defects in the immune system, leading to increased susceptibility to infections, autoimmunity, autoinflammatory diseases, allergies, and malignancies. Various diagnostic approaches, including targeted gene sequencing panels, whole exome sequencing, whole genome sequencing, RNA sequencing, or proteomics, have enabled the identification of causative genetic variants of rare diseases. These technologies not only facilitated the accurate diagnosis of IEI but also provided valuable insights into the underlying molecular mechanisms. Emerging technologies, currently mainly used in research, such as optical genome mapping, single cell sequencing or the application of artificial intelligence will allow even more insights in the aetiology of hereditary immune defects in the near future. The integration of genetic diagnostics into clinical practice significantly impacts patient care. Genetic testing enables early diagnosis, facilitating timely interventions and personalized treatment strategies. Additionally, establishing a genetic diagnosis is necessary for genetic counselling and prognostic assessments. Identifying specific genetic variants associated with inborn errors of immunity also paved the way for the development of targeted therapies and novel therapeutic approaches. This review emphasizes the challenges related with genetic diagnosis of rare diseases and provides future directions, specifically focusing on IEI. Despite the tremendous progress achieved over the last years, several obstacles remain or have become even more important due to the increasing amount of genetic data produced for each patient. This includes, first and foremost, the interpretation of variants of unknown significance (VUS) in known IEI genes and of variants in genes of unknown significance (GUS). Although genetic diagnostics have significantly contributed to the understanding and management of IEI and other rare diseases, further research, exchange between experts from different clinical disciplines, data integration and the establishment of comprehensive guidelines are crucial to tackle the remaining challenges and maximize the potential of genetic diagnostics in the field of rare diseases, such as IEI.

Integrated analyses reveal unexpected complex inversion and recombination in RH genes

AbstractPhenotype D-- is associated with severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. It is typically caused by defective RHCE genes. In this study, we identified a D-- phenotype proband and verified Rh phenotypes of other 6 family members. However, inconsistent results between the phenotypic analysis and Sanger sequencing revealed intact RHCE exons with no mutations in the D-- proband, but the protein was not expressed. Subsequent whole-genome sequencing by Oxford Nanopore Technologies of the proband revealed an inversion with ambiguous breakpoints in intron 2 and intron 7 and copy number variation loss in the RHCE gene region. Given that the RHCE gene is highly homologous to the RHD gene, we conducted a comprehensive analysis using Pacific Biosciences long-read target sequencing, Bionano optical genome mapping, and targeted next-generation sequencing. Our findings revealed that the proband had 2 novel recombinant RHCE haplotypes, RHCE∗Ce(1-2)-D(3-10) and RHCE∗Ce(1-2)-D(3-10)-Ce(10-8)-Ce(3-10), with clear-cut breakpoints identified. Furthermore, the RH haplotypes of the family members were identified and verified. In summary, we made, to our knowledge, a novel discovery of hereditary large inversion and recombination events occurring between the RHD and RHCE genes, leading to a lack of RhCE expression. This highlights the advantages of using integrated genetic analyses and also provides new insights into RH genotyping.

The clinical value of optical genome mapping in the rapid characterization of RB1 duplication and 15q23q24.2 triplication, for more appropriate prenatal genetic counselling.

Despite recent advances in prenatal genetic diagnosis, medical geneticists still face considerable difficulty in interpreting the clinical outcome of copy-number-variant duplications and defining the mechanisms underlying the formation of certain chromosomal rearrangements. Optical genome mapping (OGM) is an emerging cytogenomic tool with proved ability to identify the full spectrum of cytogenetic aberrations. Here, we report on the use of OGM in a prenatal diagnosis setting. Detailed breakpoint mapping was used to determine the relative orientations of triplicated and duplicated segments in two unrelated foetuses harbouring chromosomal aberrations: a de novo 15q23q24.2 triplication and a paternally inherited 13q14.2 duplication that overlapped partially with the RB1 gene. OGM enabled us to suggest a plausible mechanism for the triplication and confirmed that the RB1 duplication was direct oriented and in tandem. This enabled us to predict the pathogenic consequences, refine the prognosis and adapt the follow-up and familial screening appropriately. Along with an increase in diagnostic rates, OGM can rapidly highlight genotype-phenotype correlations, improve genetic counselling and significantly influence prenatal management.

Optical Genome Mapping for Cryptic Chromosomal Rearrangements Identification in Clinical Practice

Optical Genome Mapping for Cryptic Chromosomal Rearrangements Identification in Clinical Practice

Landscape of plasmids encoding β-lactamases in disinfection residual Enterobacteriaceae from wastewater treatment plants

Conventional disinfection processes, such as chlorination and UV radiation, are ineffective in controling antibiotic-resistant bacteria, especially disinfection residual Enterobacteriaceae (DRE) encoding β-lactamases, some of which have been classified as “critical priority pathogens” by the World Health Organization. However, few studies have focused on the transferability, phenotype, and genetic characteristics of DRE-derived plasmids encoding β-lactamases, especially extended-spectrum β-lactamases and carbapenemases. In this study, we isolated 10 typical DRE harboring plasmid-mediated blaNDM, blaCTX-M, or blaTEM in post-disinfection effluent from two wastewater treatment plants (WWTPs), with transfer frequency ranging from 1.69 × 10−6 to 3.02 × 10−5. According to genomic maps of plasmids, all blaNDM and blaTEM were cascaded with IS26, and blaCTX-M was adjacent to ISEcp1 or IS26, indicating the important role of these elements in the movement of β-lactamase-encoding genes. The presence of intact class 1 integrons on pWTPN-01 and pWTPC-03 suggested the ability of these DRE-derived plasmids to integrate other exogenous antibiotic resistance genes (ARGs). The coexistence of antibiotic, disinfectant, and heavy metal resistance genes on the same plasmid (e.g., pWTPT-03) implied the facilitating role of disinfectants and heavy metals in the transmission of DRE-derived ARGs. Notably, two plasmid transconjugants exhibited no discernible competitive fitness cost, suggesting a heightened environmental persistence. Furthermore, enhanced virulence induced by β-lactamase-encoding plasmids in their hosts was confirmed using Galleria mellonella infection models, which might be attributed to plasmid-mediated virulence genes. Overall, this study describes the landscape of β-lactamase-encoding plasmids in DRE, and highlights the urgent need for advanced control of DRE to keep environmental and ecological security.

The complex landscape of DMD mutations: moving towards personalized medicine.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by progressive muscle degeneration, with respiratory and cardiac complications, caused by mutations in the DMD gene, encoding the protein dystrophin. Various DMD mutations result in different phenotypes and disease severity. Understanding genotype/phenotype correlations is essential to optimize clinical care, as mutation-specific therapies and innovative therapeutic approaches are becoming available. Disease modifier genes, trans-active variants influencing disease severity and phenotypic expressivity, may modulate the response to therapy, and become new therapeutic targets. Uncovering more disease modifier genes via extensive genomic mapping studies offers the potential to fine-tune prognostic assessments for individuals with DMD. This review provides insights into genotype/phenotype correlations and the influence of modifier genes in DMD.

Plant mitochondrial genome map (PMGmap): A software tool for the comprehensive visualization of coding, noncoding and genome features of plant mitochondrial genomes.

Tools for visualizing genomes are essential for investigating genomic features and their interactions. Currently, tools designed originally for animal mitogenomes and plant plastomes are used to visualize the mitogens of plants but cannot accurately display features specific to plant mitogenomes, such as nonlinear exon arrangement for genes, the prevalence of functional noncoding features and complex chromosomal architecture. To address these problems, a software package, plant mitochondrial genome map (PMGmap), was developed using the Python programming language. PMGmap can draw genes at exon levels; draw cis- and trans-splicing gene maps, noncoding features and repetitive sequences; and scale genic regions by using the scaling of the genic regions on the mitogenome (SAGM) algorithm. It can also draw multiple chromosomes simultaneously. Compared with other state-of-the-art tools, PMGmap showed better performance in visualizing 405 plant mitogenomes, showing potential as an invaluable tool for plant mitogenome research. The web and container versions and the source code of PMGmap can be accessed through the following link: http://www.1kmpg.cn/pmgmap.

Abstract 7027: Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via TEM-seq

Abstract DNA methylation (DNAme) is an epigenetic mark that includes the modification of cytosine resides (5mC) within CpG islands. In addition to well characterized roles regulating gene expression, imprinting and silencing parasitic DNA elements, the misregulation of DNAme is implicated in multiple diseases. Evidence is emerging that DNAme is not an independent epigenetic mark but rather closely linked to the post translational modification (PTM) of histone proteins. However, examining the relationship between 5mC and PTMs are hampered by the usual approach of independent analyses that cannot establish a direct linkage. Furthermore, the traditional approach to measure 5mC relies upon harsh bisulfite chemical conversation of DNA, which introduces damage and systemic biases. To address these limitations, we developed Targeted Enzymatic Methylation-sequencing (TEM-seq): an ultra-sensitive multi-omic genomic mapping technology that delivers high resolution DNAme profiles at epitope-defined chromatin features. Importantly this assay is capable of directly examining the link between 5mC and histone PTMs and/or chromatin associated proteins (ChAPs). The TEM-seq workflow integrates CUT&RUN for genomic mapping with NEB’s enzymatic methyl-seq (EM-seq) for unbiased DNAme analysis. CUT&RUN is a highly sensitive, standards-driven assay that uses antibodies to locally tether protein A/G-micrococcal nuclease (pAG-MNase) to chromatin within intact cells or nuclei, followed by controlled activation of the MNase to cleave nearby DNA. EM-seq then enzymatically converts [5mC/5hmC] to generate single-base resolution, unbiased DNAme profiles from sub-ng samples. To determine the capabilities and limitations of this novel approach, we tested multiple chromatin targets in various cell lines. We demonstrate that TEM-seq is highly reproducible, specific, and efficient (<10% off-target binding, >90% enzymatic conversion of DNAme, <0.5% conversion of unmethylated DNA). TEM-seq is also highly-sensitive, requiring only five million short-read sequences per assay (10-50x less than whole-genome 5mC-sequencing), demonstrating the disruptive potential of the assay to enable cost-effective approach for targeted DNAme analysis. Finally, we leveraged TEM-seq to gain mechanistic insights to Rett syndrome a neurodegenerative disorder that results from mutations in the 5mC-binding domain of the MECP2 gene. Citation Format: Keith E. Maier, Vishnu U. Sunitha Kumary, Bryan J. Venters, Allison Hickman, Anup Vaidya, James Anderson, Ryan Ezell, Jonathan M. Burg, Louise Williams, Chaithanya Ponnaluri, Pierre Esteve, Isaac Meek, Zu-wen Sun, Martis W. Cowles, Sriharsa Pradhan, Michael-Christopher Keogh. Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via TEM-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7027.

Abstract 2337: Accelerated optical genome mapping analysis with Stratys Compute and Guided Assembly

Abstract Background Optical genome maps (OGM) from Bionano enable the detection of genomic structural and copy number variants that cannot be detected by next-generation sequencing (NGS) technologies and are often missed by conventional cytogenetic techniques. Bionano has developed bioinformatics pipelines for calling structural and copy number variants including the Bionano Solve de novo assembly pipeline for constitutional analysis and the Rare Variant Analysis (RVA) pipeline for low-allele-fraction cancer applications. Both pipelines are computationally intensive and currently take 5-10 hours on the latest generation of the Bionano Saphyr® Compute which is deployed as a four-node compute cluster and requires significant IT resources. Methods To increase throughput and simplify the deployment of the compute resources needed to support the Stratys™ optical genome mapping instrument, Bionano has developed Stratys™ Compute. Stratys Compute improves both compute and analytical performance by adapting compute-intensive stages in the Solve pipeline to run on GPUs and by developing the Guided Assembly pipeline. Stratys Compute is powered by state-of-the-art NVIDIA RTX 6000 Ada generation cards and CUDA-optimized refinement, alignment, and structural variation detection kernels to accelerate OGM analysis. Stratys Compute is a standalone workstation placed adjacent to Stratys instrument. This will minimize the IT footprint and bypass the integration with the customer site’s data center. We have incorporated these optimizations into the new Guided Assembly pipeline, which aims to combine the low-allele fraction detection capability of the RVA pipeline with the whole genome coverage and ability to detect smaller structural variants enabled by the de novo assembly pipeline. The Guided Assembly pipeline uses the reference genome as an initial seed followed by extension, refinement, and structural variant calling. This new analysis method has been evaluated through comparison to previous results from both existing pipelines and standard benchmarking datasets used to estimate structural variant calling performance and is deployed on Stratys Compute for both constitutional and low allele-fraction applications. Results Guided Assembly has been adapted to run on GPU hardware in a simplified compute tower that can be deployed to a lab along with the Stratys instrument without the need for a dedicated server room. We found concordance between the guided assembly results and our previous de novo and RVA pipeline results. We also found increased sensitivity at low allele fractions for detecting insertion variants smaller than 5 kb and larger than 200 kb while finding equivalent performance for other variant types with the updated methods. The accelerated Guided Assembly for constitutional and low-allele-fraction applications will be available to early access customers in Q4 2023 with full commercial release in Q2 2024. Citation Format: Damla Senol Cali, Thomas Anantharaman, Martin Muggli, Samer Al-Saffar, Charles Schoonover, Neil Miller. Accelerated optical genome mapping analysis with Stratys Compute and Guided Assembly [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2337.

Abstract 7026: Multi-omic genomic mapping with long read sequencing

Abstract Gene transcription is regulated by the complex interplay between histone post-translational modifications (PTMs), chromatin associated proteins (CAPs), and DNA methylation (DNAme). Mapping their genomic locations and examining the relationships between these chromatin elements is a powerful approach to decipher mechanisms of disease, thereby enabling discovery of novel biomarkers and therapeutics. Leading epigenomic mapping technologies (e.g., ChIP-seq, CUT&RUN) rely upon DNA fragmentation to isolate regions of interest for sequencing on short read platforms (e.g., Illumina). This strategy leads to substantial loss of contextual information regarding the surrounding DNA, precluding the identification of multiple co-occurring epigenomic features on a single DNA molecule. By contrast, long-read sequencing (LRS) platforms are capable of sequencing very long reads from a single molecule (typically >10kb), allowing relationships between features on a single molecule to be used to resolve heterogeneity within mixed populations. Here we report a robust multi-omic method that leverages LRS to simultaneously profile histone PTMs (or CAPs), DNAme, and parental haplotype in a single assay. This nondestructive, epigenomic mapping approach leverages a novel DNA methyltransferase fusion protein (pAG-M.EcoGII) to label DNA underneath antibody-targeted chromatin features, thereby marking sites of interest while preserving DNA molecules intact for LRS. Inspired by our work with state-of-the-art immunotethering-based approaches (CUT&RUN/CUT&Tag), nuclei are bound to magnetic beads to streamline and automate sample processing. Next, adenosines nearby antibody-targeted chromatin features are methylated with pAG-M.EcoGII, which are then directly read from genomic DNA using Oxford Nanopore Technologies or Pacific Biosciences LRS platforms. To determine the capabilities and limitations of this assay, we tested multiple chromatin targets in various cell lines. Importantly, this method is highly reproducible across biological replicates, and highly concordant with orthogonal SRS assays (e.g., CUT&RUN). Further, we showed that this method is a true multi-omic approach by simultaneously profiling histone PTMs, native DNAme (5mC), and parental single-nucleotide variants from single DNA molecules within a single reaction. Finally, this workflow preserves chromatin integrity for LRS, revealing heterogeneity (e.g., haplotype or paternal origin) within/between data types and providing access to previously unmappable genomic regions (e.g., centromeres). Citation Format: Bryan J. Venters, Paul W. Hook, Vishnu S. Kumary, Alli R. Hickman, James T. Anderson, Anup Vaidya, Ryan J. Ezell, Jonathan M. Burg, Zu-Wen Sun, Martis W. Cowles, Winston Timp, Michael-Christopher Keogh. Multi-omic genomic mapping with long read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7026.

Abstract 6257: Genome wide, high-throughput, high-resolution structural variation detection at low variant allele fraction for oncology samples

Abstract Structural variation detection remains an elusive undertaking. For decades, G-banded karyotyping has remained the standard practice for nearly all hematological malignancy samples. Cancers are diseases of the genome and structural variants (SVs) are a hallmark of nearly all cancers, are often drivers of oncogenicity, and can sometimes be targeted for treatment, as highlighted by the discovery of the Philadelphia chromosome and later the development of targeted kinase inhibitors. One of the most important limitations of karyotyping, its requirement for cells to divide in culture, has prevented its application for most solid tumors and therefore, there is considerably less understanding of the impact of SVs in solid tumors. Optical genome mapping is a technique that is revolutionizing cytogenomics and unlocking the understanding of SVs in the genome at a level never seen before. It not only can detect nearly all variants that a karyotype can, but it can provide 10,000x higher resolution and does not require cell culture. Because of this, it is being adopted for oncology studies across hematological malignancies and solid tumors. This study demonstrates the performance of a new high throughput Stratys instrument for generation of optical genome map data at extraordinarily high coverage depth. With this instrument, 12 samples can be loaded at once, each loaded onto its own cartridge and the instrument can collect 400x genome coverage for each of these samples in 24 hours. The instrument has random access to the cartridges so that completed samples can be swapped out to facilitate optimal instrument run efficiency. To date, this system has been demonstrated to run 60 samples in a week, each at 400x coverage. Data from the instrument can be processed by Bionano Access™ and VIA™ software to call SVs, copy number variants (CNVs), regions of loss of heterozygosity (LOH), on a genome wide basis, and to classify those variants automatically according to guidelines such as the National Comprehensive Cancer Network (NCCN), World Health Organization (WHO), and National Health Services (NHS, United Kingdom). This new high throughput system with advanced bioinformatic data analysis has the potential to further transform structural variation analysis in cancer research. Citation Format: Alex R. Hastie, Pat Lynch. Genome wide, high-throughput, high-resolution structural variation detection at low variant allele fraction for oncology samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6257.

Abstract 4895: Optical genome mapping analysis across multiple solid tumor types in Bionano VIATM software

Abstract Solid tumors can show highly complex somatic structural and copy number variants (CNVs) of multiple classes. These variants can reveal mechanisms behind carcinogenesis, including the amplification of oncogenes, deletion or inactivation of tumor suppressor genes, and fusions to create new oncogenes. High degrees of heterogeneity within solid tumors can complicate analysis. Optical Genome Mapping (OGM) is a comprehensive technology which combines the resolution of molecular-level data with the scale of cytogenomic analysis. ≈5-15mg sections of freshly frozen tumor are homogenized in buffer to release ultra-high molecular weight DNA. Long fragments are then labeled at a regular 6mer, then linearized and imaged with native long-range structure (≥150kbp) preserved. In silico molecules are assembled into maps and aligned to a reference genome to detect structural variants with sensitivity to ≥5% allele fraction. Bionano VIATM software provides tools for visualization and interpretation of structural variants detected with OGM. VIA also provides CNV calling and detection of Absence of Heterozygosity (AOH) and allelic imbalance using SNP-FASST3, a segmentation algorithm for detecting mosaic events in OGM and other types of data. VIA calculates a B-Allele frequency (BAF) distribution from OGM data and adds a genome-wide BAF track to the applications suite, enabling calling of allelic imbalance events such as AOH. Visualized together, the overlay of structural variant information on copy number data provides context for chromosome fusions underlying copy number change. Additionally, balanced fusions are revealed consistently in cases where the impact is copy neutral. To demonstrate the utility of integrative analysis of structural, copy number, and allelic imbalance variants in solid tumor datasets, we processed a pilot set of six diverse solid tumors through Bionano software. Brain, breast, kidney, lung, ovarian, and prostate tumor data which had undergone previous analysis in Bionano Solve 3.7 were freshly analyzed in Solve 3.8 and VIA 7.0. In the lung tumor dataset there are clear allelic imbalance segments spanning most of chromosome 4 with no concomitant copy number change. In VIA, these segments can be merged on examination, converted to AOH calls, and further specified to reflect mosaic copy neutral loss of heterozygosity (CN-LOH). In the complex kidney tumor dataset, it is difficult to resolve ploidy state based on copy number data alone. This ambiguity is resolvable with VIA, by leveraging the copy number and BAF-based allelic imbalance distributions and recentering probes to a manually defined diploid region. The comprehensive OGM data approach facilitated by VIA can streamline analysis in challenging solid tumor datasets. Structural variant visualization, CNV detection and AOH detection are available in VIA 7.0. Citation Format: Samer I. Al-Saffar, Benjamin Clifford, Aliz Raksi, Alex Hastie, Neil Miller, Alka Chaubey. Optical genome mapping analysis across multiple solid tumor types in Bionano VIATM software [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4895.

Abstract 2271: Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts

Abstract Background: Extrachromosomal DNA (ecDNA) is a driver of tumor heterogeneity and is associated with poor survival in many different adult and childhood cancer types. Patient-derived xenograft (PDX) models facilitate preclinical drug testing in an in vivo environment that enables analysis of tumor growth and survival and molecular tumor profiling. These models, combined with single-cell profiling, can be used to study intratumoral heterogeneity under therapeutic pressure and the emergence of drug resistance. Here we investigate the abundance and conservation of ecDNA in PDX models with the aim of assessing the faithful representation of ecDNA compared to the tumors from which they are derived. Methods: Whole-genome sequencing (WGS) was applied to 27 medulloblastoma (MB) PDX models and their tumors of origin using Amplicon Architect, a tool used to reconstruct focally amplified regions of DNA to provide insights into the landscape of cancer genomes. The frequency of ecDNA in these models was also compared to that of ecDNA in the MB patient population. Optical genome mapping enabled accurate and reliable ecDNA sequence reconstructions. Multiome single-cell RNA and ATAC-sequencing of a PDX tumor enabled analysis of ecDNA conservation and intratumoral heterogeneity compared to similar data previously obtained from the primary tumor. Results: Comparative analyses revealed that ecDNA is largely conserved in PDX models, emphasizing their relevance as faithful models of the primary tumors. In addition, we find that ecDNA is significantly more frequent in MB PDX models (16 out of 27 analyzed patient-derived tumors) as compared to their frequency in the overall patient population (~18% of all medulloblastoma tumors contain ecDNA), suggesting ecDNA as a driver for successful establishment of MB PDX models. Single-cell analysis revealed that ecDNA is omnipresent in almost all PDX tumor cells, while its presence was limited to only ~9% of tumor cells in the primary tumor. Conclusion: Analysis of ecDNA in medulloblastoma primary tumors and in PDX models revealed that ecDNA is preserved in PDX models. This is an important characteristic that renders patient-derived mouse xenografts as faithful models of their primary tumors. These results emphasize the relevance of PDX models for preclinical in vivo studies aimed at analyzing the role of ecDNA under therapeutic pressure and for analyzing clonal selection along with molecular evolution of ecDNA during the development of therapeutic resistance. Citation Format: Rishaan Kenkre, Owen Chapman, Jens Luebeck, Vineet Bafna, Robert Wechsler-Reya, Jill Mesirov, Lukas Chavez. Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2271.

Structural rearrangements as a recurrent pathogenic mechanism for SETBP1 haploinsufficiency

Chromosomal structural rearrangements consist of anomalies in genomic architecture that may or may not be associated with genetic material gain and loss. Evaluating the precise breakpoint is crucial from a diagnostic point of view, highlighting possible gene disruption and addressing to appropriate genotype–phenotype association. Structural rearrangements can either occur randomly within the genome or present with a recurrence, mainly due to peculiar genomic features of the surrounding regions. We report about three non-related individuals, harboring chromosomal structural rearrangements interrupting SETBP1, leading to gene haploinsufficiency. Two out of them resulted negative to Chromosomal Microarray Analysis (CMA), being the rearrangement balanced at a microarray resolution. The third one, presenting with a complex three-chromosome rearrangement, had been previously diagnosed with SETBP1 haploinsufficiency due to a partial gene deletion at one of the chromosomal breakpoints. We thoroughly characterized the rearrangements by means of Optical Genome Mapping (OGM) and Whole Genome Sequencing (WGS), providing details about the involved sequences and the underlying mechanisms. We propose structural variants as a recurrent event in SETBP1 haploinsufficiency, which may be overlooked by laboratory routine genomic analyses (CMA and Whole Exome Sequencing) or only partially determined when associated with genomic losses at breakpoints. We finally introduce a possible role of SETBP1 in a Noonan-like phenotype.

Prospective Investigation of Optical Genome Mapping for Prenatal Genetic Diagnosis.

Optical genome mapping (OGM) is a novel assay for detecting structural variants (SVs) and has been retrospectively evaluated for its performance. However, its prospective evaluation in prenatal diagnosis remains unreported. This study aimed to prospectively assess the technical concordance of OGM with standard of care (SOC) testing in prenatal diagnosis. A prospective cohort of 204 pregnant women was enrolled in this study. Amniotic fluid samples from these women were subjected to OGM and SOC testing, which included chromosomal microarray analysis (CMA) and karyotyping (KT) in parallel. The diagnostic yield of OGM was evaluated, and the technical concordance between OGM and SOC testing was assessed. OGM successfully analyzed 204 cultured amniocyte samples, even with a cell count as low as 0.24 million. In total, 60 reportable SVs were identified through combined OGM and SOC testing, with 22 SVs detected by all 3 techniques. The diagnostic yield for OGM, CMA, and KT was 25% (51/204), 22.06% (45/204), and 18.14% (37/204), respectively. The highest diagnostic yield (29.41%, 60/204) was achieved when OGM and KT were used together. OGM demonstrated a concordance of 95.56% with CMA and 75.68% with KT in this cohort study. Our findings suggest that OGM can be effectively applied in prenatal diagnosis using cultured amniocytes and exhibits high concordance with SOC testing. The combined use of OGM and KT appears to yield the most promising diagnostic outcomes.