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The recommended practice for individuals suspected of a genetic etiology for disorders including unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA) involves a genetic testing workflow including chromosomal microarray (CMA), Fragile-X testing, karyotype analysis, and/or sequencing-based gene panels. Since genomic imbalances are often found to be causative, CMA is recommended as first tier testing for many indications. Optical genome mapping (OGM) is an emerging next generation cytogenomic technique that can detect not only copy number variants (CNVs), triploidy and absence of heterozygosity (AOH) like CMA, but can also define the location of duplications, and detect other structural variants (SVs), including balanced rearrangements and repeat expansions/contractions. This study compares OGM to CMA for clinically reported genomic variants, some of these samples also have structural characterization by fluorescence in situ hybridization (FISH). OGM was performed on IRB approved, de-identified specimens from 55 individuals with genomic abnormalities previously identified by CMA (61 clinically reported abnormalities). SVs identified by OGM were filtered by a control database to remove polymorphic variants and against an established gene list to prioritize clinically relevant findings before comparing with CMA and FISH results. OGM results showed 100% concordance with CMA findings for pathogenic variants and 98% concordant for all pathogenic/likely pathogenic/variants of uncertain significance (VUS), while also providing additional insight into the genomic structure of abnormalities that CMA was unable to provide. OGM demonstrates equivalent performance to CMA for CNV and AOH detection, enhanced by its ability to determine the structure of the genome. This work adds to an increasing body of evidence on the analytical validity and ability to detect clinically relevant abnormalities identified by CMA. Moreover, OGM identifies translocations, structures of duplications and complex CNVs intractable by CMA, yielding additional clinical utility. [ABSTRACT FROM AUTHOR]

Certain cancer treatments have been shown to be effective in killing cancer cells exhibiting genome instability signatures indicative of homologous recombination deficiency (HRD). There are three measurements of HRD signatures commonly employed: loss of heterozygosity (HRD-LOH), telomeric allelic imbalance (TAI) and large-scale state transition (LST). Current genomic technologies lack sensitivity to capture the full extent of somatic variants related to HRD and this limits their sensitivity. Here, optical genome mapping (OGM) was used to detect large structural variants (SVs) and calculate a HRD score. OGM captures high molecular weight DNA to call SVs by aligning these molecules to the public reference genome. Subsequently, an automated algorithm was developed to compute the HRD score, which is the summation of the three HRD signatures: HRD-LOH, the number of regions with a loss >15 Mbp but shorter than the whole chromosome; TAI, the number of regions of gain and loss >10Mbp that extend to a subtelomere but do not cross the centromere; and LST, the number of chromosomal breakpoints whose SV size >10Mb but not the whole chromosome. We applied this novel algorithm to 20 samples from solid tumors and myeloid neoplasms, and the automated scores are concordant with expertly curated scores on the same OGM data. Further, compared with NGS or CMA, OGM data analyzed with this automated algorithm is able to detect many more HRD related variant compared to other approaches. [ABSTRACT FROM AUTHOR]

Genomic structural variation (SV) analysis is fundamental to understanding cancer genetics, but its detection remains elusive. Standard-of-care tests such as karyotyping are low-throughput and labor-intensive, while chromosomal microarrays (CMA) cannot find copy-neutral events. Advances in sequencing and Bionano optical genome mapping (OGM) can address these shortcomings. The OGM workflow includes extraction of megabases-long DNA, labeling at specific motifs, and linearization in nanochannels for imaging. These molecules (>150kbp) are assembled or aligned to the human reference assembly to capture large SVs. Here, we aim to generate a set of reference somatic SVs in a well-described matched breast tumor-normal cell line pair (HCC1395 and HCC13395BL) using multiple technologies and to compare their performance. The technologies used were Illumina, 10X Genomics, PacBio, Nanopore, Dovetail Hi-C, and Bionano OGM. CMA and PCR were used for validation. Overall, SV sensitivity is dependent on technology used. While small SVs are consistent across platforms. SV calls >50kbp are the least discoverable, and OGM has the highest performance. Whereas smaller SVs called by OGM are more consistent with other platforms, suggesting that the platform is robust, large OGM calls are uniquely captured by ultra-long molecules. Bionano excelled at finding large DNA gains and fusion events - typical insertion is 2-orders of magnitude larger than others. Furthermore, Bionano uniquely called a ∼40Mb somatic terminal 5q duplication. Clinical genomic research requires SNV and SV analysis to comprehensively evaluate genomes, and this study shows that OGM is required to find all SV classes and that sequencing and OGM are complementary technologies. [ABSTRACT FROM AUTHOR]

Solid tumors are often characterized by a high degree of complex somatic structural variants of multiple classes, especially rearrangements and copy number variants. Characterizing this genomic complexity is crucial for understanding the biology behind carcinogenesis but is challenging as a result of limitations of current genomic technology classes: cytogenic (low resolution) and molecular (poor sensitivity for structural variation). Accurate assessment of genomic structural variation is important because some tumors acquire growths advantage by amplifying or creating oncogenes by fusing otherwise non-pathogenic genes and by deleting/inactivating tumor suppressor genes. Fusions are generally detected through targeted assays like NGS panels, PCR and FISH, or through karyotyping. However, high-resolution genome-wide approaches to detect fusions are needed. Optical Genome Mapping (OGM) is able fill this gap, providing high resolution (∼3kbp breakpoint precision) and ability to span repetitive and complicated genomic regions. Here we demonstrate a simple OGM workflow for the analysis of tumor biopsies, applying it to varying types (bladder, brain, breast, colon, kidney, liver, lung, ovary, prostate, tongue, thyroid). High-molecular weight DNA is isolated from snap-frozen tissue from 6.5-18 mg biopsies, then processed for OGM. The resulting analyses produced variants annotated and filtered within Bionano Access? software to enrich for somatic variants by filtering against a control database (?1% presence) and for those that overlap with gene(s). Based on this analysis, we have identified rearrangements and CNVs involving oncogenes and tumor suppressors (ERBB2, CDKN2A, NF1; many others) as well as numerous novel fusions in our comprehensive characterization of these complex genomes. [ABSTRACT FROM AUTHOR]

Structural variations (SV) play a key role in the pathogenesis of hematologic malignancies. Standard-of-care cytogenetic methods (SOC) including chromosome (karyotyping) and fluorescence in situ hybridization (FISH) analysis have inherent limitations, while NGS technologies have limited ability to detect most SVs. Optical genome mapping (OGM) is a high-resolution SV detection method that overcomes multiple issues including culture or amplification biases. This study describes the validation of OGM as a laboratory developed test (LDT) for hematologic malignancies conducted at Bionano Laboratories. A total of 77 datasets from blood or bone marrow specimens from 57 patients harboring a broad spectrum of SVs (30 with SOC results), 18 normal controls and two cancer cell lines were included in this validation. DNA isolation and labeling were performed using manufacturer's instructions. OGM data generated on the Bionano Saphyr© system was analyzed utilizing Bionano Access v1.7.2 and filtered based on disease or pan-cancer specific guideline files to identify variants known to be diagnostically or prognostically relevant. Accuracy studies showed high concordance with SOC results; sensitivity was 100% and specificity was 100% for guideline-based variants (NCCN, WHO, and NHS). Repeatability and reproducibility were 100% and 96%, respectively. Variant-specific lower limit of detection was also determined for 6 different SV types and ranged from 4.1% (inversions) to 11% (trisomy). In conclusion, in strong agreement with recent publications, this study validates the clinical utility of OGM for diagnostic evaluation of hematologic malignancies and its implementation is predicted to enhance accurate diagnosis, classification, prognostication, therapy selection, and disease monitoring. [ABSTRACT FROM AUTHOR]

Simple Summary: Whilst association of genetic mutations with targeted therapies is common in leukemia, to our knowledge, no one has associated genomic structural variants with drug sensitivities. Here we use optical genome mapping as an unbiased, genome-wide detection method for structural variants and show that some of these events are associated with sensitivity or resistance to clinically relevant anti-cancer drugs. Acute leukemia is a particularly problematic collection of hematological cancers, and, while somewhat rare, the survival rate of patients is typically abysmal without bone marrow transplantation. Furthermore, traditional chemotherapies used as standard-of-care for patients cause significant side effects. Understanding the evolution of leukemia to identify novel targets and, therefore, drug treatment regimens is a significant medical need. Genomic rearrangements and other structural variations (SVs) have long been known to be causative and pathogenic in multiple types of cancer, including leukemia. These SVs may be involved in cancer initiation, progression, clonal evolution, and drug resistance, and a better understanding of SVs from individual patients may help guide therapeutic options. Here, we show the utilization of optical genome mapping (OGM) to detect known and novel SVs in the samples of patients with leukemia. Importantly, this technology provides an unprecedented level of granularity and quantitation unavailable to other current techniques and allows for the unbiased detection of novel SVs, which may be relevant to disease pathogenesis and/or drug resistance. Coupled with the chemosensitivities of these samples to FDA-approved oncology drugs, we show how an impartial integrative analysis of these diverse datasets can be used to associate the detected genomic rearrangements with multiple drug sensitivity profiles. Indeed, an insertion in the gene MUSK is shown to be associated with increased sensitivity to the clinically relevant agent Idarubicin, while partial tandem duplication events in the KMT2A gene are related to the efficacy of another frontline treatment, Cytarabine. [ABSTRACT FROM AUTHOR]

Homologous recombination deficiency (HRD) is characterized by the inability of a cell to repair the double-stranded breaks using the homologous recombination repair (HRR) pathway. The deficiency of the HRR pathway results in defective DNA repair, leading to genomic instability and tumorigenesis. The presence of HRD has been found to make tumors sensitive to ICL-inducing platinum-based therapies and poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitors (PARPi). However, there are no standardized methods to measure and report HRD phenotypes. Herein, we compare optical genome mapping (OGM), chromosomal microarray (CMA), and a 523-gene NGS panel for HRD score calculations. This retrospective study included the analysis of 196 samples, of which 10 were gliomas, 176 were hematological malignancy samples, and 10 were controls. The 10 gliomas were evaluated with both CMA and OGM, and 30 hematological malignancy samples were evaluated with both the NGS panel and OGM. To verify the scores in a larger cohort, 135 cases were evaluated with the NGS panel and 71 cases with OGM. The HRD scores were calculated using a combination of three HRD signatures that included loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transitions (LST). In the ten glioma cases analyzed with OGM and CMA using the same DNA (to remove any tumor percentage bias), the HRD scores (mean ± SEM) were 13.2 (±4.2) with OGM compared to 3.7 (±1.4) with CMA. In the 30 hematological malignancy cases analyzed with OGM and the 523-gene NGS panel, the HRD scores were 7.6 (±2.2) with OGM compared to 2.6 (±0.8) with the 523-gene NGS panel. OGM detected 70.8% and 66.8% of additional variants that are considered HRD signatures in gliomas and hematological malignancies, respectively. The higher sensitivity of OGM to capture HRD signature variants might enable a more accurate and precise correlation with response to PARPi and platinum-based drugs. This study reveals HRD signatures that are cryptic to current standard of care (SOC) methods used for assessing the HRD phenotype and presents OGM as an attractive alternative with higher resolution and sensitivity to accurately assess the HRD phenotype. [ABSTRACT FROM AUTHOR]

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality. [ABSTRACT FROM AUTHOR]

The Hawaiian monk seal (HMS) is the single extant species of tropical earless seals of the genus Neomonachus. The species survived a severe bottleneck in the late 19th century and experienced subsequent population declines until becoming the subject of a NOAA-led species recovery effort beginning in 1976 when the population was fewer than 1000 animals. Like other recovering species, the Hawaiian monk seal has been reported to have reduced genetic heterogeneity due to the bottleneck and subsequent inbreeding. Here, we report a chromosomal reference assembly for a male animal produced using a variety of methods. The final assembly consisted of 16 autosomes, an X, and portions of the Y chromosomes. We compared variants in this animal to other HMS and to a frequently sequenced human sample, confirming about 12% of the variation seen in man. To confirm that the reference animal was representative of the HMS, we compared his sequence to that of 10 other individuals and noted similarly low variation in all. Variation in the major histocompatibility (MHC) genes was nearly absent compared to the orthologous human loci. Demographic analysis predicts that Hawaiian monk seals have had a long history of small populations preceding the bottleneck, and their current low levels of heterozygosity may indicate specialization to a stable environment. When we compared our reference assembly to that of other species, we observed significant conservation of chromosomal architecture with other pinnipeds, especially other phocids. This reference should be a useful tool for future evolutionary studies as well as the long-term management of this species. [ABSTRACT FROM AUTHOR]

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease. [ABSTRACT FROM AUTHOR]

Genomic structural variants comprise a significant fraction of somatic mutations driving cancer onset and progression. However, such variants are not readily revealed by standard next-generation sequencing. Optical genome mapping (OGM) surpasses short-read sequencing in detecting large (>500 bp) and complex structural variants (SVs) but requires isolation of ultra-high-molecular-weight DNA from the tissue of interest. We have successfully applied a protocol involving a paramagnetic nanobind disc to a wide range of solid tumors. Using as little as 6.5 mg of input tumor tissue, we show successful extraction of high-molecular-weight genomic DNA that provides a high genomic map rate and effective coverage by optical mapping. We demonstrate the system's utility in identifying somatic SVs affecting functional and cancer-related genes for each sample. Duplicate/triplicate analysis of select samples shows intra-sample reliability but also intra-sample heterogeneity. We also demonstrate that simply filtering SVs based on a GRCh38 human control database provides high positive and negative predictive values for true somatic variants. Our results indicate that the solid tissue DNA extraction protocol, OGM and SV analysis can be applied to a wide variety of solid tumors to capture SVs across the entire genome with functional importance in cancer prognosis and treatment. [ABSTRACT FROM AUTHOR]