Detection of Genomic Structural Variations Associated with Drug Sensitivity and Resistance in Acute Leukemia.

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.

Analytic Validation of Optical Genome Mapping in Hematological Malignancies.

Structural variations (SVs) play a key role in the pathogenicity of hematological malignancies. Standard-of-care (SOC) methods such as karyotyping and fluorescence in situ hybridization (FISH), which have been employed globally for the past three decades, have significant limitations in terms of resolution and the number of recurrent aberrations that can be simultaneously assessed, respectively. Next-generation sequencing (NGS)-based technologies are now widely used to detect clinically significant sequence variants but are limited in their ability to accurately detect SVs. Optical genome mapping (OGM) is an emerging technology enabling the genome-wide detection of all classes of SVs at a significantly higher resolution than karyotyping and FISH. OGM requires neither cultured cells nor amplification of DNA, addressing the limitations of culture and amplification biases. This study reports the clinical validation of OGM as a laboratory-developed test (LDT) according to stringent regulatory (CAP/CLIA) guidelines for genome-wide SV detection in different hematological malignancies. In total, 60 cases with hematological malignancies (of various subtypes), 18 controls, and 2 cancer cell lines were used for this study. Ultra-high-molecular-weight DNA was extracted from the samples, fluorescently labeled, and run on the Bionano Saphyr system. A total of 215 datasets, Inc.luding replicates, were generated, and analyzed successfully. Sample data were then analyzed using either disease-specific or pan-cancer-specific BED files to prioritize calls that are known to be diagnostically or prognostically relevant. Sensitivity, specificity, and reproducibility were 100%, 100%, and 96%, respectively. Following the validation, 14 cases and 10 controls were run and analyzed using OGM at three outside laboratories showing reproducibility of 96.4%. OGM found more clinically relevant SVs compared to SOC testing due to its ability to detect all classes of SVs at higher resolution. The results of this validation study demonstrate the superiority of OGM over traditional SOC methods for the detection of SVs for the accurate diagnosis of various hematological malignancies.

Comparative Benchmarking of Optical Genome Mapping and Chromosomal Microarray Reveals High Technological Concordance in CNV Identification and Additional Structural Variant Refinement.

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.

Optical Genome Mapping: Integrating Structural Variations for Precise Homologous Recombination Deficiency Score Calculation.

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.

Clinical Utility of Optical Genome Mapping and 523-Gene Next Generation Sequencing Panel for Comprehensive Evaluation of Myeloid Cancers.

The standard-of-care (SOC) for genomic testing of myeloid cancers primarily relies on karyotyping/fluorescent in situ hybridization (FISH) (cytogenetic analysis) and targeted gene panels (usually ≤54 genes) that harbor hotspot pathogenic variants (molecular genetic analysis). Despite this combinatorial approach, ~50% of myeloid cancer genomes remain cytogenetically normal, and the limited sequencing variant profiles obtained from targeted panels are unable to resolve the molecular etiology of many myeloid tumors. In this study, we evaluated the performance and clinical utility of combinatorial use of optical genome mapping (OGM) and a 523-gene next-generation sequencing (NGS) panel for comprehensive genomic profiling of 30 myeloid tumors and compared it to SOC cytogenetic methods (karyotyping and FISH) and a 54-gene NGS panel. OGM and the 523-gene NGS panel had an analytical concordance of 100% with karyotyping, FISH, and the 54-gene panel, respectively. Importantly, the IPSS-R cytogenetic risk group changed from very good/good to very poor in 22% of MDS (2/9) cases based on comprehensive profiling (karyotyping, FISH, and 54-gene panel vs. OGM and 523-gene panel), while additionally identifying six compound heterozygous events of potential clinical relevance in six cases (6/30, 20%). This cost-effective approach of using OGM and a 523-gene NGS panel for comprehensive genomic profiling of myeloid cancers demonstrated increased yield of actionable targets that can potentially result in improved clinical outcomes.

Clinical Validation and Diagnostic Utility of Optical Genome Mapping in Prenatal Diagnostic Testing.

The standard-of-care diagnostic prenatal testing includes a combination of cytogenetic methods, such as karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA), using either direct or cultured amniocytes or chorionic villi sampling. However, each technology has its limitations: karyotyping has a low resolution (>5 Mb), FISH is targeted, and CMA does not detect balanced structural variations (SVs). These limitations necessitate the use of multiple tests, either simultaneously or sequentially, to reach a genetic diagnosis. Optical genome mapping (OGM) is an emerging technology that can detect several classes of SVs in a single assay, but it has not been evaluated in the prenatal setting. This validation study analyzed 114 samples that were received in our laboratory for traditional cytogenetic analysis with karyotyping, FISH, and/or CMA. OGM was 100% concordant in identifying the 101 aberrations that included 29 interstitial/terminal deletions, 28 duplications, 26 aneuploidies, 6 absence of heterozygosity regions, 3 triploid genomes, 4 isochromosomes, and 1 translocation; and the method revealed the identity of 3 marker chromosomes and 1 chromosome with additional material not determined by karyotyping. In addition, OGM detected 64 additional clinically reportable SVs in 43 samples. OGM has a standardized laboratory workflow and reporting solution that can be adopted in routine clinical laboratories and demonstrates the potential to replace the current standard-of-care methods for prenatal diagnostic testing.

Genetic Predisposition to Neurological Complications in Patients with COVID-19.

Several studies have identified rare and common genetic variants associated with severe COVID-19, but no study has reported genetic determinants as predisposition factors for neurological complications. In this report, we identified rare/unique structural variants (SVs) implicated in neurological functions in two individuals with neurological manifestations of COVID-19. This report highlights the possible genetic link to the neurological symptoms with COVID-19 and calls for a collective effort to study these cohorts for a possible genetic linkage.

Structural variant analysis of a cancer reference cell line sample using multiple sequencing technologies.

BACKGROUND: The cancer genome is commonly altered with thousands of structural rearrangements including insertions, deletions, translocation, inversions, duplications, and copy number variations. Thus, structural variant (SV) characterization plays a paramount role in cancer target identification, oncology diagnostics, and personalized medicine. As part of the SEQC2 Consortium effort, the present study established and evaluated a consensus SV call set using a breast cancer reference cell line and matched normal control derived from the same donor, which were used in our companion benchmarking studies as reference samples. RESULTS: We systematically investigated somatic SVs in the reference cancer cell line by comparing to a matched normal cell line using multiple NGS platforms including Illumina short-read, 10X Genomics linked reads, PacBio long reads, Oxford Nanopore long reads, and high-throughput chromosome conformation capture (Hi-C). We established a consensus SV call set of a total of 1788 SVs including 717 deletions, 230 duplications, 551 insertions, 133 inversions, 146 translocations, and 11 breakends for the reference cancer cell line. To independently evaluate and cross-validate the accuracy of our consensus SV call set, we used orthogonal methods including PCR-based validation, Affymetrix arrays, Bionano optical mapping, and identification of fusion genes detected from RNA-seq. We evaluated the strengths and weaknesses of each NGS technology for SV determination, and our findings provide an actionable guide to improve cancer genome SV detection sensitivity and accuracy. CONCLUSIONS: A high-confidence consensus SV call set was established for the reference cancer cell line. A large subset of the variants identified was validated by multiple orthogonal methods.

Clinical Validation and Diagnostic Utility of Optical Genome Mapping for Enhanced Cytogenomic Analysis of Hematological Neoplasms.

The current standard-of-care cytogenetic techniques for the analysis of hematological malignancies include karyotyping, fluorescence in situ hybridization, and chromosomal microarray, which are labor intensive and time and cost prohibitive, and they often do not reveal the genetic complexity of the tumor, demonstrating the need for alternative technology for better characterization of these tumors. Herein, we report the results from our clinical validation study and demonstrate the utility of optical genome mapping (OGM), evaluated using 92 sample runs (including replicates) that included 69 well-characterized unique samples (59 hematological neoplasms and 10 controls). The technical performance (quality control metrics) resulted in 100% first-pass rate, with analytical performance (concordance) showing a sensitivity of 98.7%, a specificity of 100%, and an accuracy of 99.2%. OGM demonstrated robust technical, analytical performance, and interrun, intrarun, and interinstrument reproducibility. The limit of detection was determined to be at 5% allele fraction for aneuploidy, translocation, interstitial deletion, and duplication. OGM identified several additional structural variations, revealing the genomic architecture in these neoplasms that provides an opportunity for better tumor classification, prognostication, risk stratification, and therapy selection. Overall, OGM has outperformed the standard-of-care tests in this study and demonstrated its potential as a first-tier cytogenomic test for hematologic malignancies.

Optical Genome Mapping and Single Nucleotide Polymorphism Microarray: An Integrated Approach for Investigating Products of Conception.

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, as well as microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from formalin-fixed paraffin embedded (FFPE) tissue with macro-dissection for chorionic villi to enrich fetal tissue component for single nucleotide polymorphism microarray (SNPM) analysis. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC-negative cases or SNPM with histological assessment for MCC-positive cases. A result was obtained in 93% (29/31) of cases with a diagnostic yield of 45.1% (14/31) with the proposed workflow, compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

A high-quality bonobo genome refines the analysis of hominid evolution.

The divergence of chimpanzee and bonobo provides one of the few examples of recent hominid speciation1,2. Here we describe a fully annotated, high-quality bonobo genome assembly, which was constructed without guidance from reference genomes by applying a multiplatform genomics approach. We generate a bonobo genome assembly in which more than 98% of genes are completely annotated and 99% of the gaps are closed, including the resolution of about half of the segmental duplications and almost all of the full-length mobile elements. We compare the bonobo genome to those of other great apes1,3-5 and identify more than 5,569 fixed structural variants that specifically distinguish the bonobo and chimpanzee lineages. We focus on genes that have been lost, changed in structure or expanded in the last few million years of bonobo evolution. We produce a high-resolution map of incomplete lineage sorting and estimate that around 5.1% of the human genome is genetically closer to chimpanzee or bonobo and that more than 36.5% of the genome shows incomplete lineage sorting if we consider a deeper phylogeny including gorilla and orangutan. We also show that 26% of the segments of incomplete lineage sorting between human and chimpanzee or human and bonobo are non-randomly distributed and that genes within these clustered segments show significant excess of amino acid replacement compared to the rest of the genome.

Identification of Somatic Structural Variants in Solid Tumors by Optical Genome Mapping.

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.

Population genomics of the critically endangered kakapo.

The kakapo is a flightless parrot endemic to New Zealand. Once common in the archipelago, only 201 individuals remain today, most of them descending from an isolated island population. We report the first genome-wide analyses of the species, including a high-quality genome assembly for kakapo, one of the first chromosome-level reference genomes sequenced by the Vertebrate Genomes Project (VGP). We also sequenced and analyzed 35 modern genomes from the sole surviving island population and 14 genomes from the extinct mainland population. While theory suggests that such a small population is likely to have accumulated deleterious mutations through genetic drift, our analyses on the impact of the long-term small population size in kakapo indicate that present-day island kakapo have a reduced number of harmful mutations compared to mainland individuals. We hypothesize that this reduced mutational load is due to the island population having been subjected to a combination of genetic drift and purging of deleterious mutations, through increased inbreeding and purifying selection, since its isolation from the mainland ∼10,000 years ago. Our results provide evidence that small populations can survive even when isolated for hundreds of generations. This work provides key insights into kakapo breeding and recovery and more generally into the application of genetic tools in conservation efforts for endangered species.

Fully Phased Sequence of a Diploid Human Genome Determined de Novo from the DNA of a Single Individual.

In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable.

Haplotype-resolved genomes provide insights into structural variation and gene content in Angus and Brahman cattle.

Inbred animals were historically chosen for genome analysis to circumvent assembly issues caused by haplotype variation but this resulted in a composite of the two genomes. Here we report a haplotype-aware scaffolding and polishing pipeline which was used to create haplotype-resolved, chromosome-level genome assemblies of Angus (taurine) and Brahman (indicine) cattle subspecies from contigs generated by the trio binning method. These assemblies reveal structural and copy number variants that differentiate the subspecies and that variant detection is sensitive to the specific reference genome chosen. Six genes with immune related functions have additional copies in the indicine compared with taurine lineage and an indicus-specific extra copy of fatty acid desaturase is under positive selection. The haplotyped genomes also enable transcripts to be phased to detect allele-specific expression. This work exemplifies the value of haplotype-resolved genomes to better explore evolutionary and functional variations.

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.

Long-read single-molecule maps of the functional methylome.

We report on the development of a methylation analysis workflow for optical detection of fluorescent methylation profiles along chromosomal DNA molecules. In combination with Bionano Genomics genome mapping technology, these profiles provide a hybrid genetic/epigenetic genome-wide map composed of DNA molecules spanning hundreds of kilobase pairs. The method provides kilobase pair-scale genomic methylation patterns comparable to whole-genome bisulfite sequencing (WGBS) along genes and regulatory elements. These long single-molecule reads allow for methylation variation calling and analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell second-generation sequencing. The method is applied here to study facioscapulohumeral muscular dystrophy (FSHD), simultaneously recording the haplotype, copy number, and methylation status of the disease-associated, highly repetitive locus on Chromosome 4q.

Genome maps across 26 human populations reveal population-specific patterns of structural variation.

Large structural variants (SVs) in the human genome are difficult to detect and study by conventional sequencing technologies. With long-range genome analysis platforms, such as optical mapping, one can identify large SVs (>2 kb) across the genome in one experiment. Analyzing optical genome maps of 154 individuals from the 26 populations sequenced in the 1000 Genomes Project, we find that phylogenetic population patterns of large SVs are similar to those of single nucleotide variations in 86% of the human genome, while ~2% of the genome has high structural complexity. We are able to characterize SVs in many intractable regions of the genome, including segmental duplications and subtelomeric, pericentromeric, and acrocentric areas. In addition, we discover ~60 Mb of non-redundant genome content missing in the reference genome sequence assembly. Our results highlight the need for a comprehensive set of alternate haplotypes from different populations to represent SV patterns in the genome.

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

Author Correction: The axolotl genome and the evolution of key tissue formation regulators.

In the originally published version of this Article, the sequenced axolotl strain (the homozygous white mutant) was denoted as 'D/D' rather than 'd/d' in Fig. 1a and the accompanying legend, the main text and the Methods section. The original Article has been corrected online.