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DNA copy number variants (CNVs) account for approximately 300 Mb of sequence variation in the normal human genome. Significant numbers of pathogenic CNVs contribute toward human genetic disorders. Recent studies suggest a higher diagnostic and clinical significance of low-pass genome sequencing (LP-GS) compared with chromosomal microarrays (CMAs). The performance metrics of the 5X LP-GS was compared with CMA to validate a low-cost and high-throughput method. LP-GS test performed on 409 samples (including 78 validation and 331 clinical) was evaluated using American College of Medical Genetics and Genomics guidelines. The CNV accuracy, precision, specificity, and sensitivity were calculated to be 100% for all previously characterized CNVs by CMA. Samples (n = 6) run at both approximately 30X GS and approximately 5X GS (LP-GS) average depth detected a concordance of 89.43% to 91.8% and 77.42% to 89.86% for overall single-nucleotide variants and insertions/deletions, respectively. In the 331 clinical samples, 17.2% each were classified as pathogenic/likely pathogenic and uncertain clinical significance. In addition, several cases with pathogenic CNVs were detected that were missed by CMA. This study demonstrates that LP-GS (5X GS) was able to reliably detect absence of heterozygosity, microdeletion/microduplication syndromes, and intragenic CNVs with higher coverage and resolution over the genome. Because of lower cost, higher resolution, and greater sensitivity of this test, our study in combination with other reports could be used in an evidence-based review by professional societies to recommend replacing CMAs.

The prognostic role of cytogenetic analysis is well-established in B-cell chronic lymphocytic leukemia (CLL). Approximately 80% of patients have a cytogenetic aberration. Interphase FISH panels have been the gold standard for cytogenetic evaluation, but conventional cytogenetics allows detection of additional abnormalities, including translocations, complex karyotypes and multiple clones. Whole genome copy number assessment, currently performed by chromosomal microarray analysis (CMA), is particularly relevant in CLL for the following reasons: (1) copy number alterations (CNAs) represent key events with biologic and prognostic significance; (2) DNA from fresh samples is generally available; and (3) the tumor burden tends to be relatively high in peripheral blood. CMA also identifies novel copy number variants and copy-neutral loss-of-heterozygosity (CN-LOH), and can refine deletion breakpoints. The Cancer Genomics Consortium (CGC) Working Group for CLL has performed an extensive literature review to describe the evidence-based clinical utility of CMA in CLL. We provide suggestions for the integration of CMA into clinical use and list recurrent copy number alterations, regions of CN-LOH and mutated genes to aid in interpretation.

We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/μl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.

This review discusses the current testing methodologies for COVID-19 diagnosis and explores next-generation sequencing (NGS) technology for the detection of SARS-CoV-2 and monitoring phylogenetic evolution in the current COVID-19 pandemic. The review addresses the development, fundamentals, assay quality control and bioinformatics processing of the NGS data. This article provides a comprehensive review of the obstacles and opportunities facing the application of NGS technologies for the diagnosis, surveillance, and study of SARS-CoV-2 and other infectious diseases. Further, we have contemplated the opportunities and challenges inherent in the adoption of NGS technology as a diagnostic test with real-world examples of its utility in the fight against COVID-19.

Global medical associations (ACOG, ISUOG, ACMG) recommend diagnostic prenatal testing for the detection and prevention of genetic disorders. Historically, cytogenetic methods such as karyotype analysis, fluorescent in situ hybridization (FISH), and chromosomal microarray (CMA) are utilized worldwide to diagnose common syndromes. However, the limitations of each of these methods, either performed in tandem or simultaneously, demonstrates the need of a revolutionary technology that can alleviate the need of multiple technologies. Optical genome mapping (OGM) is a novel technology that fills this void by being able to detect all classes of structural variations (SVs), including copy number variations (CNVs). OGM is being adopted by laboratories as a next-generation cytogenomic tool for both postnatal constitutional genetic disorders and hematological malignancies. This commentary highlights the potential of OGM to become a standard of care in prenatal genetic testing by its ability to identify large balanced and unbalanced SVs (currently the strength of karyotyping and metaphase FISH), CNVs (by CMA), repeat contraction disorders (by Southern blotting) and multiple repeat expansion disorders (by PCR based methods or Southern blotting). Also, next-generation sequencing (NGS) methods are excellent at detecting sequence variants but are unable to accurately detect the repeat regions of the genome which limits the ability to detect all classes of SVs. Notably, multiple molecular methods are used to identify repeat expansion and contraction disorders in routine clinical laboratories around the world. With non-invasive prenatal screening test (NIPT) as the standard of care screening assay for all global pregnancies, we anticipate OGM as a high-resolution cytogenomic diagnostic tool employed following a positive NIPT screen or for high-risk pregnancies with an abnormal ultrasound. Accurate detection of all types of genetic disorders by OGM, such as liveborn aneuploidies, sex chromosome anomalies, microdeletion/microduplication syndromes, repeat expansion/contraction disorders is key to reducing the global burden of genetic disorders.

Molecular diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) involves a two-tiered approach for detection of deletions/duplications using MLPA or array CGH, followed by sequencing of coding and flanking intronic regions to detect sequence variants, which is time-consuming and expensive. We have developed a comprehensive next-generation sequencing (NGS)-based single-step assay to sequence the entire 2.2 Mb of the DMD gene to detect all copy number and sequence variants in both index males and carrier females. Assay validation was 100% concordant with other methodologies. A total of 772 samples have been tested, of which 62% (N = 480) were index cases with a clinical suspicion of DMD. Carrier testing females account for 38% (N = 292). Molecular diagnosis was confirmed in 86% (N = 413) of the index cases. Intragenic deletions and duplications (single-exon or multi-exon) were detected in 60% (N = 247) and 14% (N = 58) of the index cases, respectively. Full-sequence analysis of the entire gene allows for detection of deep intronic pathogenic variants and accurate breakpoint detection of CNVs involving similar exons, which could have an impact on the outcome of clinical trials. This comprehensive assay is highly sensitive for diagnostic testing for DMD and is also suitable for confirmatory testing for newborn screening for DMD.

BackgroundThe varied clinical manifestations and outcomes in patients with SARS-CoV-2 infections implicate a role of host-genetics in the predisposition to disease severity. This is supported by evidence that is now emerging, where initial reports identify common risk factors and rare genetic variants associated with high risk for severe/ life-threatening COVID-19. Impressive global efforts have focused on either identifying common genetic factors utilizing short-read sequencing data in Genome-Wide Association Studies (GWAS) or whole-exome and genome studies to interrogate the human genome at the level of detecting single nucleotide variants (SNVs) and short indels. However, these studies lack the sensitivity to accurately detect several classes of variants, especially large structural variants (SVs) including copy number variants (CNVs), which account for a substantial proportion of variation among individuals. Thus, we investigated the host genomes of individuals with severe/life-threatening COVID-19 at the level of large SVs (500bp-Mb level) to identify events that might provide insight into the inter-individual clinical variability in clinical course and outcomes of COVID-19 patients.MethodsOptical genome mapping using Bionano’s Saphyr® system was performed on thirty-seven severely ill COVID-19 patients admitted to intensive care units (ICU). To extract candidate SVs, three distinct analyses were undertaken. First, an unbiased whole-genome analysis of SVs was performed to identify rare/unique genic SVs in these patients that did not appear in population datasets to determine candidate loci as decisive predisposing factors associated with severe COVID-19. Second, common SVs with a population frequency filter was interrogated for possible association with severe COVID-19 based on literature surveys. Third, genome-wide SV enrichment in severely ill patients versus the general population was investigated by calculating odds ratios to identify top-ranked genes/loci. Candidate SVs were confirmed using qPCR and an independent bioinformatics tool (FaNDOM).ResultsOur patient-centric investigation identified 11 SVs involving 38 genes implicated in three key host-viral interaction pathways: (1) innate immunity and inflammatory response, (2) airway resistance to pathogens, and (3) viral replication, spread, and RNA editing. These included seven rare/unique SVs (not present in the control dataset), identified in 24.3% (9/37) of patients, impacting up to 31 genes, of whichSTK26andDPP4are the most promising candidates. A duplication partially overlappingSTK26was corroborated with data showing upregulation of this gene in severely ill patients. Further, using a population frequency filter of less than 20% in the Bionano control dataset, four SVs involving seven genes were identified in 56.7% (21/37) of patients.ConclusionThis study is the first to systematically assess and highlight SVs’ potential role in the pathogenesis of COVID-19 severity. The genes implicated here identify novel SVs, especiallySTK26, and extend previous reports involving innate immunity and type I interferon response in the pathogenesis of COVID-19. Our study also shows that optical genome mapping can be a powerful tool to identify large SVs impacting disease outcomes with split survival and add valuable genomic information to the existing sequencing-based technology databases to understand the inter-individual variability associated with SARS-CoV-2 infections and COVID-19 mortality.

Chromosomal aberrations and structural variations (SVs) are major causes of human genetic diseases and cancer. Their detection is essential for diagnosis, prognosis, patient management, and cytogenomic basic research. Current cytogenetic workflows rely on the combination of multiple technologies [(molecular) karyotyping, chromosome microarray] to interrogate the genome for the presence of SVs. Each approach presents its own limitation in terms of size resolution, detection of balanced events, or restriction to predefined loci of clinical interest. Next-generation sequencing technologies have improved genetic diagnosis of disorders resulting from detection of single-nucleotide variants (SNVs) and “indels” but have failed to efficiently call SV found by cytogenetic methodologies. Therefore, cytogenetic methods are still essential for detecting anomalies in many disorders. Optical genome mapping (OGM) leverages direct visualization of extremely long DNA molecules to allow for the accurate automatic identification of clinically significant balanced and unbalanced SVs in clinical samples. We describe different applications of OGM for the study of complex repeat disorders, prenatal and postnatal cases, hematologic, and solid tumor genomes. Moreover, we describe how OGM accurately identifies compelling leads for patients with no genetic diagnosis based on standard of care tests, resolving unsolved cases and shedding light on SVs in region inaccessible to other technologies. Last, we report the results of different benchmarking studies highlighting the outstanding concordance between OGM and classical cytogenetic workflows, with the addition of clinically relevant additional structural alterations previously missed by gold standard tests. Taken together, OGM shows the ability to overwhelmingly replace traditional cytogenetic methodologies as well as dramatically improve sensitivity to a wide range of SV between SNV and larger indels.