The Journal of Molecular Diagnostics

BackgroundLiquid biopsy (LBx) assays are transforming precision oncology by the screening of genomic alterations in cfDNA. These assays provide a less invasive alternative to tissue biopsies, which are not always feasible. Molecular pathology laboratories require LBx assays that detect variants at low allele frequencies using standardized methods. MethodsThis study evaluated the Hedera Profiling 2 ctDNA test panel (HP2) (Hedera Dx, Epalinges, Switzerland), a hybrid capture-based NGS assay for the detection of somatic alterations from cfDNA. Covering 32 genes, HP2 enables the detection of SNVs, Indels, Fusions, CNVs, and MSI status from a single DNA-only workflow. The analytical performance was assessed using reference standards and a diverse cohort of 137 clinical samples pre-characterized by orthogonal methods. ResultsIn reference standards at 0.5% VAF, detection sensitivity and specificity for SNVs/Indels were 96.92% and 99.67%, respectively, and 100% each for Fusions. For MSI with VAFs of [≥]1% and CNVs with VAFs of [≥] 2% both achieved 100% sensitivity. ConclusionThis international, multicenter analytical performance evaluation study across a large number of hospital laboratories demonstrated high concordance of HP2 assay with orthogonal methods, confirming its significant potential as a highly sensitive, and efficient Pan-Cancer test for future decentralized LBx testing.

Evaluation of the test performance of the targeted enhanced whole-genome sequencing (TE-WGS) assay for comprehensive oncology genomic profiling. The analytical validation of the assay included sensitivity and specificity for single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs), revealing a revealed a sensitivity of 99.8% for SNVs and 99.2% for indels. The PPV was 99.3% for SNVs and 98.7% for indels. Clinical validation was benchmarked against established orthogonal methods and demonstrated high concordance with reference methods in variant characterization. The TE-WGS assay enhances personalized cancer treatment by offering detailed genomic insights and the adaptability to include emerging biomarkers.

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), employed globally for the past three decades have significant limitations in the resolution or 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 neither requires cultured cells nor amplification of DNA and hence addresses the limitations of culture and amplification biases. This study reports the clinical validation of OGM as a laboratory developed test (LDT), according to CLIA guidelines, for genome-wide SV detection in different hematological malignancies. In total, 68 cases with hematological malignancies (of various subtypes), 27 controls and two 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 Genomics Saphyr system. A total of 207 datasets, including replicates, were generated and 100% could be 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. Accuracy, precision, PPV and NPV were all 100% against standard of care results. Sensitivity, specificity, and reproducibility were 100%, 100% and 96%, respectively. Following the validation, 11 cases were run and analyzed using OGM at three additional sites. OGM found more clinically relevant SVs compared to SOC testing due to its ability to detect all classes of SVs at much higher resolution. The results of this validation study demonstrate OGMs superiority over traditional SOC methods for the detection of SVs for the accurate diagnosis of various hematological malignancies.

BackgroundSpinal Muscular Atrophy (SMA) is one of the most common recessive disorders for which several life-saving treatment options are currently available. It is essential to establish universal SMA screening and diagnostic programs using scalable, cost-effective and accessible platforms to accurately identify all variation types, which is complicated by homologous SMN1 and SMN2 genes. MethodsWe developed a dual-mode PCR-based target enrichment that generates 2.7 to 11.2 kb amplicons spanning SMN1 and SMN2 genes for any-length nanopore sequencing. We trained a variant calling model that utilizes paralog-specific sequences and read-depth data to accurately detect sequence and copy number variants specific to each gene. ResultsWe present results from the development, optimization, and external evaluation of this assay using over 750 samples, including cell lines, residual presumed normal blood donors, and patients with known SMN1 and SMN2 genotypes. The assay detects SNVs, indels, and CNVs with >98% accuracy across all sample sets, with a highly dynamic throughput range, relatively fast turnaround time, and limited hands-on-time. Together with the modest capital investment and consumable costs per sample, this assay can help increase access to SMA testing in low- and middle-income settings. ConclusionWe describe a PCR/Nanopore sequencing assay and a customized analysis pipeline for the comprehensive and accurate detection of variation at the SMA locus and demonstrate its scalability, cost-effectiveness, and potential for the universal implementation of SMA screening and diagnostic programs. Human GenesSMN1 survival of motor neuron 1, telomeric HGNC:11117 SMN2 survival of motor neuron 2, centromeric HGNC:11118 CFTR CF transmembrane conductance regulator HGNC:1884

PurposeCarrier screening for hereditary conditions is challenged by genes with complex genomic architecture, where short-read sequencing can fail to detect clinically relevant variants. This study evaluated a unified, amplification-based nanopore sequencing workflow across multiple laboratories for comprehensive analysis of such loci. MethodsA modular long-read sequencing assay was evaluated across five laboratories using targeted PCR enrichment, Oxford Nanopore sequencing, and automated variant analysis. The workflow interrogated genes associated with spinal muscular atrophy, thalassemia, cystic fibrosis, fragile X syndrome, congenital adrenal hyperplasia, Gaucher disease, and hemophilia A. Performance was assessed against orthogonal methods for single nucleotide variants (SNVs), indels, copy-number variants, repeat expansions, and structural rearrangements. ResultsAcross 882 unique samples (1,266 tests), overall agreement with comparator methods exceeded 96% for variant-level detection and 97% for genotype status classification. Long-read sequencing enabled phasing of paralogous loci, integrated sizing and interruption analysis for FMR1 repeats, and simultaneous detection of SNVs and structural variants in globin loci and CYP21A2-TNXB region, reducing reliance on multiple workflows. ConclusionThis multisite evaluation suggests that targeted long-read sequencing can consolidate complex variant detection into a single workflow, improving analytical completeness and operational efficiency for carrier screening.

BackgroundNext-generation sequencing (NGS) comprehensive genomic profiling panels provide targeted genome-wide detection of the somatic variant landscape of various cancer types. Recently, an innovative sequencing technology, Avidity Sequencing from Element Biosciences, has emerged to provide economical option for mid-throughput sequencing, with high quality (Q40). AimsThe aim of this study was to evaluate the performance characteristics of AVITI sequencing by avidity on detection of somatic variants from reference samples, and to compare the variant detection concordance between AVITI and NovaSeq sequencing platforms by evaluating the variant callings of 518 cancer-related genes using Burning Rocks OncoScreenTM Plus panel. MethodsContrived commercial reference control samples (harboring known variants) and clinical Formalin-Fixed Paraffin-Embedded tissue (FFPE) samples were examined to evaluate the variant detection performance of two sequencing platforms, with respects to Single Nucleotide Variant (SNV), Insertion-Deletion (Indel), Structure Variant (SV/Fusion), Copy Number Variation (CNV), Microsatellite Instability (MSI), and Tumor Mutation Burden (TMB). Variant specific QC metrics were developed to evaluate sequencing-level and variant calling level accuracy. All samples were processed utilizing the OncoScreenTM Plus assay, including library preparation and bioinformatics post sequencing data analysis. ResultsThe OncoScreenTM Plus assay is compatible with the AVITI system. Samples prepared with the OncoScreenTM Plus panel and sequenced on the AVITI system has successfully detected cancer related variants including SNV/Indels, SV/Fusion, CNVs, MSIs, and TMB, which were highly concordant with reference controls. In addition, the AVITI system produced a higher overall quality score, index assignment rate, mean target coverage, and lower optical duplication rate AVITI than NovaSeq system. ConclusionThe new sequencing technology Avidity from AVITI system can be applied seamlessly to current high-performance targeted oncology assays to reliably identify somatic variants in a flexible and cost-effective manner.

Accurate diagnosis and risk stratification of hematological malignancies require disease-specific laboratory testing procedures involving the use of hematopathology, flow cytometry, molecular, and cytogenetic testing. While individual laboratories develop unique workflows to accommodate volume, clinical needs, and staffing, cytogenetic laboratories generally require a multitude of targeted and genome-wide tests that detect clinically relevant aberrations in hematologic malignancies. Specifically, the frequent use of multiple FISH panels coupled with concurrent chromosome analysis, can be both labor, and resource intensive. Optical Genome Mapping (OGM) is a comprehensive cytogenetic solution for detecting structural variants with high resolution and increased accuracy for hematological malignancy subtypes at the DNA level without need of any cell culture regimens. A new software tool for analysis of OGM data called VIA (Variant Intelligence Applications), provides an integrative analysis, interpretation, and reporting solution for OGM and other datatypes. In this study, we performed retrospective review of 56 datasets, representing 10 unique myeloid cases to assess multi-user (technologist and laboratory director) analyses and classification. Interpretation and reporting of OGM results were 100% concordant between reviewers for four cases with negative results by standard of care (SOC) testing. For the other six cases, five pathognomonic gene fusions identified by SOC assays were unanimously reported as Tier 1A classification was unanimous for five sentinel gene fusion rearrangements identified by SOC. OGM also found additional structural variants of clinical relevance in five of the six cases that were not found by SOC methods. Leveraging automatic pre-classification of variants and a custom decision tree, the VIA software enabled complete analysis with a mean technologist review time (variant analysis and initial tier determination) of 30.7 minutes. The analysis, interpretation, and reporting workflow described in this pilot study provides a framework for standardized and streamlined reporting of clinically significant variant in myeloid malignancies using VIA.

BackgroundSensorineural hearing impairment (SNHI) is a common disorder with a significant genetic component. Genetic testing for SNHI often involves next-generation sequencing (NGS), but SNHI-related pathogenic STRC variants cannot be directly addressed by conventional NGS due to the complex genomic scenario derived from large genomic rearrangements and a highly homologous pseudogene. Long-read sequencing (LRS) offers an unprecedented resolution to these challenges. MethodsWe developed a comprehensive workflow that integrates the PacBio-based LRS approach with marker-mediated refinements to effectively address pseudogene contamination. This methodology was applied to analyze the STRC gene in a cohort of 100 unrelated Taiwanese patients diagnosed with SNHI of unknown genetic cause after first-tier NGS testing. ResultsWe identified bi-allelic STRC variants in 11 patients (11% diagnostic yield), including homozygous deletions, compound heterozygous deletions and conversions, and compound heterozygous SNVs and CNVs. In total, we detected STRC variants in 27 patients, with 81.6% of these variants occurring in patients with mild to moderate SNHI. ConclusionsThis study represents the first large-scale clinical investigation utilizing LRS technology for the genetic diagnosis of SNHI. Our study highlights the diagnostic capabilities of LRS in detecting complex variants within the STRC and advancing our understanding of the genetic etiology of SNHI that remains unresolved by conventional NGS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/24316795v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1fae5aforg.highwire.dtl.DTLVardef@fd6d2eorg.highwire.dtl.DTLVardef@b18c1org.highwire.dtl.DTLVardef@86364f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Liquid biopsy comprehensive genomic profiling (LB-CGP) testing is performed on circulating tumor DNA to detect tumor recurrence, predict prognosis, and select therapeutic agents. Pathogenic variants of germline origin in genes associated with hereditary tumor syndrome (HTS) can be simultaneously detected by CGP testing. Nonetheless, it is often challenging to differentiate whether the variants are of somatic or germline origin. The differentiation criteria were primarily based on the variant allele frequencies (VAFs). However, more evidence is needed to establish clear criteria, and it is often difficult to differentiate between variants based on VAF alone. In this study, using the national database of the Center for Cancer Genomics and Advanced Therapeutics, which accumulates real-world data on CGP testing in Japan, we analyzed 169,370 variants detected in 11,399 patients registered with FoundationOne Liquid CDx testing. By extracting the predominantly presumed somatic and germline variants, we established a criterion for VAF that could achieve high specificity and sensitivity. Further investigation into the detection status of other variants led to the development of an algorithm for differentiating somatic/germline variants of genes associated with HTSs. Based on this algorithm, 726 variants were extracted as presumed germline pathogenic variants among the 26 genes with high germline conversion rates in 710 patients in the study. This algorithm should help to discriminate with high accuracy whether the variants detected in LB-CGP tests are of somatic or germline origin, although further analyses are required to confirm the validity of this algorithm. Highlights- The highly accurate VAF criterion for differentiating somatic and germline variants was determined using real-world data from more than 11,000 patients who underwent liquid biopsy CGP testing. - To improve the specificity of the PGPV extraction, an additional criterion was defined: checking the status of other genomic alterations detected. - Criteria for considering information other than VAFs associated with the variant under PGPV consideration were developed to improve the sensitivity of PGPV extraction. - By integrating these criteria and previous evidence on germline conversion rates, a GeMSort algorithm was established. - Based on this algorithm, 726 variants from 710 patients were extracted as PGPVs among 26 hereditary tumor syndrome-associated genes with high germline conversion rates.

Currently, DNA and RNA are used separately to capture different types of gene mutations. DNA is commonly used for the detection of SNVs, indels and CNVs; RNA is used for analysis of gene fusion and gene expression. To perform both DNA sequencing (DNA-seq) and RNA-seq, material is divided into two copies, and two different procedures are required for sequencing. Due to overconsumption of samples and experimental process complexity, it is necessary to create an experimental method capable of analyzing SNVs, indels, fusions and expression. We developed an RNA-based hybridization capture panel targeting actionable driver oncogenes in solid tumors and corresponding sample preparation and bioinformatics workflows. Analytical validation with an RNA standard reference containing 16 known fusion mutations and 6 SNV mutations demonstrated a detection specificity of 100.0% [95% CI 88.7%~100.0%] for SNVs and 100.0% [95% CI 95.4%~100.0%] for fusions. The targeted RNA panel achieved a 0.73-2.63 copies/ng RNA lower limit of detection (LOD) for SNVs and 0.21-6.48 copies/ng RNA for fusions. Gene expression analysis revealed a correlation greater than 0.9 across all 15 cancer-related genes between the RNA-seq results and targeted RNA panel. Among 1253 NSCLC FFPE tumor samples, multiple mutation types were called from DNA- and RNA-seq data and compared between the two assays. The DNA panel detected 103 fusions and 21 METex14 skipping events; 124 fusions and 26 METex14 skipping events were detected by the target RNA panel; 21 fusions and 4 METex14 skipping events were only detected by the target RNA panel. Among the 173 NSCLC samples negative for targetable mutations by DNA-seq, 15 (15/173, 8.67%) showed targetable gene fusions that may change clinical decisions with RNA-seq. In total, 226 tier I and tier II missense variants for NSCLC were analyzed at genomic (DNA-seq) and transcriptomic (RNA-seq) levels. The positive percent agreement (PPA) was 97.8%, and the positive predictive value (PPV) was 98.6%. Interestingly, variant allele frequencies were generally higher at the RNA level than at the DNA level, suggesting relatively dominant expression of mutant alleles. PPA was 97.6% and PPV 99.38% for EGFR 19del and 20ins variants. We also explored the relationship of RNA expression with gene copy number and protein expression. The RPKM of EGFR transcripts assessed by the RNA panel showed a linear relationship with copy number quantified by the DNA panel, with an R of 0.8 in 1253 samples. In contrast, MET gene expression is regulated in a more complex manner. In IHC analysis, all 3+ samples exhibited higher RPKM levels; IHC level of 2+ and below showed lower RNA expression. Parallel DNA- and RNA-seq and systematic analysis demonstrated the accuracy and robustness of the RNA sequencing panel in identifying multiple types of variants for cancer therapy. Contact: zhaojia0327@126.com

Homologous Recombination Repair (HRR) testing has become increasingly important in clinical genomic labs due to the use of poly-ADP-ribose polymerase (PARP) inhibitor therapy for epithelial ovarian, fallopian tube, or peritoneum cancer. While sequencing and copy number variation analysis can identify patients with a pathogenic mutation in BRCA1 or BRCA2 who can benefit from PARPi therapy, there are also patients who may benefit but do not have these mutations. To address this, our lab has developed a test called HRD-One, in partnership with SOPHiA GENETICS, that can detect sequence variants in genes involved in HRR, as well as genomic scars that indicate Homologous Recombination Deficiency (HRD), which may be present even when a pathogenic variant is not detected. We tested 59 high-grade serous epithelial ovarian cancer samples using HRD-One and found that it had an overall categorical concordance of 94.74% with Myriads myChoice(R) score, which is a commercial HRD test. 12 out of 13 samples that carried a pathogenic or likely pathogenic variant in BRCA1/2 also had a positive HRD-One score, and 9 samples in which a pathogenic variant in BRCA1/2 was not identified had a positive score in both HRD-One and myChoice(R). Of the samples that passed quality control, we observed an average of 1.62 points variation between replicates on a scale from -25.0 to +25.0. We also found that low-confidence results were associated with a low DNA input and the age of FFPE blocks, while the estimated tumor percentage in the block, NGS library yield, and score of genomic instability did not have a significant association. We determined that blocks older than 3 years or with a DNA input of less than 25ng are not reliable for producing high-quality results. Finally, we validated the HRD-One test with SOPHiA Homologous Recombination Solution (Library Prep kit II) and correlated it to myChoice(R), and found that the AmoyDx(R) HRD Focus Panel had the same sensitivity but a higher number of false positive samples and therefore lower specificity. Overall, we have shown that HRD-One can provide a reproducible and concordant score for inferring HRD, and an HRD-One score of 2.0 or greater predicts HRD and correlates to Myriads myChoice(R) score of 42 in high-grade serous epithelial ovarian cancers samples that meet our minimum quality criteria.

BackgroundMicrosatellite instability (MSI) indicates DNA mismatch repair deficiency in cancers like colorectal cancer. The current gold standard technique, PCR/capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. Methods101 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared to the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical MSI-H samples. Analytical sensitivity, specificity, limit of detection and input requirements were assessed using synthetic commercial reference standards. ResultsVarTrace demonstrated 100% test success rate, 100% sensitivity and 98% specificity compared to OncoMate across 101 clinical FFPE samples. It detected MSI-H with 97% accuracy down to 10% tumor percentage. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. ConclusionsThis study validates VarTrace as a swift, accurate and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTraces capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical workflows and broadening the availability of this crucial test.

RNA sequencing is emerging as a powerful technique to detect a diverse array of fusions in human neoplasia, but few clinically validated assays have been described to date. We designed and validated a hybrid-capture RNAseq assay for FFPE tissue (Fusion-STAMP). It fully targets the transcript isoforms of 43 genes selected for their known impact as actionable targets of existing and emerging anti-cancer therapies (especially in lung adenocarcinomas), prognostic features, and/or utility as diagnostic cancer biomarkers (especially in sarcomas). 57 fusion results across 34 samples were evaluated. Fusion-STAMP demonstrated high overall accuracy with 98% sensitivity and 94% specificity for fusion detection. There was high intra- and inter-run reproducibility. Detection was sensitive to approximately 10% tumor, though this is expected to be impacted by fusion transcript expression levels, hybrid capture efficiency, and RNA quality. Challenges of clinically validating RNA sequencing for fusion detection include a low average RNA quality in FFPE specimens, and variable RNA total content and expression profile per cell. These challenges contribute to highly variable on-target rates, total read pairs, and total mapped read pairs. False positive results may be caused by intergenic splicing, barcode hopping / index hopping, or misalignment. Despite this, Fusion-STAMP demonstrates high overall performance metrics for qualitative fusion detection and is expected to provide clinical utility in identifying actionable fusions.

This study evaluates the performance of the RareVision Whole Genome Sequencing (WGS) assay for comprehensive genomic profiling in rare genetic diseases. The analytical validation assessed the assays sensitivity and positive predictive values (PPV) for single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs), revealing a sensitivity of 99.4% for SNVs and 98.7% for indels, with PPVs of 99.3% for SNVs and 98.7% for indels. Clinical validation involved benchmarking against established orthogonal methods, demonstrating high concordance in variant detection with reference laboratories. The assays reproducibility was confirmed with 100% inter-precision and intra-precision concordance. The RareVision WGS assay provides detailed genomic insights, enhancing the diagnosis and management of rare genetic disorders by offering a comprehensive and accurate genomic profiling tool.

Commercial liquid biopsy assays are routinely used by oncologists to monitor disease response and resistance to therapy. Additionally, in cases where tumor tissue is not available, clinicians may rely on cell-free DNA (cfDNA) testing as a surrogate for comprehensive tumor testing. While some gene rearrangements are well detected, current commercial liquid biopsy assays exhibit low sensitivity for fibroblast growth factor receptor (FGFR) rearrangements. FGFRs are altered in [~]2.5% of all cancers, including FGFR2 rearrangements in 10% of intrahepatic cholangiocarcinoma and FGFR3 point mutations and rearrangements in 10-15% of urothelial carcinoma. Therefore, we developed and analytically validated FGFR-Dx, an FGFR-focused cfDNA assay with improved sensitivity for FGFR rearrangements. FGFR-Dx comprehensively targets the introns in FGFR1-3 previously shown to be involved in gene fusions as well as all coding exons. Custom FGFR synthetic reference standards representing both single nucleotide variants (SNVs) and gene rearrangements were utilized at a range of variant frequencies and revealed a detection limit of 0.5% with sensitivities of 97.2% and 92.9% for SNVs and rearrangements, respectively. Furthermore, FGFR-Dx detected rearrangements and identified the intronic breakpoints from cfDNA collected from 13 of 15 patients with known FGFR fusions.

BRAF is a serine/threonine protein kinase whose mutations lead to unregulated cell growth and cause different types of cancers. Since V600E is a major BRAF mutation and V600E detection as a companion diagnostic test (CDx) is stipulated in the labeling of the BRAF V600 inhibitors. Traditional Sanger sequencing cannot accurately detect mutations lower than 15% variant allele frequency (VAF) due to its limited sensitivity. Here we applied our patented XNA molecular clamping technology to modify Sanger sequencing template preparation by enriching the mutation population. We found that the use of our mutation-enriched template enhanced the analytical sensitivity of Sanger sequencing to 0.04% VAF. The method is verified to detect V600E, V600K, and V600R mutants and is validated for the known BRAF mutation status in clinical samples. Our streamlined protocol can be used for easy validation of the highly sensitive target-enrichment method for detecting BRAF V600 mutations using Sanger sequencing in clinical labs. In addition to BRAF V600 mutations, this method can be extended to the detection of other clinically important actionable mutations for cancer diagnostics.

Acute myeloid leukemia (AML) is the most common leukemia in adults and current methods rely on cytogenic and molecular profiling for AML classification and risk stratification. Recent advances have demonstrated that next generation sequencing (NGS) may improve prognostic prediction and risk stratification of AML patients. A tumor-only high coverage ([~]220x) whole genome sequencing (WGS) method was developed and its analytical performance (limit of detection (LoD), sensitivity, and precision) was evaluated with clinical samples. Overall, the assay observed analytical sensitivity of 97.6%, 89.5% and 92.9% for small variants, Structural Variants (SV) and Copy Number Alterations (CNA), respectively comparing against reference sets from multiple modalities. LoD was evaluated as a function of sequence coverage and somatic variant allele frequency (VAF). At a given sequence depth of 140X, small variants (SNVs and indels) and SV achieved 95% detection rates when VAFs were 5% and 7.3%, respectively. CNA were detected with copy number fold change of 1.09 and 0.87 for duplication and deletion events, respectively. Further, loss of heterozygosity was detected with tumor purity of 17%. In Summary, the results demonstrate the WGS tumor-only (WGS TO) pipeline has high sensitivity in all variant types with a fast turnaround time of [~]5 days and can be used to identify variants indicative for AML treatment options.

Optical genome mapping (OGM) is an emerging technology with great potential for prenatal diagnosis. OGM can identify and resolve all types of balanced and unbalanced cytogenomic abnormalities in a single test, which are typically assessed by multiple standard of care (SOC) methods including karyotyping, fluorescence in situ hybridization and chromosomal microarray. To assess OGMs viability as an alternative to conventional SOC testing, a comprehensive clinical research study was conducted across multiple sites, operators, and instruments to evaluate its accuracy and clinical utility. This report provides an update for the phase 2 results of the ongoing multisite evaluation and validation study evaluating OGM for prenatal applications. In phase 1, 123 prenatal cases were assessed by OGM, and in phase 2, 219 retrospective and prospective prenatal cases have been evaluated. For 71% of cases, at least two SOC tests were performed. The study found that OGM had an overall accuracy of 99.6% and positive predictive value of 100% when compared to all cytogenetic SOC results. With its standardized workflow, cost-effectiveness, and high-resolution cytogenomic analysis, OGM shows great promise as an alternative technology that uses a single assay to consolidate the multiple SOC tests usually used for prenatal cytogenetic diagnosis.

DNA labelling fluorescent dyes such as ethidium bromide have long been considered to be highly mutagenic during DNA replication. While recent studies have pushed back on this narrative, the intercalative nature of these dyes continues to raise the possibility that these dyes can induce mutations. The iconPCR instrument by n6tec uses fluorescent dyes to measure amplification in real time and to adjust cycling conditions. However, since this use of qPCR is preparative and not analytical, mutations introduced by fluorescent dyes would be propagated into the sequencing reaction. To address the impact of these dyes on downstream analyses, we have performed routine mutation calling as well as mutational signature analysis on samples amplified using the iconPCR in the presence of either SYBR or EvaGreen. Sequence analysis revealed very minimal impacts of dyes on the reactions, largely within the noise regimen with only subtle changes in mutation rates seen. Mutational signature analysis was unable to identify any key signatures assignable to the dyes in either substitutions or indel domains. The mutational impact of intercalating dyes during fluorescence-guided amplification is therefore minimal and can be disregarded in all but the most sensitive NGS applications.

The standard-of-care (SOC) for genomic testing of myeloid cancers primarily relies on karyotyping and fluorescent in situ hydridization (FISH) (cytogenetic analysis) and targeted gene panels ([≤]54 genes) that harbor hotspot pathogenic variants (molecular genetic analysis). Both cytogenetic and molecular testing workup is necessary for the identification and detection of large structural variants (SVs) and small variants like single nucleotide variants (SNV) and indels, respectively. 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 genetic etiology of these myeloid tumors. In this study, we evaluated the performance and clinical utility of optical genome mapping (OGM) and a 523-gene next-generation sequencing (NGS) panel for comprehensive genomic profiling of 15 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 were found to have an analytical concordance of 100% with karyotyping, FISH, and the 54-gene panel, respectively. Additionally, OGM better characterized and resolved the structural variants previously reported by karyotyping in five cases, such as identifying the genomic content of marker and ring chromosomes. OGM also identified several additional translocations and eleven copy number variations (CNVs), of which the CNVs were validated/confirmed by the 523-gene panel. The 523-gene panel identified seven additional clinically relevant SNVs (two tier 1A variants and five tier 2C variants, as per the ACMG/AMP guidelines) in four cases. The simultaneous visualization of SVs and small NGS detected sequence variants (SNVs and small indels) from OGM and 523-gene NGS panel, respectively in the NxClinical software v6.1 identified two clinically relevant compound heterozygous events in two samples. This study demonstrates the higher sensitivity, resolution, accuracy, and ability to reveal cryptic and clinically relevant novel variants in myeloid cancers as compared to SOC methodologies. Our cost-effective approach of using OGM and a 523-gene NGS panel for comprehensive genomic profiling of myeloid cancers will not only increase the yield of actionable targets leading to improved clinical outcomes but also help resolve our ongoing conundrum of apparently genomically normal myeloid cancers by providing more answers.