The Journal of Molecular Diagnostics

BackgroundThe standard of care (SOC) cytogenetic testing methods, such as chromosomal microarray (CMA) and Fragile-X syndrome (FXS) testing, have been employed for the detection of copy number variations (CNVs), and tandem repeat expansions/contractions that contribute towards a sizable portion of genetic abnormalities in constitutional disorders. However, CMA is unable to detect balanced structural variations (SVs) or determine the precise location or orientation of copy number gains. Karyotyping, albeit with lower resolution, has been used for the detection of balanced SVs. Other molecular methods such as PCR and Southern blotting, either simultaneously or in a tiered fashion have been used for FXS testing, adding time, cost, and complexity to reach an accurate diagnosis in affected individuals. Optical genome mapping (OGM), innovative technology in the cytogenomics arena enables a direct, high-resolution view of ultra-long DNA molecules (more than 150 kbp), which are then assembled de novo to detect germline SVs ranging from 500 bp insertions and deletions to complex chromosomal rearrangements. The purpose of this study was to evaluate the performance of OGM in comparison to the current SOC methods and assess the intra- and inter-site reproducibility of the OGM technique. We report the largest retrospective dataset to date on OGM performed at five laboratories (multi-site) to assess the robustness, QC performance, and analytical validation (multi-operator, and multi-instrument) in detecting SVs and CNVs associated with constitutional disorders compared to SOC technologies. MethodsThis multi-center IRB-approved, double-blinded, study includes a total of 331 independent flow cells run (including replicates), representing 202 unique retrospective samples, including but not limited to pediatric-onset neurodevelopmental disorders. This study included affected individuals with either a known genetic abnormality or no known genetic diagnosis. Control samples (n=42) were also included. Briefly, OGM was performed on either peripheral blood samples or cell lines using the Saphyr system. The OGM assay results were compared to the human reference genome (GRCh38) to detect different types of SVs (CNV, insertions, inversions, translocations). A unique coverage-based CNV calling algorithm was also used to complement the SV calls. Analysis of heterozygous SVs was performed to assess the absence of heterozygosity (AOH) regions in the genome. For specific clinical indications of FSHD1 and FXS, the EnFocus FXS and FSHD1 tools were used to generate the region-specific reports. OGM data was analyzed and visualized using Access software (version 1.7), where the SVs were filtered using an OGM specific internal control database. The samples were analyzed by laboratory analysts at each site in a blinded fashion using ACMG guidelines for SV interpretation and further reviewed by expert geneticists to assess concordance with SOC testing results. ResultsOf the first 331 samples run between five sites, 99.1% of sample runs were completed successfully. Of the 331 datasets, 219 were assessed for concordance by the time of this publication; these were samples that harbored known variants, of which 214/219 were detected by OGM resulting in a concordance of 97.7% compared to SOC testing. 47 samples were also run in intra- and inter-site replicate and showed 100% concordance for pathogenic CNVs and SVs and 100% concordance for pathogenic FMR1 repeat expansions. ConclusionThe results from this study demonstrate the potential of OGM as an alternative to existing SOC methods in detecting SVs of clinical significance in constitutional postnatal genetic disorders. The outstanding technical performance of OGM across multiple sites demonstrates the robustness and reproducibility of the OGM technique as a rapid cytogenomics testing tool. Notably, OGM detected all classes of SVs in a single assay, which allows for a faster result in cases with diverse and heterogeneous clinical presentations. OGM demonstrated 100% concordance for pathogenic variants previously identified including FMR1 repeat expansions (full mutation range), pathogenic D4Z4 repeat contractions (FSHD1 cases), aneuploidies, interstitial deletions, interstitial duplications, intragenic deletions, balanced translocations, and inversions. Based on our large dataset and high technical performance we recommend OGM as an alternative to the existing SOC tests for the rapid detection and diagnosis of postnatal constitutional disorders.

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.

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.

Screening for colorectal cancer (CRC) using plasma methylation is challenging due to the low abundance of cell-free DNA (cfDNA). Therefore, the development of signal amplification assays based on appropriate markers is essential to increase sensitivity. In this study, we employed an epigenome-wide approach for de novo identification differentially methylated CpGs (DMCs) common to CRC and adenoma using 17 public datasets. A sense-antisense and dual MGB probe (SADMP) assay was then developed. Subsequently, the biomarkers were validated in 712 plasma samples based on SADMP. A total of 2237 DMCs showed overlap between CRC and adenoma. Of these, 75 were hypomethylated in 30 other non-CRC cancers. Following LASSO regression, WBC validation and primer/probe design evaluation, NTMT1 and MAP3K14-AS1 were identified as the most informative candidate biomarkers. At preset template concentrations, the SADMP assay for NTMT1 or MAP3K14-AS1 could reduce the cycle threshold by 1. The NTMT1 and MAP3K14-AS1 dual-target SADMP assay demonstrated a sensitivity of 84.8% for CRC (stage I: 75.0%), a sensitivity of 32.0% for advanced adenomas (AA), and a specificity of 91.5% in controls. The dual-target assay showed high performance for CRC early detection in plasma, suggesting that it may serve as a promising noninvasive tool for CRC screening.

To battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy. We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as "2019-nCoV not detected", whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as "Invalid Result" and the procedure should be repeated. In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

PurposeClinical analysis and reporting of somatically acquired copy number abnormalities (CNAs) detected through next-generation sequencing (NGS) is time consuming and requires significant expertise. Interpretation is complicated by other classes of variants such as coding mutations and gene fusions. Recent guidelines for the clinical assessment of tumor CNAs harmonize and simplify the reporting criteria but did not directly address NGS-specific concerns or the need for a standardized and scalable protocol for CNA analysis. MethodsWe developed a scalable NGS-derived CNA analysis protocol paired with a novel interactive web application, CNA Explorer and anaLyzer (CNAEL), to facilitate the rapid, scalable, and reproducible analysis and reporting of complex tumor-derived CNA profiles https://CNAEL.sema4.com. ResultsNovel features of CNAEL include on-the-fly data rescaling to account for tumor ploidy, purity, and modal chromosomal copy number; integration of gene expression, coding, and fusion variants into review and automated genome-wide summarization to enable rapid reporting. We found that case curation times were significantly reduced when using CNAEL [median:7 mins, IQR = 4, 10.25] compared with our previous laboratory standard operating procedure [median: 61 mins, IQR = 23.75, 176,25] with p=4.631e-05. ConclusionCNAEL enables efficient and accurate clinical review and reporting of complex NGS-derived tumor copy number profiles.

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.

BackgroundThe rapid emergence of new vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires an equally rapid deployment of diagnostic tests to specifically identify each VOC as soon as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) Alpha/Beta/Gamma RT-PCR melting temperature (Tm) signature-based assay, which now includes modifications that allow specific detection of Delta (B.1.617.2) and Omicron (B.1.529) VOCs. MethodsWe developed a dual SMB assay (SMB-452), which targeted the T22917G (L452R) mutation in the SARS-CoV-2 spike protein to specifically detect the Delta VOC. We also identified a Tm profile in our existing SMB-501 and SMB-484 assays (which detect mutations in codons 501 and 484 of the SARS-CoV-2 spike protein, respectively) that differentiate the Omicron-specific N501Y (A23063T) and E484A (A23013C) mutations from both wild type (WT) and other VOCs. The entire six SMB three-codon assay was tested using reference SARS-CoV-2 RNAs. The assay was then validated using clinical samples from COVID-19 patients tested with a LightCycler 480 (LC480) (74 samples), Bio-Rad CFX96 (34 samples), Rotor-Gene Q (Qiagen) (34 samples) and an ABI-7500 (34 samples) RT-PCR instruments. Six SMB Tm results were then inputted into an Excel Analysis tool to generate specific VOC identifications. ResultsThe limit of detection (LOD) for the new SMB-452 assay, which specifically identified the Delta variant was 1 genomic equivalent (GE) per reaction. The LODs of the SMB-501 and SMB-484 assays, which detect Omicron were 100 and 103 GE respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% of the samples as WT or VOCs in clinical samples and 6% of the tests producing indeterminate results. None of the samples were incorrectly identified as WT or as a different VOC. Thus, excluding samples with indeterminant results, the assay was 100% sensitive and 100% specific compared to sequencing. There was also 100% concordance between the LC480, BioRad, ABI and Qiagen results, excluding negative or indeterminate results; however, the Qiagen assay had significantly more indeterminates than the other assays. ConclusionThis new assay can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

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.

Background & AimsCell-free DNA (cfDNA) has advanced cancer genetic profiling through liquid biopsy. While plasma is traditionally the primary source, emerging evidence highlights urinary cfDNA as a novel and noninvasive alternative. This study aimed to comprehensively assess transrenal DNA (trDNA) as a novel noninvasive biomarker source in HCC patients, compared to blood-based liquid biopsy. Approach & ResultsHBV DNA was used as a biomarker for trDNA. HBV-targeted and HCC-focused next generation sequencing (NGS) and whole genome sequencing (WGS) were used to compare fragment insert-sizes, the genome coverage, and germline genotyping accuracy. Urinary cfDNA overall exhibited a predominantly mononucelosomal pattern similar to plasma cfDNA, but with shorter fragments, broader size distribution and a more pronounced 10-bp periodicity. In contrast, trDNA were shorter and more variable among all patients. In HCC patients, trDNA was even shorter, with distinct 4-mer end motifs, compared to non-HCC trDNA. Higher concentrations of HCC-distinctive 4-mer end motif and TP53 mutations were found in urine compared to plasma. The overall genome coverage breadth by WGS was similar between urine and plasma cfDNA, with a higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. In 101 HCC patients, there was a 78% overall concordance of HCC-associated mutations (TP53, CTNNB1, and hTERT) and in select 15 patients, 97% overall position-level concordance by targeted NGS between plasma and urine cfDNA. ConclusionUrine cfDNA has comparable features with distinct characteristics to plasma cfDNA and is a promising tool for liver cancer studies.

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.

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.

Rapid classification and detection of SARS-CoV-2 variants have been critical in comprehending the viruss transmission dynamics. Clinical manifestation of the infection is influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management may differ for new variants. For example, some monoclonal antibody treatments are variant-specific. Yet, an FDA-approved test for detecting the SARS-CoV-2 variant is unavailable. A laboratory-developed test (LDT) remains a viable option for reporting the infecting variant for clinical intervention or epidemiological purposes. Accordingly, we have validated the Illumina COVID-Seq assay as an LDT according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The limit of detection (LOD) of this test is Ct<30 ([~]15 viral copies) and >200X genomic coverage, and the test is 100% specific in the detection of existing variants. The test demonstrated 100% precision in inter-day, intra-day, and intra-laboratory reproducibility studies. It is also 100% accurate, defined by reference strain testing and split sample testing with other CLIA laboratories. Advanta Genetics LDT COVID Seq has been reviewed by CAP inspectors and is under review by FDA for Emergency Use Authorization.

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

BackgroundThe sequencing-by-synthesis technology by Illumina, Inc. enables efficient and scalable readouts of mutations from genomic data. To enhance sequencing speed and efficiency, Illumina has shifted from the four-color base calling chemistry of the HiSeq series to a two-color fluorescent dye chemistry in the NovaSeq series. Benchmarking sequencing artifacts due to biases in the newer chemistry is important to evaluate the quality of identified mutations. ResultsWe re-analyzed a series of whole-genome sequencing experiments in which the same samples were sequenced on the NovaSeq 6000 (two-color) and HiSeq X10 (four-color) platforms by independent groups. In several samples, we observed a higher frequency of T-to-G and A-to-C substitutions ("T>G") at the read level for NovaSeq 6000 versus HiSeq X10. As the per-base error rate is still low, the artifactual substitutions have a negligible effect in identifying germline or high variant allele frequency (VAF) somatic mutations. However, such errors can confound the detection of low-VAF somatic variants in high-depth sequencing samples, particularly in studies of mosaic mutations in normal tissues, where variants have low read support and are called without a matched normal. The artifactual T>G variant calls disproportionately occur at NT[TG] trinucleotides, and we leveraged this observation to bioinformatically reduce the T>G excess in somatic mutation callsets. ConclusionsWe identified a recurrent artifact specific to the Illumina two-color chemistry platform on the NovaSeq 6000 with the potential to contaminate low-VAF somatic mutation calls. Thus, an unexpected enrichment of T>G mutations in mosaicism studies warrants caution.

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.

BackgroundRecent advances in molecular genetics have dramatically improved our understanding of the pathophysiology and classification of salivary gland tumors. The identification of recurrent oncogenic fusions has been especially helpful in distinguishing entities with overlapping histomorphology. MethodsChromogenic RNA in situ hybridization (RNA-ISH) using BaseScope technology was performed to detect gene fusions associated with microsecretory adenocarcinoma (MSA), MEF2C::SS18, and mucoepidermoid carcinoma (MEC), CRTC1::MAML2, using probes specific to the exon junctions of the MEF2C::SS18 (exon 7 of MEF2C to exon 4 of SS18) and CRTC1::MAML2 (exon 1 of CRTC1 to exon 2 of MAML2) fusion transcripts. Sixteen cases of MEF2C::SS18 fusion-positive MSA, six cases of CRTC1::MAML2 fusion-positive MEC, three cases of fusion-unknown MEC, and one case of fusion-negative MEC were included in the test cohort. Positive signal strength was assessed using a semi-quantitative scoring method as per manufacturer guidelines. ResultsFusion transcripts were detected by RNA-ISH results in 14/16 cases (88%) of fusion-positive MSAs and 3/6 cases (50%) of fusion-positive MEC. Interestingly, 2 cases (67%) of fusion-unknown MEC were also positive by RNA-ISH for CRTC1::MAML2 while the fusion-negative MEC was also negative by RNA-ISH. Positivity ranged between 1+ (one dot per cell in [&ge;]5% of tumor cells in one 40X field) and 2+ (two to three dots per cell in [&ge;]5% of tumor cells in one 40X field). ConclusionHere, we provide the first assessment of chromogenic RNA-ISH to detect gene fusions associated with microsecretory adenocarcinoma, MEF2C::SS18, and mucoepidermoid carcinoma, CRTC1::MAML2. Our results highlight the potential for ultrasensitive RNA-ISH to be used as an alternative method of fusion detection for salivary gland malignancies with highly conserved fusion transcript exon junctions. While additional studies are needed to validate the clinical utility of the assay and to determine optimal testing conditions, RNA-ISH may provide a means for restricted fusion analysis in cases with limited material and for pathologists without easy access to conventional molecular diagnostic testing.

Short tandem repeats (STRs) are amongst the most abundant class of variations in human genomes and are meiotically and mitotically unstable which leads to expansions and contractions. STR expansions are frequently associated with genetic disorders, with the size of expansions often correlating with the severity and age of onset. Therefore, being able to accurately detect the total repeat expansion length and to identify potential somatic repeat instability is important. Current standard of care (SOC) diagnostic assays include laborious repeat-primed PCR-based tests as well as Southern blotting, which are unable to precisely determine long repeat expansions and/or require a separate set-up for each locus. Sequencing-based assays have proven their potential for the genome-wide detection of repeat expansions but have not yet replaced these diagnostic assays due to their inaccuracy to detect long repeat expansions (short-read sequencing) and their costs (long-read sequencing). Here, we tested whether optical genome mapping (OGM) can efficiently and accurately identify the STR length and assess the stability of known repeat expansions. We performed OGM for 85 samples with known clinically relevant repeat expansions in DMPK, CNBP and RFC1, causing myotonic dystrophy type 1 and 2 and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), respectively. After performing OGM, we applied three different repeat expansion detection workflows, i.e. manual de novo assembly, local guided assembly (local-GA) and molecule distance script of which the latter two were developed as part of this study. The first two workflows estimated the repeat size for each of the two alleles, while the third workflow was used to detect potential somatic instability. The estimated repeat sizes were compared to the repeat sizes reported after the SOC and concordance between the results was determined. All except one known repeat expansions above the pathogenic repeat size threshold were detected by OGM, and allelic differences were distinguishable, either between wildtype and expanded alleles, or two expanded alleles for recessive cases. An apparent strength of OGM over current SOC methods was the more accurate length measurement, especially for very long repeat expansion alleles, with no upper size limit. In addition, OGM enabled the detection of somatic repeat instability, which was detected in 9/30 DMPK, 23/25 CNBP and 4/30 RFC1 samples, leveraging the analysis of intact, native DNA molecules. In conclusion, for tandem repeat expansions larger than [~]300 bp, OGM provides an efficient method to identify exact repeat lengths and somatic repeat instability with high confidence across multiple loci simultaneously, enabling the potential to provide a significantly improved and generic genome-wide assay for repeat expansion disorders.

Cytogenetic studies represent a critical component of prenatal genetic testing. Prenatal diagnostic testing of amniotic fluid, chorionic villus sampling, or more rarely, fetal cord blood, is recommended following a positive or unreportable NIPT, maternal serum screen, abnormal ultrasound or increased genetic risk based on family history. While chromosomal microarray is the recommended first-tier prenatal diagnostic test for the detection of sub-microscopic copy number variants, in practice, multiple assays are often assessed, in concert, to achieve a final diagnostic result. The use of multiple methodologies is costly, time consuming, and labor intensive. Optical genome mapping is an emerging technique with application for prenatal diagnosis because of its ability to detect and resolve, in a single assay, all classes of pathogenic cytogenetic aberrations detectable by karyotyping, FISH, and microarray. In an effort to characterize the potential of optical genome mapping as a novel alternative to conventional testing, a multi-site, multi-operator, multi-instrument clinical research study was conducted to demonstrate its analytic validity and clinical utility. In the first phase a total of 200 specimens representing 123 unique cases demonstrated 100% concordance with standard of care methods and 100% reproducibility between sites, operators, and instruments. Analysis and interpretation of cases with incidental findings of potential clinical significance also were performed.

Systematic performance comparing the results of exome-sequencing as a single test replacing Sanger-sequencing of targeted gene(s) is still lacking. In this study we compared Sanger-sequencing results of 258 genes to those obtained from next generation sequencing (NGS) using two exome-sequencing enrichment kits: Agilent-SureSelectQXT and Illumina-Nextera. Sequencing was performed on leukocytes and buccal-derived DNA from a single individual, and all 258 genes were sequenced a total of 11 times (using different sequencing methods and DNA sources). Sanger-sequencing was completed for all exons, including flanking {+/-}8bp regions. For the 258 genes, NGS mean coverage was >20x for >98% and >91% of the regions targeted by SureSelect and Nextera, respectively. Overall, 449 variants were identified in at least one experiment, and 407/449 (90.6%) were detected by all. Of the 42 discordant variants, 23 were determined as true calls, summing-up to a truth set of 430 variants. Sensitivity of true-variant detection was 99% for Sanger-sequencing and 97%-100% for the NGS experiments. Mean false-positive rates were 3.7E-6 for Sanger-sequencing, 2.5E-6 for SureSelect-NGS and 5.2E-6 for Nextera-NGS. Our findings suggest a high overall concordance between Sanger-sequencing and NGS. Both methods demonstrated false positive and false negative calls and similar performances. Consequently, high clinical suspicion for a specific diagnosis should override negative results of either Sanger-sequencing or NGS.