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Journal of Molecular Diagnostics: JMD

Evelina Mocci, Marija Debeljak, Alison P Klein, James R Eshleman
We developed a novel phasing approach, based solely on molecules and genotype frequency, and that does not rely on inference of new alleles. We initiated the project because of errors that were detected in the phased 1000 genomes data. The algorithm first combined identical genotypes into clusters and ranked them by descending frequency. Using alleles defined in homozygotes, it combined them to produce expected genotypes that were dismissed, and subtracted them from remaining genotypes to define additional new putative alleles...
March 11, 2019: Journal of Molecular Diagnostics: JMD
Maryke Schoonen, Izelle Smuts, Roan Louw, Joanna L Elson, Etresia van Dyk, Lindi-Maryn Jonck, Richard J T Rodenburg, Francois H van der Westhuizen
Mitochondrial disease (MD) is a group of rare inherited disorders with clinical heterogeneous phenotypes. Recent advances in next-generation sequencing (NGS) allows for rapid genetic diagnostics in patients who suffer from MD, resulting in significant strides into determining the aetiology of the disease. This, however, has not been the case in many patient populations. We report on a molecular diagnostic study using mitochondrial DNA (mtDNA) and targeted nuclear DNA (nDNA) NGS of an extensive cohort of predominantly sub-Saharan African paediatric patients with clinical and biochemically defined MD...
March 11, 2019: Journal of Molecular Diagnostics: JMD
Evgeniya Volkova, Emilia Sippert, Meihong Liu, Teresita Mercado, Gregory A Denomme, Orieji Illoh, Zhugong Liu, Maria Rios
Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic due to a disease state or recent transfusions. Additionally, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed...
March 11, 2019: Journal of Molecular Diagnostics: JMD
Maristela L Onozato, Clarence Yapp, Douglas Richardson, Tilak Sundaresan, Varun Chahal, Jesse Lee, James P Sullivan, Marisa W Madden, Hyo Sup Shim, Mathew Liebers, Quan Ho, Shyamala Maheswaran, Daniel A Haber, Zongli Zheng, Brian Clancy, Hunter L Elliott, Jochen K Lennerz, A John Iafrate
The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent "barcode" system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised...
March 9, 2019: Journal of Molecular Diagnostics: JMD
Walter S Campbell, Alexis B Carter, Allison M Cushman-Vokoun, Timothy C Greiner, Rajesh C Dash, Mark Routbort, Monica E de Baca, James R Campbell
Incorporating genetic variant data into the electronic health record (EHR) in discrete computable fashion has vexed the informatics community for years. Genetic sequence test results are typically communicated by the molecular laboratory and stored in the EHR as textual documents. Although text documents are useful for human readability and initial use, they are not conducive for data retrieval and reuse. As a result, clinicians often struggle to find historical gene sequence results on a series of oncology patients within the EHR that might influence the care of the current patient...
February 20, 2019: Journal of Molecular Diagnostics: JMD
Jennifer Tyler, Lorie Kumer, Carolyn Fisher, Heather Casey, Hiroko Shike
Chimerism testing is used to monitor engraftment and risk of relapse after allogeneic hematopoietic stem cell transplant for hematological malignancy. Although short tandem repeat (STR) method is widely used among clinical laboratories, quantitative PCR (qPCR) provides better sensitivity (0.1%) than STR (1% to 5%), but is less accurate than STR for patients in mixed chimerism. qPCR chimerism test allows evaluation of residual recipient cells as a surrogate of measurable residual disease. To achieve higher sensitivity and accuracy, we applied qPCR or STR, based on patient chimerism status (recipient alleles <5% or ≥5%, respectively)...
February 20, 2019: Journal of Molecular Diagnostics: JMD
Ryan J Schmidt, Allison Macleay, Long Phi Le
Accurate genetic variant representation through nomenclature and annotation is essential for understanding functional consequence and properly noting the presence of variants across time, assays, and laboratories. Current variant calling algorithms detect single deletion-insertion variants as multiple indel and/or substitution variants from next-generation sequencing data. Consequently, these variants are separately annotated in bioinformatics pipelines, leading to inaccurate variant representation. We developed a bioinformatic solution to this problem - VarGrouper - that automatically recognizes individual variants that arise from a deletion-insertion variant and aggregates them into a single variant that can be properly annotated...
February 19, 2019: Journal of Molecular Diagnostics: JMD
Marelize Swart, Wesley M Stansberry, Victoria M Pratt, Elizabeth B Medeiros, Patrick J Kiel, Fei Shen, Bryan P Schneider, Todd C Skaar
The Clinical Laboratory Improvement Amendments (CLIA) of 1988 requires that pharmacogenetic genotyping methods need to be established according to technical standards and laboratory practice guidelines before testing can be offered to patients. Testing methods for variants in ABCB1, CBR3, COMT, CYP3A7, C8ORF34, FCGR2A, FCGR3A, HAS3, NT5C2, NUDT15, SBF2, SEMA3C, SLC16A5, SLC28A3, SOD2, TLR4, and TPMT were validated in a CLIA-accredited laboratory. As no known reference materials were available, DNA samples that were from Coriell Cell Repositories (Camden, NJ) were used for the analytical validation studies...
February 19, 2019: Journal of Molecular Diagnostics: JMD
Saradhi Mallampati, Dzifa Y Duose, Michael A Harmon, Meenakshi Mehrotra, Rashmi Kanagal-Shamanna, Stephanie Zalles, Ignacio I Wistuba, Xiaoping Sun, Rajyalakshmi Luthra
The emergence of highly sensitive molecular diagnostic approaches such as droplet digital PCR has allowed the accurate identification of low-frequency variant alleles in clinical specimens; however, the multiplex capabilities of droplet digital PCR for variant detection are inadequate. The incorporation of molecular barcodes or unique IDs into next-generation sequencing libraries through PCR has enabled the detection of low-frequency variant alleles across multiple genomic regions. However, rational library preparation and sequencing data analytical strategies that integrate molecular barcodes have rarely been applied to clinical settings...
February 19, 2019: Journal of Molecular Diagnostics: JMD
Amanda Vannitamby, Shona Hendry, Tanvi Makadia, Janine Danks, John Slavin, Louis Irving, Daniel Steinfort, Steven Bozinovski
Multiple biomarkers are under evaluation to guide the use of immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC), including programed death ligand 1 (PD-L1) tumor cell staining. We have developed a new approach that accurately quantifies PD-L1 status and identifies multiple mutations by using a single bronchoscopy specimen. A novel molecular marker was identified to detect the presence of malignant cells in radial endobronchial ultrasound bronchial brushings from NSCLC (n = 15) and benign (n = 13) nodules by quantitative real-time RT-PCR (RT-qPCR)...
February 8, 2019: Journal of Molecular Diagnostics: JMD
Elin S Gray, Tom Witkowski, Michelle Pereira, Leslie Calapre, Karl Herron, Darryl Irwin, Brett Chapman, Muhammad A Khattak, Jeanette Raleigh, Athena Hatzimihalis, Jonathan Cebon, Shahneen Sandhu, Grant A McArthur, Michael Millward, Melanie Ziman, Alexander Dobrovic, Stephen Q Wong
The analysis of circulating tumor DNA (ctDNA) provides a minimally-invasive molecular interrogation that has the potential to guide treatment selection and disease monitoring. Here, we evaluated a custom UltraSEEK melanoma panel for the MassARRAY system, probing for 61 mutations over 13 genes. The analytical sensitivity and clinical accuracy of the UltraSEEK melanoma panel was compared to droplet digital PCR. The blinded analysis of 68 mutations detected in 48 plasma samples from stage IV melanoma patients revealed a concordance of 88% between the two platforms...
February 4, 2019: Journal of Molecular Diagnostics: JMD
Hussein Daoud, Mahdi Ghani, Landry Nfonsam, Ryan Potter, Shelley Ordorica, Virginia Haslett, Nathaniel Santos, Heather Derksen, Donelda Lahey, Martha McGill, Vanessa Trudel, Brittany Antoniuk, Nasim Vasli, Caitlin Chisholm, Gabrielle Mettler, Elizabeth Sinclair-Bourque, Jean McGowan-Jordan, Amanda Smith, Robert Roberts, Olga Jarinova
Inherited cardiomyopathies (ICs) are a major cause of heart disease. Given their marked clinical and genetic heterogeneity, the content and clinical utility of IC multi-gene panels has been the topic of continuous debate. Our genetics diagnostic laboratory has been providing clinical diagnostic testing for ICs since 2012. We began by testing nine genes, and expanded our panel by 5-fold in 2015. Here, we describe the implementation of a cost-effective next-generation sequencing (NGS)-based assay for testing of IC genes, including a protocol that minimizes the amount of Sanger sequencing required to confirm variants identified via NGS, which reduces the cost and time of testing...
February 4, 2019: Journal of Molecular Diagnostics: JMD
Lucie Coppin, Pascal Plouvier, Michel Crépin, Anne-Sophie Jourdain, Emilie Ait Yahya, Stéphane Richard, Brigitte Bressac-de Paillerets, Catherine Cardot-Bauters, Sophie Lejeune, Julie Leclerc, Pascal Pigny
Von Hippel Lindau disease (VHL) is a monogenic disorder characterized by the development of tumors affecting the central nervous system, kidney, pancreas, or adrenal glands, and due to germline mutations in the VHL tumor suppressor gene. About 5% of patients harboring a typical VHL phenotype have no mutation detected by conventional techniques, so a postzygotic VHL mosaicism can be suspected. The aim of this study was therefore to implement a next-generation sequencing (NGS) strategy for VHL mosaic mutation detection, including an optimization of the original Personal Genome Machine design by enrichment with oligonucleotides corresponding to amplicons with insufficient depth of coverage...
February 4, 2019: Journal of Molecular Diagnostics: JMD
Alexis B Carter
Next-generation sequencing produces large amounts of data. The complexity and data management issues associated with next-generation sequencing have led many laboratories to turn to cloud services, especially when internal information technology infrastructure is inadequate to support data requirements. In addition, public cloud repositories of variants are being increasingly utilized, and their data sets are being populated through crowdsourcing submissions of human genetic variation identified within laboratories...
January 28, 2019: Journal of Molecular Diagnostics: JMD
Broderick C Corless, Gregory A Chang, Samantha Cooper, Mahrukh M Syeda, Yongzhao Shao, Iman Osman, George Karlin-Neumann, David Polsky
Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients...
March 2019: Journal of Molecular Diagnostics: JMD
(no author information available yet)
No abstract text is available yet for this article.
March 2019: Journal of Molecular Diagnostics: JMD
Jeong E Kim, Sung-Min Chun, Yong S Hong, Kyu-Pyo Kim, Sun Y Kim, Jihun Kim, Chang Ohk Sung, Eun J Cho, Tae W Kim, Se Jin Jang
Next-generation sequencing (NGS) panels are widely used for defining tumor mutation profiles and determining treatment approaches. We performed targeted NGS with 382 colorectal cancer genes with known microsatellite instability (MSI). After exclusion of germline alterations, the load of somatic mutations and small insertion/deletion (indel) alterations were determined. In the test set, 79 patients with 41 microsatellite-stable (MSS) and 38 MSI tumors were included. There were 120 MSS and eight MSI-high tumors in the validation set...
March 2019: Journal of Molecular Diagnostics: JMD
Lauren L Ritterhouse
This commentary highlights the article by Blidner et al that describes a novel assay for detection of chimeric RNAs from gene fusions and exon-skipping events in non-small-cell lung cancer.
January 19, 2019: Journal of Molecular Diagnostics: JMD
Stephen E Lincoln, Rebecca Truty, Chiao-Feng Lin, Justin M Zook, Joshua Paul, Vincent H Ramey, Marc Salit, Heidi L Rehm, Robert L Nussbaum, Matthew S Lebo
Orthogonal confirmation of next-generation sequencing (NGS)-detected germline variants has been standard practice, although published studies have suggested that confirmation of the highest quality calls may not always be necessary. The key question is how laboratories can establish criteria that consistently identify those NGS calls that require confirmation. Most prior studies addressing this question have limitations: These studies are generally small, omit statistical justification, and explore limited aspects of the underlying data...
January 2, 2019: Journal of Molecular Diagnostics: JMD
Keyur P Patel, Roberto Ruiz-Cordero, Wei Chen, Mark J Routbort, Kristen Floyd, Sergio Rodriguez, John Galbincea, Bedia A Barkoh, David Hatfield, Haitham Khogeer, Rashmi Kanagal-Shamanna, C Cameron Yin, Zhuang Zuo, Sanam Loghavi, Chi Young Ok, Courtney D DiNardo, Rajyalakshmi Luthra, L Jeffrey Medeiros
Next-generation sequencing (NGS)-based mutation panels profile multiple genes simultaneously, allowing the reporting of numerous genes while saving labor and resources. However, one drawback of using NGS is that the turnaround time is often longer than conventional single gene tests. This delay can be problematic if molecular results are required to guide therapy in patients with clinically aggressive diseases, such as acute myeloid leukemia. To overcome this limitation, we developed a novel custom platform designated as Ultra-rapid Reporting of GENomic Targets (URGENTseq), an integrated solution that includes workflow optimization and an innovative custom bioinformatics pipeline to provide targeted NGS results on fresh peripheral blood and bone marrow samples within an actionable time period...
January 2019: Journal of Molecular Diagnostics: JMD
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