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Journal of Molecular Diagnostics: JMD

Weihua Huang, Changhong Yin, Guiqing Wang, Jeremy Rosenblum, Sankaran Krishnan, Nevenka Dimitrova, John T Fallon
Compared to conventional serological, culture-, and molecular-based diagnostic tests, next-generation sequencing (NGS) provides sequence-evidenced detection of various microbes, without prior knowledge, and is thus becoming a novel diagnostic approach. We describe here a RNA-based metatranscriptomic NGS (mtNGS) protocol for culture-independent detection of potential infectious pathogens, using clinical bronchoalveolar lavage specimens as an example. We present both an optimized workflow for experimental sequence data collection and a simplified pipeline for bioinformatics sequence data processing...
October 30, 2018: Journal of Molecular Diagnostics: JMD
Jeong Eun Kim, Sung-Min Chun, Yong Sang Hong, Kyu-Pyo Kim, Sun Young Kim, Jihun Kim, Chang-Ohk Sung, Eun Jeong Cho, Tae Won 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 genes in colorectal cancer with known microsatellite instability (MSI) status. After exclusion of germline alterations, 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...
October 30, 2018: Journal of Molecular Diagnostics: JMD
Jacqueline Maier, Thoralf Lange, Michael Cross, Kathrin Wildenberger, Dietger Niederwieser, Georg-Nikolaus Franke
Quantitative real-time-PCR methods are commonly used to monitor BCR-ABL transcript levels in patients with chronic myelogenous leukemia. However, standard techniques involve separate measurements of target and reference DNAs, require standard curves, and are susceptible to PCR inhibition. An optimized duplex droplet digital (dd)PCR should provide absolute quantification without the need for standard curves. The combination of high sensitivity and low background is particularly important for reliable monitoring of minimal residual disease...
October 19, 2018: Journal of Molecular Diagnostics: JMD
Michael J Kluk, Adam Bagg
This commentary highlights the article by Patel et al that reports a novel custom next-generation sequencing platform for fast detection of select genes in hematological malignancies.
October 19, 2018: Journal of Molecular Diagnostics: JMD
Borahm Kim, Hyeonah Lee, Saeam Shin, Seung-Tae Lee, Jong Rak Choi
The application of next-generation sequencing (NGS) technology in clinical diagnostics should proceed with care. We have evaluated the clinical validity of two commercially available RNA fusion panels, the TruSight RNA fusion panel (Illumina) and FusionPlex Pan-Heme Kit (ArcherDx), to detect recurrent translocations in hematologic malignancies. Twenty-four bone marrow samples taken at the initial diagnosis of patients with acute leukemia and chronic myeloid leukemia were included. To assess the limit of detection, serial dilutions of BCR-ABL1 (e1a2) positive RNAs were prepared using a commercial reference material...
October 19, 2018: Journal of Molecular Diagnostics: JMD
Silvia R Vitale, Anieta M Sieuwerts, Nick Beije, Jaco Kraan, Lindsay Angus, Bianca Mostert, Esther A Reijm, Ngoc M Van, Ronald van Marion, Luc Y Dirix, Paul Hamberg, Felix E de Jongh, Agnes Jager, John A Foekens, Paolo Vigneri, Stefan Sleijfer, Maurice P H M Jansen, John W M Martens
The detection of mutated genes in cell-free DNA (cfDNA) in plasma has emerged as an important minimally invasive way to obtain detailed information regarding tumor biology. Reliable determination of circulating tumor-derived DNA, often present at a low quantity amidst an excess of normal DNA in plasma, would be of added value for screening and monitoring of cancer patients and for hypothesis-generating studies in valuable retrospective cohorts. Our aim was to establish a workflow to simultaneously assess four hotspot estrogen receptor mutations (mESR1) in cfDNA isolated from only 200 μL of plasma by means of uniplex or multiplex pre-amplification combined with digital PCR...
October 6, 2018: Journal of Molecular Diagnostics: JMD
Jenny L Kerschner, Sujana Ghosh, Alekh Paranjapye, Wilmel R Cosme, Marie-Pierre Audrézet, Miyuki Nakakuki, Hiroshi Ishiguro, Claude Férec, Johanna Rommens, Ann Harris
It is estimated that up to 5% of cystic fibrosis transmembrane conductance regulator (CFTR) pathogenic alleles are unidentified. Some of these errors may lie in non-coding regions of the locus and impact gene expression. To identify regulatory element variants in the CFTR locus, SureSelect targeted enrichment of 460kb encompassing the gene was optimized to deep-sequence genomic DNA from 80 CF patients with an unequivocal clinical diagnosis but only one or no CFTR-coding region pathogenic variants. Bioinformatics tools were used to identify sequence variants and predict their impact, which was then assayed in transient reporter gene luciferase assays...
October 5, 2018: Journal of Molecular Diagnostics: JMD
Erik Delsing Malmberg, Anna Rehammar, Mariana B Pereira, Jonas Abrahamsson, Tore Samuelsson, Sara Ståhlman, Julia Asp, Anne Tierens, Lars Palmqvist, Erik Kristiansson, Linda Fogelstrand
Minimal residual disease (MRD) in acute myeloid leukemia (AML) is of major prognostic importance. The genetic landscape of AML is characterized by numerous somatic mutations, which constitute potential MRD markers. Leukemia-specific mutations can be identified with exome sequencing at diagnosis and assessed during follow-up at low frequencies by using targeted deep sequencing. Our aim was to further validate this patient-tailored assay for substitution mutations. By applying a statistical model, which corrects for position-specific errors, a limit of detection for single nucleotide variations of variant allele frequency (VAF) of 0...
September 29, 2018: Journal of Molecular Diagnostics: JMD
Sushant A Patil, Ibro Mujacic, Lauren L Ritterhouse, Jeremy P Segal, Sabah Kadri
Lack of reliable reference samples containing different mutations of interest across large sets of disease-relevant loci limits the extensive validation clinical next-generation sequencing (NGS) assays and their associated bioinformatics pipelines. Here, we have created a publicly available, highly flexible tool, in silico Mutator (insiM) to introduce point mutations, insertions, deletions, and duplications of any size into real datasets of amplicon-based or hybrid-capture NGS assay. insiM accepts an alignment file along with target territory and produces paired-end FASTQ files containing specified mutations via modification of original sequencing reads...
September 28, 2018: Journal of Molecular Diagnostics: JMD
William R Jeck, Jesse Lee, Hayley Robinson, Long P Le, A John Iafrate, Valentina Nardi
Structural chromosomal rearrangements leading to gene fusions are strong driver mutations in a variety of tumors. Identification of specific gene fusions can be essential for distinguishing benign from malignant conditions and for recognizing specific subtypes of neoplasms that can have different management and prognosis. Rapid identification of gene fusions is particularly critical for patients with acute leukemia who cannot wait more than a few days before initiating treatment and for whom treatment can be dramatically different depending on the leukemia subtype...
September 28, 2018: Journal of Molecular Diagnostics: JMD
Yin-Hung Lin, Chen-Chi Wu, Yi-Hsin Lin, Ying-Chang Lu, Chih-Shan Chen, Tien-Chen Liu, Pei-Lung Chen, Chuan-Jen Hsu
Enlarged vestibular aqueduct (EVA) is an inner-ear malformation associated with sensorineural hearing impairment. Most EVAs are associated with Pendred syndrome and nonsyndromic DFNB4, two autosomal-recessive disorders caused by mutations in SLC26A4. However, many EVA patients cannot have a confirmed diagnosis by screening common SLC26A4 mutations, constituting an enigma in genetic diagnosis. To enable comprehensive genetic examination and explore the etiologies of EVA, we designed a next-generation sequencing panel targeting the entire length of 3 Pendred syndrome/DFNB4 genes (SLC26A4, FOXI1, and KCNJ10) and exons of 10 other genes related to EVA and performed genetic testing in 50 EVA families without confirmative results on screening for SLC26A4 hotspots (c...
September 28, 2018: Journal of Molecular Diagnostics: JMD
Fanny Leon, Albert Meyer, Robin Reynier, Emilie Blanc, Lilian Bruyère-Ostells, Jean-Charles Brès, Yannick Simonin, Sara Salinas, Pierre Gallian, Isabelle Leparc-Goffart, Antoine Biron, Myrielle Dupont-Rouzeyrol, François Morvan, Jean-Jacques Vasseur, Vincent Foulongne, Philippe Van de Perre, Jean-François Cantaloube, Chantal Fournier-Wirth
Nucleic acid testing during the preseroconversion viremic phase is required to differentially diagnose arboviral infections. The continuing emergence of arboviruses such as Zika (ZIKV), dengue (DENV), and chikungunya (CHIKV) viruses necessitates the development of a flexible diagnostic approach. Similar clinical signs and the priority to protect pregnant women from ZIKV infection indicate that the differential diagnosis of arboviruses is essential for effective patient management, clinical care, and epidemiological surveillance...
September 27, 2018: Journal of Molecular Diagnostics: JMD
Daniel E Sabath
No abstract text is available yet for this article.
September 27, 2018: Journal of Molecular Diagnostics: JMD
Turner A Conrad, Chien-Chi Lo, Jeffrey W Koehler, Amanda S Graham, Christopher P Stefan, Adrienne T Hall, Christina E Douglas, Patrick Sam Guy Chain, Timothy D Minogue
Next-generation sequencing (NGS) for infectious disease diagnostics is a relatively new and under-developed concept. If this technology is to become a regulatory-grade clinical diagnostic, standardization in the form of locked-down assays and firmly established underlying processes is necessary. Targeted sequencing, specifically by amplification of genomic signatures, has the potential to bridge the gap between PCR- and NGS-based diagnostics; however, existing NGS assay panels lack validated analytical techniques to adjudicate high background and error prone NGS data...
September 27, 2018: Journal of Molecular Diagnostics: JMD
Coralie Derrieux, Amélie Trinquand, Julie Bruneau, Virginie Verkarre, Ludovic Lhermitte, Marion Alcantara, Patrick Villarese, Bertrand Meresse, David Sibon, Olivier Hermine, Nicole Brousse, Thierry Molina, Christophe Cellier, Nadine Cerf-Bensussan, Georgia Malamut, Elizabeth Macintyre
Celiac disease is a chronic inflammation of the small intestine with villous atrophy that can become refractory to a gluten-free diet. Two categories of refractory celiac disease (I and II) can be distinguished by the phenotype of intra-epithelial lymphocytes and the status of TRG genes. Their distinction is important because 30% to 50% of type II, but only 0% to 14% of type I, evolve to an aggressive enteropathy-associated T-cell lymphoma and therefore require intensive treatment. Currently, differential diagnosis integrates immunohistochemistry, immunophenotyping, and TRG clonality analyses but each have limitations...
September 27, 2018: Journal of Molecular Diagnostics: JMD
Hana Jaworek, Vladimira Koudelakova, Jiri Drabek, Jana Vrbkova, Blazena Zborilova, Ivana Oborna, Jana Brezinova, Radim Marek, Karel Huml, Peter Vanek, Marian Hajduch
High-risk human papillomavirus (hrHPV) infection is a cause of cervical cancer development. The addition of hrHPV testing to cervical cancer screening and monitoring of cervical intraepithelial neoplasia treatment improves the efficacy of screening and treatment, respectively. Self-sampling for hrHPV testing seems a promising tool for increasing patient participation in cervical cancer screening. In this project, 1198 cervical swabs obtained by physicians and 176 cervicovaginal swabs obtained by self-sampling (not collected in parallel) were analyzed for the presence of 14 hrHPV genotypes using three commercially available assays in comparison...
August 28, 2018: Journal of Molecular Diagnostics: JMD
Viola Paradiso, Andrea Garofoli, Nadia Tosti, Manuela Lanzafame, Valeria Perrina, Luca Quagliata, Matthias S Matter, Stefan Wieland, Markus H Heim, Salvatore Piscuoglio, Charlotte K Y Ng, Luigi M Terracciano
Commercially available targeted panels miss genomic regions frequently altered in hepatocellular carcinoma (HCC). We sought to design and benchmark a sequencing assay for genomic screening in HCC. We designed an AmpliSeq custom panel targeting all exons of 33 protein-coding and two long noncoding RNA genes frequently mutated in HCC, TERT promoter, and nine genes with frequent copy number alterations. By using this panel, the profiling of DNA from fresh-frozen (n = 10, 1495×) and/or formalin-fixed, paraffin-embedded (FFPE) tumors with low-input DNA (n = 36, 530×) from 39 HCCs identified at least one somatic mutation in 90% of the cases...
August 22, 2018: Journal of Molecular Diagnostics: JMD
Rebecca F McClure, Mark D Ewalt, Jennifer Crow, Robyn L Temple-Smolkin, Mrudula Pullambhatla, Rachel Sargent, Annette S Kim
To address the clinical relevance of small DNA variants in chronic myeloid neoplasms (CMNs), an Association for Molecular Pathology Working Group comprehensively reviewed published literature, summarized key findings that support clinical utility, and defined critical gene inclusions for high-throughput sequencing testing panels. This review highlights the biological complexity of CMNs [including myelodysplastic syndromes, myeloproliferative neoplasms, entities with overlapping features (myelodysplastic syndromes/myeloproliferative neoplasms), and systemic mastocytosis], the genetic heterogeneity within diagnostic categories, and similarities between apparently disparate diagnostic entities...
August 20, 2018: Journal of Molecular Diagnostics: JMD
Matthew C Hiemenz, Dejerianne G Ostrow, Tracy M Busse, Jonathan Buckley, Dennis T Maglinte, Moiz Bootwalla, James Done, Jianling Ji, Gordana Raca, Alex Ryutov, Xinjie Xu, Chao Jie Zhen, Jeffrey M Conroy, Florette K Hazard, Joshua L Deignan, Beverly Rogers, Amanda L Treece, David M Parham, Xiaowu Gai, Alexander R Judkins, Timothy J Triche, Jaclyn A Biegel
The OncoKids panel is an amplification-based next-generation sequencing assay designed to detect diagnostic, prognostic, and therapeutic markers across the spectrum of pediatric malignancies, including leukemias, sarcomas, brain tumors, and embryonal tumors. This panel uses low input amounts of DNA (20 ng) and RNA (20 ng) and is compatible with formalin-fixed, paraffin-embedded and frozen tissue, bone marrow, and peripheral blood. The DNA content of this panel covers the full coding regions of 44 cancer predisposition loci, tumor suppressor genes, and oncogenes; hotspots for mutations in 82 genes; and amplification events in 24 genes...
August 20, 2018: Journal of Molecular Diagnostics: JMD
Fang Wei, Charles M Strom, Jordan Cheng, Chien-Chung Lin, Ching-Yun Hsu, Guy W Soo Hoo, David Chia, Yong Kim, Feng Li, David Elashoff, Tristan Grognan, Michael Tu, Wei Liao, Rena Xian, Wayne W Grody, Wu-Chou Su, David T W Wong
Previously, we detected circulating tumor DNA that contained two EGFR mutations (p.L858R and exon19 del) in plasma of patients with late-stage non-small-cell lung carcinoma (NSCLC) using the electric field-induced release and measurement (EFIRM) platform. Our aim was to determine whether EFIRM technology can detect these mutations in patients with early-stage NSCLC. Prospectively, 248 patients with radiographically determined pulmonary nodules were recruited. Plasma was collected before biopsy and histologic examination of the nodule...
November 2018: Journal of Molecular Diagnostics: JMD
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