Method for definition of CIS-TRANS status in double CEBPA mutations.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e18511-e18511
Author(s):  
Miguel Mitne-Neto ◽  
Caroline Mônaco Moreira ◽  
Ana Maria Fraga
Keyword(s):  
Gene Cloning ◽  
Cost Effective ◽  
Digital Pcr ◽  
Turnaround Time ◽  
Sequencing Analysis ◽  
Coding Region ◽  
Dna Molecule ◽  
System A ◽  
Cebpa Mutations ◽  
Definition Of

e18511 Background: Acute myeloid leukemia (AML) is characterized by infiltration of blood and bone marrow by proliferative, clonal and abnormally differentiated hematopoietic cells, affecting 3 in 100.000 adults. CEBPA is a transcription factor and a biomarker for cytogenetically normal (CN) patients. Somatic CEBPA mutations occur in 10% of CN patients and recent studies have shown that a favorable outcome may only be obtained in those carrying CEBPA mutations in different alleles ( trans). The evaluation of double mutations and the definition of bi-allelic ( trans) versus mono-allelic ( cis) status applies long protocols, involving gene cloning and sequencing, which are challenging for the diagnostic routine and negatively impacts the turnaround time (TAT). The present study aimed the development of an alternate method for the identification of double mutations in CEBPA and its CIS- TRANScondition. Methods: A previously screened sample carrying double CEBPA mutations was selected and its DNA was extracted. This sample and a DNA control were quantified and diluted in order to obtain ½ DNA molecule per well for amplification, using digital PCR rationale. Samples were distributed in 40 wells of a 96 well plate. A pair of primers was used to amplify the entire CEBPA coding region in a Real-Time PCR using SYBR green-based detection in QuantStudio System. A subset of primers was used to amplify and to sequence the regions of interest. The entire process was repeated three times, in order to prove its reproducibility. Results: The above described dilution allowed an estimation that half of the wells had no DNA and half contained a single DNA molecule. An average of 18 wells presented amplification. Five of the positive wells were used as templates for a nested PCR followed by Sanger sequencing analysis. Since each mutation was identified as the result of distinct well amplification, we concluded that the mutations were in trans. Conclusions: Our results show that it is possible to determine the CIS- TRANS allelic configuration of CEBPA dual-mutation in a cost-effective process and with a short TAT, avoiding gene cloning systems. Additionally, the process is reproducible, can be easily implemented as a routine analysis and has a great impact for AML patients’ diagnosis.

scholarly journals Automated PCR detection of BRAF mutations in colorectal adenocarcinoma: a diagnostic test accuracy study

2015 ◽  
Vol 69 (5) ◽  
pp. 398-402 ◽  
Author(s):  
Richard Colling ◽  
Lai Mun Wang ◽  
Elizabeth Soilleux
Keyword(s):  
Digital Pcr ◽  
Turnaround Time ◽  
Test Accuracy ◽  
Braf Mutations ◽  
System A ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Accuracy Study ◽  
Automated Platform ◽  
Formalin Fixed

BackgroundTesting for BRAF mutations in colorectal carcinoma (CRC) is important in the screening pathway for Lynch syndrome and is of prognostic value to guide management. This is a diagnostic accuracy study of the Idylla system, a novel and automated alternative PCR system.Methods100 consecutive formalin-fixed, paraffin-embedded CRC resection cases were tested for BRAF mutations using the Idylla automated platform and compared with standard (Cobas) PCR.ResultsThe sensitivity of the Idylla BRAF test was 100% and the specificity was 96%. Only one discordant Idylla positive/standard PCR negative result occurred and on Droplet Digital PCR demonstrated a mutation not identified by traditional PCR in this case.ConclusionThis study has validated the Idylla system for BRAF testing in CRC and demonstrated a possibly greater sensitivity, in addition to cost effectiveness and shorter turnaround time, when compared with standard PCR.

scholarly journals Nested pool testing strategy for the diagnosis of infectious diseases

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Inés Armendáriz ◽  
Pablo A. Ferrari ◽  
Daniel Fraiman ◽  
José M. Martínez ◽  
Hugo G. Menzella ◽  
...  
Keyword(s):  
Large Scale ◽  
Cost Effective ◽  
Digital Pcr ◽  
Practical Implementation ◽  
Testing Procedures ◽  
Effective Strategies ◽  
Rna Quantification ◽  
Pooling Strategy ◽  
Definition Of ◽  
Self Consistency

AbstractThe progress of the SARS-CoV-2 pandemic requires the design of large-scale, cost-effective testing programs. Pooling samples provides a solution if the tests are sensitive enough. In this regard, the use of the gold standard, RT-qPCR, raises some concerns. Recently, droplet digital PCR (ddPCR) was shown to be 10–100 times more sensitive than RT-qPCR, making it more suitable for pooling. Furthermore, ddPCR quantifies the RNA content directly, a feature that, as we show, can be used to identify nonviable samples in pools. Cost-effective strategies require the definition of efficient deconvolution and re-testing procedures. In this paper we analyze the practical implementation of an efficient hierarchical pooling strategy for which we have recently derived the optimal, determining the best ways to proceed when there are impediments for the use of the absolute optimum or when multiple pools are tested simultaneously and there are restrictions on the throughput time. We also show how the ddPCR RNA quantification and the nested nature of the strategy can be combined to perform self-consistency tests for a better identification of infected individuals and nonviable samples. The studies are useful to those considering pool testing for the identification of infected individuals.

scholarly journals Validation of CRC and NSCLC somatic mutations detected by NGS using ddPCR

2019 ◽  
Vol 3 (1) ◽  
pp. 71
Author(s):  
Lana Salihefendić ◽  
Dino Pećar ◽  
Rijad Konjhodžić
Keyword(s):  
Lung Cancer ◽  
Somatic Mutations ◽  
Low Frequency ◽  
High Sensitivity ◽  
Cost Effective ◽  
Digital Pcr ◽  
Turnaround Time ◽  
Genetic Alterations ◽  
Wild Type ◽  
Good Tool

Next Generation Sequencing (NGS) has become powerful tool in molecular oncology. It allows multiparallel targeted sequencing that enables comprehensive assessment of tumor heterogeneity. Detection of mutations in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) defines patients diagnosis, therapy and prognosis. Multiple genes, their somatic mutations to be precise, carry different degrees of importance for any of these stages. Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2, which was used in this study, allows detection of hotspot mutations on 22 genes in a single reaction. Droplet digital PCR (ddPCR) has a unique advantage in low frequency mutation detection and it has been used as a validation method for mutations that were detected with NGS. It has high sensitivity and enables accurate detection of mutant allele in a background of abundant wild type alleles. For this study 35 samples of CRC and NSCLC were sequenced and same samples were analysed on ddPCR for KRAS, NRAS, EGFR and BRAF genes. All processed samples were successfully sequenced and had average base coverage >500X. NGS sequencing proved itself to be cost effective, has shorter turnaround time and is highly sensitive. Out of 35 samples, 25 had genetic alterations, while 10 samples are reported as wild type but were still tested on ddPCR as controls. In three samples low frequency somatic mutations were detected by NGS and verified using ddPCR, which leads us to conclusion that ddPCR is a good tool for verification of somatic mutations in CRC and NSCLC.

Parallel Computing for Tire Simulations

2011 ◽  
Vol 39 (3) ◽  
pp. 193-209 ◽  
Author(s):  
H. Surendranath ◽  
M. Dunbar
Keyword(s):  
High Performance ◽  
Effective Means ◽  
Cost Effective ◽  
Turnaround Time ◽  
Careful Consideration ◽  
Rolling Contact ◽  
Element Analysis ◽  
Parallel Solvers ◽  
Design Changes ◽  
Highly Nonlinear

Abstract Over the last few decades, finite element analysis has become an integral part of the overall tire design process. Engineers need to perform a number of different simulations to evaluate new designs and study the effect of proposed design changes. However, tires pose formidable simulation challenges due to the presence of highly nonlinear rubber compounds, embedded reinforcements, complex tread geometries, rolling contact, and large deformations. Accurate simulation requires careful consideration of these factors, resulting in the extensive turnaround time, often times prolonging the design cycle. Therefore, it is extremely critical to explore means to reduce the turnaround time while producing reliable results. Compute clusters have recently become a cost effective means to perform high performance computing (HPC). Distributed memory parallel solvers designed to take advantage of compute clusters have become increasingly popular. In this paper, we examine the use of HPC for various tire simulations and demonstrate how it can significantly reduce simulation turnaround time. Abaqus/Standard is used for routine tire simulations like footprint and steady state rolling. Abaqus/Explicit is used for transient rolling and hydroplaning simulations. The run times and scaling data corresponding to models of various sizes and complexity are presented.

scholarly journals Current and Future Pathotyping Platforms for Plasmodiophora brassicae in Canada

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1446
Author(s):  
Heather H. Tso ◽  
Leonardo Galindo-González ◽  
Stephen E. Strelkov
Keyword(s):  
Crop Production ◽  
Plasmodiophora Brassicae ◽  
Cost Effective ◽  
Turnaround Time ◽  
Molecular Techniques ◽  
Differential Set ◽  
Management Plans ◽  
Increased Sensitivity ◽  
Phenotypic Methods ◽  
Important Disease

Clubroot, caused by Plasmodiophora brassicae, is one of the most detrimental threats to crucifers worldwide and has emerged as an important disease of canola (Brassica napus) in Canada. At present, pathotypes are distinguished phenotypically by their virulence patterns on host differential sets, including the systems of Williams, Somé et al., the European Clubroot Differential set, and most recently the Canadian Clubroot Differential set and the Sinitic Clubroot Differential set. Although these are frequently used because of their simplicity of application, they are time-consuming, labor-intensive, and can lack sensitivity. Early, preventative pathotype detection is imperative to maximize productivity and promote sustainable crop production. The decreased turnaround time and increased sensitivity and specificity of genotypic pathotyping will be valuable for the development of integrated clubroot management plans, and interest in molecular techniques to complement phenotypic methods is increasing. This review provides a synopsis of current and future molecular pathotyping platforms for P. brassicae and aims to provide information on techniques that may be most suitable for the development of rapid, reliable, and cost-effective pathotyping assays.

scholarly journals Zur exakten Behandlung von kollektiven Rotationen Teil A: Theorie / On the Exact Treatment of Collective Rotations Part A: Theory

1973 ◽  
Vol 28 (2) ◽  
pp. 206-215
Author(s):  
Hanns Ruder
Keyword(s):  
Coordinate System ◽  
Perturbation Expansion ◽  
Rotational Energy ◽  
The Body ◽  
System A ◽  
N Body Problem ◽  
Definition Of ◽  
Fixed System ◽  
Body Fixed Coordinate System ◽  
Groundstate Energy

Basic in the treatment of collective rotations is the definition of a body-fixed coordinate system. A kinematical method is derived to obtain the Hamiltonian of a n-body problem for a given definition of the body-fixed system. From this exact Hamiltonian, a consequent perturbation expansion in terms of the total angular momentum leads to two exact expressions: one for the collective rotational energy which has to be added to the groundstate energy in this order of perturbation and a second one for the effective inertia tensor in the groundstate. The discussion of these results leads to two criteria how to define the best body-fixed coordinate system, namely a differential equation and a variational principle. The equivalence of both is shown.

scholarly journals Nanopore Targeted Sequencing for Rapid Gene Mutations Detection in Acute Myeloid Leukemia

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1026 ◽  
Author(s):  
Cumbo ◽  
Minervini ◽  
Orsini ◽  
Anelli ◽  
Zagaria ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Low Cost ◽  
Gene Mutations ◽  
Predictive Biomarkers ◽  
Molecular Testing ◽  
Sequencing Analysis ◽  
Cebpa Mutations ◽  
Targeted Gene Sequencing ◽  
Acute Myeloid

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

XV.—The Placoderm Fish Rhachiosteus pterygiatus Gross and its Relationships

1966 ◽  
Vol 66 (15) ◽  
pp. 377-392 ◽  
Author(s):  
Roger S. Miles
Keyword(s):  
Sensory System ◽  
System A ◽  
The Family ◽  
Definition Of

SynopsisThe holotype and only known specimen of Rhachiosteus pterygiatus Gross is partially redescribed and new restorations are given. Attention is drawn to important points in its osteology and the possible development of a cutaneous sensory system. A definition of the family Rhachiosteidsæ Stensiö is given. This family differs from all other described groups of euarthrodires in the lack of posterior lateral and posterior dorsolateral flank plates. Rhachiosteus is a pachyosteomorph brachythoracid, as defined in the text, and may be fairly closely related in some way to the (coccosteomorph) family Coccosteidsæ. There is no indication that it is closely related to any other known pachyosteomorph, or to other groups of arthrodires, such as the Rhenanida and Ptyctodontida, in which there are no posterior flank plates.

The Use of Photogrammetry in Piping Design

1983 ◽  
Vol 197 (2) ◽  
pp. 125-138 ◽  
Author(s):  
P A Bracewell ◽  
U R Klement
Keyword(s):  
Information Retrieval ◽  
Mathematical Formulation ◽  
Three Dimensional ◽  
Cost Effective ◽  
Process Industry ◽  
Process Plant ◽  
Definition Of ◽  
High Degree ◽  
Positional Data

Piping design for ‘revamp’ projects in the process industry requires the retrieval of large amounts of ‘as-built’ data from existing process plant installations. Positional data with a high degree of accuracy are required. Photogrammetry, the science of measurement from photographs, was identified in Imperial Chemical Industries plc (ICI) as a suitable tool for information retrieval. The mathematical formulation enabling the definition of three-dimensional positions from photographic information is described. The process of using ICI's photogrammetric system for the definition of complete objects such as structures and pipes is illustrated. The need for specialized photogrammetric software for design purposes is explained. A case study describing how the photogrammetric system has been applied is described and graphical outputs from this exercise are shown. It is concluded that this particular photogrammetric system has proved to be a cost effective and accurate tool for the retrieval of ‘as-built’ information.

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