scholarly journals A simple and cost-effective approach for technical validation of next generation methylation sequencing data

2019 ◽  
Author(s):  
Ali Javadmanesh ◽  
Afsaneh Mojtabanezhad Shariatpanahi ◽  
Ehsan Shams Davodly ◽  
Marjan Azghandi ◽  
Maryam Yassi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Resolution ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Next Generation ◽  
Sequencing Data ◽  
Cost Effective Approach ◽  
Technical Validation ◽  
R Programming ◽  
And Control

Abstract Background DNA methylation is a fundamental epigenetic process that, in most cases, modulates genetic expression levels. Changes in DNA methylation, either hypo- or hypermethylation, have a key role in many biological processes and several human diseases such as cancer. In the current study, we offered an approach to validate the next generation methylation sequencing data.Methods Genomic DNA was extracted from target and control samples (6 in each group), followed by bisulfite conversion. Next generation methylation sequencing and methylation sensitive high-resolution melting assay were carried out. The primers for methylation sequencing validation were designed by R programming language.Results In the current study, two groups, case and control, were discriminated based on methylation sequencing results and the real time PCR-based results were in accordance with the next generation methylation sequencing.Discussion Methylation sensitive high-resolution melting validation assay is a simple and cost-effective method, which confirmed next generation methylation sequencing results.

scholarly journals Genotyping of GATA4 Gene Variant (G296S) in Malaysian Congenital Heart Disease Subjects by Real-Time PCR High Resolution Melting Analysis

2013 ◽  
Vol 32 (2) ◽  
pp. 152-157
Author(s):  
Nora Fawzi ◽  
Ramachandran Vasudevan ◽  
Patimah Ismail ◽  
Mazeni Alwi ◽  
Ahmad Fazli Abdul Aziz ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
High Resolution ◽  
Congenital Heart ◽  
High Resolution Melting ◽  
New Technology ◽  
Cost Effective ◽  
Study Cohort ◽  
Hrm Analysis ◽  
Melting Analysis

Summary Background: Congenital heart disease (CHD) is the most common birth defect; however, the underlying etiology is unrecognized in the majority of cases. GATA-binding protein 4 (GATA4), a cardiac transcription factor gene, has a crucial role in the cardiogenesis process; hence, a number of heterozygote sequence variations were identified as a cause of CHD. G296S heterozygote variant is the most frequently reported GATA4 gene sequence alteration. This study aims to investigate the role of G296S variant of the GATA4 gene in Malaysian CHD subjects. Methods: We have investigated 86 Malaysian CHD subjects with cardiac septation defects for the presence of the GATA4 gene heterozygote variant (G296S) by the new technology of high resolution melting (HRM) analysis. Results: Genotyping of G296S (c.886G>A) by HRM analysis shows that all the sample genotypes were of the wild GG type genotype and the heterozygote mutant GA genotype was totally absent from this study cohort. Conclusions: The results of our study showed that the G296S variant of the GATA4 gene was not associated with the development of CHD in Malaysian subjects. The use of HRM analysis proved a cost-effective, high-throughput, specific and sensitive genotyping technique which eliminates the need for unnecessary sequencing.

scholarly journals Microsatellite marker development for the tetraploid Veronica aragonensis (Plantaginaceae) using next-generation sequencing and high-resolution melting analyses

2018 ◽  
Vol 6 (5) ◽  
pp. e01154
Author(s):  
Nélida Padilla-García ◽  
Teresa Malvar-Ferreras ◽  
Josie Lambourdière ◽  
M. Montserrat Martínez-Ortega ◽  
Nathalie Machon
Keyword(s):  
Next Generation Sequencing ◽  
High Resolution ◽  
Microsatellite Marker ◽  
High Resolution Melting ◽  
Marker Development ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals High-Resolution Melting Is a Sensitive, Cost-Effective, Time-Saving Technique for BRAF V600E Detection in Thyroid FNAB Washing Liquid: A Prospective Cohort Study

2015 ◽  
Vol 4 (2) ◽  
pp. 73-81 ◽  
Author(s):  
Marco Marino ◽  
Maria Laura Monzani ◽  
Giulia Brigante ◽  
Katia Cioni ◽  
Bruno Madeo ◽  
...  
Keyword(s):  
Cohort Study ◽  
High Resolution ◽  
Molecular Analysis ◽  
Prospective Cohort Study ◽  
Prospective Cohort ◽  
Large Scale ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Braf V600e Mutation ◽  
Braf V600e

Objective: The diagnostic accuracy of thyroid fine needle aspiration biopsy (FNAB) can be improved by the combination of cytological and molecular analysis. In this study, washing liquids of FNAB (wFNAB) were tested for the BRAF V600E mutation, using the sensitive and cost-effective technique called high-resolution melting (HRM). The aim was to demonstrate the feasibility of BRAF analysis in wFNAB and its diagnostic utility, combined with cytology. Design: Prospective cohort study. Methods: 481 patients, corresponding to 648 FNAB samples, were subjected to both cytological (on cells smeared onto a glass slide) and molecular analysis (on fluids obtained washing the FNAB needle with 1 ml of saline) of the same aspiration. BRAF V600E analysis was performed by HRM after methodological validation for application to wFNAB (technique sensitivity: 5.4%). Results: The cytological results of the FNAB were: 136 (21%) nondiagnostic (THY1); 415 (64%) benign (THY2); 80 (12.4%) indeterminate (THY3); 9 (1.4%) suspicious for malignancy (THY4); 8 (1.2%) diagnostic of malignancy (THY5). The BRAF V600E mutation was found in 5 THY2, 2 THY3, 6 THY4 and 6 THY5 samples. Papillary carcinoma diagnosis was histologically confirmed in all BRAF+ thyroidectomized patients. BRAF combined with cytology improved the diagnostic value compared to cytology alone in a subgroup of 74 operated patients. Conclusions: HRM was demonstrated to be a feasible method for BRAF analysis in wFNAB. Thanks to its sensitivity and cost-effectiveness, it might be routinely used on a large scale in clinical practice. In perspective, standby wFNAB samples could be analyzed a posteriori in case of indeterminate cytology and/or suspicious findings on ultrasound.

High-resolution root zone soil moisture-based indices for drought monitoring in the Ebro basin

2021 ◽  
Author(s):  
Vivien-Georgiana Stefan ◽  
Maria-José Escorihuela ◽  
Pere Quintana-Seguí
Keyword(s):  
Remote Sensing ◽  
Soil Moisture ◽  
High Resolution ◽  
Root Zone ◽  
Cost Effective ◽  
Strong Relationship ◽  
Drought Indices ◽  
Ebro Basin ◽  
Small Areas ◽  
Cost Effective Approach

<h3>Agriculture is an important factor on water resources, given the constant population growth and the strong relationship between water availability and food production. In this context, root zone soil moisture (RZSM) measurements are used by modern irrigators in order to detect the onset of crop water stress and to trigger irrigations. Unfortunately, in situ RZSM measurements are costly; combined with the fact they are available only over small areas and that they might not be representative at the field scale, remote sensing is a cost-effective approach for mapping and monitoring extended areas. A recursive formulation of an exponential filter was used in order to derive 1 km resolution RZSM estimates from SMAP (Soil Moisture Active Passive) surface soil moisture (SSM) over the Ebro basin. The SMAP SSM was disaggregated to a 1 km resolution by using the DISPATCH (DISaggregation based on a Physical And Theoretical scale CHange) algorithm. The pseudodiffusivity parameter of the exponential filter was calibrated per land cover type, by using ISBA-DIF (Interaction Soil Biosphere Atmosphere) surface and root zone soil moisture data as an intermediary step. The daily 1 km RZSM estimates were then used to derive 1 km drought indices such as soil moisture anomalies and soil moisture deficit indices (SMDI), on a weekly time-scale, covering the entire 2020 year. Results show that both drought indices are able to capture rainfall and drying events, with the weekly anomaly being more responsive to sudden events such as heavy rainfalls, while the SMDI is slower to react do the inherent inertia it has. Moreover, a quantitative comparison with drought indices derived from a model-based RZSM estimates has also been performed, with results showing a strong correspondence between the different indices. For comparison purposes, the weekly soil moisture anomalies and SMDI derived using 1 km SMAP-derived SSM were also estimated. The analysis shows that the anomalies and SMDI based on the RZSM are more representative of the hydric stress level of the plants, given that the RZSM is better suited than the SSM to describe the moisture conditions at the deeper layers, which are the ones used by plants during growth and development.</h3><h3>The study provides an insight into obtaining robust, high-resolution remote-sensing derived drought indices based on remote-sensing derived RZSM estimates. The 1 km resolution proves an improvement from other currently available drought indices, such as the European Drought Observatory’s 5 km resolution drought index, which is not able to capture as well the spatial variability present within heterogeneous areas. Moreover, the SSM-derived drought indices are currently used in a drought observatory project, covering a region in the Tarragona province of Catalonia, Spain. The project aims at offering irrigation recommendations to water agencies, and the introduction of RZSM-derived drought indices will further improve such advice.</h3>

scholarly journals High Resolution Melting and Insertion Site-Based Polymorphism Markers for Wheat Variability Analysis and Candidate Genes Selection at Drought and Heat MQTL Loci

Agronomy ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1294 ◽  
Author(s):  
Rosa Mérida-García ◽  
Sergio Gálvez ◽  
Etienne Paux ◽  
Gabriel Dorado ◽  
Laura Pascual ◽  
...  
Keyword(s):  
Quantitative Trait Loci ◽  
High Resolution ◽  
Quantitative Trait ◽  
High Resolution Melting ◽  
Insertion Site ◽  
Cost Effective ◽  
Wheat Breeding ◽  
Reference Sequence ◽  
Detection Techniques ◽  
Trait Loci

The practical use of molecular markers is facilitated by cost-effective detection techniques. In this work, wheat insertion site-based polymorphisms (ISBP) markers were set up for genotyping using high-resolution melting analysis (HRM). Polymorphic HRM-ISBP assays were developed for wheat chromosomes 4A and 3B and used for wheat variability assessment. The marker sequences were mapped against the wheat genome reference sequence, targeting interesting genes. Those genes were located within or in proximity to previously described quantitative trait loci (QTL) or meta-quantitative trait loci (MQTL) for drought and heat stress tolerance, and also yield and yield related traits. Eighteen of the markers used tagged drought related genes and, interestingly, eight of the genes were differentially expressed under different abiotic stress conditions. These results confirmed HRM as a cost-effective and efficient tool for wheat breeding programs.

scholarly journals High-Resolution Melting-Based Quantitative Analysis of RASSF1 Methylation in Breast Cancer

Medicina ◽  
2013 ◽  
Vol 49 (2) ◽  
pp. 14 ◽  
Author(s):  
Kristina Stuopelytė ◽  
Kristina Daniūnaitė ◽  
Aida Laurinavičienė ◽  
Valerijus Ostapenko ◽  
Sonata Jarmalaitė
Keyword(s):  
Breast Cancer ◽  
Polymerase Chain Reaction ◽  
High Resolution ◽  
Quantitative Methods ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Breast Tissues

Background and Objective. Breast cancer is the leading cause of death from cancer among women worldwide. The aberrant promoter methylation of tumor suppressor genes is a typical epigenetic alteration for breast cancer and can be detected in early carcinogenesis. High-throughput and cost-effective methods are needed for the early and sensitive detection of epigenetic changes in clinical material. The main purpose of our study was to optimize a high-resolution melting (HRM) assay for the reliable and quantitative assessment of RASSF1 gene methylation, which is considered one of the earliest epigenetic alterations in breast cancer. Material and Methods. A total of 76 breast carcinomas and 10 noncancerous breast tissues were studied by means of HRM and compared with the results obtained by means of quantitative methylation-specific polymerase chain reaction (QMSP) and methylation-specific polymerase chain reaction (MSP). Results. Both quantitative methods, HRM and QMSP, showed a similar specificity and sensitivity for the detection of RASSF1 methylation in breast cancer (about 80% and 70%, respectively). In breast cancer, the mean methylation intensity of RASSF1 was 42.5% and 48.6% according to HRM and QMSP, respectively. Both methods detected low levels of methylation (less than 5%) in noncancerous breast tissues. In comparison with quantitative methods, MSP showed a lower sensitivity (70%), but a higher specificity (80%) for the detection of RASSF1 methylation in breast cancer. Conclusions. HRM is as a simple, cost-effective method for the reliable high-throughput quantification of DNA methylation in clinical material.

A Cost-effective High-resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

2010 ◽  
Vol 52 (12) ◽  
pp. 1036-1042 ◽  
Author(s):  
Yi-Dan Li ◽  
Zhi-Zhan Chu ◽  
Xiang-Guo Liu ◽  
Hai-Chun Jing ◽  
Yao-Guang Liu ◽  
...  
Keyword(s):  
High Resolution ◽  
Dna Polymorphism ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Polymorphism Detection

Enabling Precision Oncology Through Precision Diagnostics

2020 ◽  
Vol 15 (1) ◽  
pp. 97-121 ◽  
Author(s):  
Noah A. Brown ◽  
Kojo S.J. Elenitoba-Johnson
Keyword(s):  
Next Generation Sequencing ◽  
Cancer Patients ◽  
Cancer Genomics ◽  
Cost Effective ◽  
Routine Clinical Practice ◽  
Precision Oncology ◽  
Next Generation ◽  
Sequencing Data ◽  
Molecular Alterations ◽  
Generation Sequencing

Genomic testing enables clinical management to be tailored to individual cancer patients based on the molecular alterations present within cancer cells. Genomic sequencing results can be applied to detect and classify cancer, predict prognosis, and target therapies. Next-generation sequencing has revolutionized the field of cancer genomics by enabling rapid and cost-effective sequencing of large portions of the genome. With this technology, precision oncology is quickly becoming a realized paradigm for managing the treatment of cancer patients. However, many challenges must be overcome to efficiently implement the transition of next-generation sequencing from research applications to routine clinical practice, including using specimens commonly available in the clinical setting; determining how to process, store, and manage large amounts of sequencing data; determining how to interpret and prioritize molecular findings; and coordinating health professionals from multiple disciplines.

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