scholarly journals Epitachophoresis is a novel versatile total nucleic acid extraction method

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vladimira Datinska ◽  
Pantea Gheibi ◽  
Keynttisha Jefferson ◽  
Jaeyoung Yang ◽  
Sri Paladugu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Extraction Method ◽  
Acid Extraction ◽  
Total Nucleic Acid ◽  
Next Generation ◽  
Nucleic Acid Extraction ◽  
Dna And Rna ◽  
Generation Sequencing

AbstractEpitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt–290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing.

scholarly journals Formalin-Fixed and Paraffin-Embedded Samples for Next Generation Sequencing: Problems and Solutions

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1472
Author(s):  
Gerardo Cazzato ◽  
Concetta Caporusso ◽  
Francesca Arezzo ◽  
Antonietta Cimmino ◽  
Anna Colagrande ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Material ◽  
Growth Factor Receptor ◽  
Acid Extraction ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Tissue Samples ◽  
Dna And Rna ◽  
Sequencing Problems ◽  
Generation Sequencing

Over the years, increasing information has been asked of the pathologist: we have moved from a purely morphological diagnosis to biomolecular and genetic studies, which have made it possible to implement the use of molecular targeted therapies, such as anti-epidermal growth factor receptor (EGFR) molecules in EGFR-mutated lung cancer, for example. Today, next generation sequencing (NGS) has changed the approach to neoplasms, to the extent that, in a short time, it has gained a place of absolute importance and diagnostic, prognostic and therapeutic utility. In this scenario, formaldehyde-fixed and paraffin-embedded (FFPE) biological tissue samples are a source of clinical and molecular information. However, problems can arise in the genetic material (DNA and RNA) for use in NGS due to fixation, and work is being devoted to possible strategies to reduce its effects. In this paper, we discuss the applications of FFPE tissue samples in the execution of NGS, we focus on the problems arising with the use of this type of material for nucleic acid extraction and, finally, we consider the most useful strategies to prevent and reduce single nucleotide polymorphisms (SNV) and other fixation artifacts.

scholarly journals Cell-Free Total Nucleic Acid-Based Genotyping of Aggressive Lymphoma: Comprehensive Analysis of Gene Fusions and Nucleotide Variants by Next-Generation Sequencing

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3032
Author(s):  
Attila Mokánszki ◽  
Réka Bicskó ◽  
Lajos Gergely ◽  
Gábor Méhes
Keyword(s):  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Prospective Study ◽  
Aggressive Lymphoma ◽  
Gene Fusions ◽  
Total Nucleic Acid ◽  
Next Generation ◽  
Invasive Approach ◽  
Non Invasive ◽  
Generation Sequencing

Chromosomal translocations and pathogenic nucleotide variants both gained special clinical importance in lymphoma diagnostics. Non-invasive genotyping from peripheral blood (PB) circulating free nucleic acid has been effectively used to demonstrate cancer-related nucleotide variants, while gene fusions were not covered in the past. Our prospective study aimed to isolate and quantify PB cell-free total nucleic acid (cfTNA) from patients diagnosed with aggressive lymphoma and to compare with tumor-derived RNA (tdRNA) from the tissue sample of the same patients for both gene fusion and nucleotide variant testing. Matched samples from 24 patients were analyzed by next-generation sequencing following anchored multiplexed polymerase chain reaction (AMP) for 125 gene regions. Eight different gene fusions, including the classical BCL2, BCL6, and MYC genes, were detected in the corresponding tissue biopsy and cfTNA specimens with generally good agreement. Synchronous BCL2 and MYC translocations in double-hit high-grade B-cell lymphomas were obvious from cfTNA. Besides, mutations of 29 commonly affected genes, such as BCL2, MYD88, NOTCH2, EZH2, and CD79B, could be identified in matched cfTNA, and previously described pathogenic variants were detected in 16/24 cases (66.7%). In 3/24 cases (12.5%), only the PB sample was informative. Our prospective study demonstrates a non-invasive approach to identify frequent gene fusions and variants in aggressive lymphomas. cfTNA was found to be a high-value representative reflecting the complexity of the lymphoma aberration landscape.

Optimization of total nucleic acid extraction method for detecting Coconut cadang-cadang viroid variants in oil palm

2017 ◽  
Vol 46 (3) ◽  
pp. 235-237
Author(s):  
R. Kanavedee ◽  
G. Vadamalai ◽  
W. H. Lau ◽  
N. D. Roslan ◽  
S. Sundram
Keyword(s):  
Nucleic Acid ◽  
Oil Palm ◽  
Extraction Method ◽  
Acid Extraction ◽  
Total Nucleic Acid ◽  
Nucleic Acid Extraction

scholarly journals Guideline-Adherent Clinical Validation of a Comprehensive 170-Gene DNA/RNA Panel for Determination of Small Variants, Copy Number Variations, Splice Variants, and Fusions on a Next-Generation Sequencing Platform in the CLIA Setting

2021 ◽  
Vol 12 ◽  
Author(s):  
Theresa A. Boyle ◽  
Ashis K. Mondal ◽  
Daryoush Saeed-Vafa ◽  
Sudha Ananth ◽  
Pankaj Ahluwalia ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Limit Of Detection ◽  
Splice Variants ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Clinical Validation ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Dna And Rna ◽  
Generation Sequencing

We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/μl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.

The applications of next-generation sequencing (NGS) in drug development for cancer therapy

2020 ◽  
Vol 16 ◽  
Author(s):  
Pelin Telkoparan-Akillilar ◽  
Dilek Cevik
Keyword(s):  
Next Generation Sequencing ◽  
Drug Discovery ◽  
Cancer Therapy ◽  
Target Identification ◽  
Rapid Development ◽  
Next Generation ◽  
Biomedical Sciences ◽  
Dna And Rna ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Background: Numerous sequencing techniques have been progressed since the 1960s with the rapid development of molecular biology studies focusing on DNA and RNA. Methods: a great number of articles, book chapters, websites are reviewed, and the studies covering NGS history, technology and applications to cancer therapy are included in the present article. Results: High throughput next-generation sequencing (NGS) technologies offer many advantages over classical Sanger sequencing with decreasing cost per base and increasing sequencing efficiency. NGS technologies are combined with bioinformatics software to sequence genomes to be used in diagnostics, transcriptomics, epidemiologic and clinical trials in biomedical sciences. The NGS technology has also been successfully used in drug discovery for the treatment of different cancer types. Conclusion: This review focuses on current and potential applications of NGS in various stages of drug discovery process, from target identification through to personalized medicine.

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