scholarly journals Molecular Diagnostic of Solid Tumor Using a Next Generation Sequencing Custom-Designed Multi-Gene Panel

Diagnostics ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 250 ◽  
Author(s):  
Dario de Biase ◽  
Giorgia Acquaviva ◽  
Michela Visani ◽  
Viviana Sanza ◽  
Chiara M. Argento ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Solid Tumors ◽  
Single Gene ◽  
Gene Panel ◽  
Molecular Diagnostic ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Gene Characterization ◽  
Generation Sequencing ◽  
Oncosuppressor Genes

Next generation sequencing (NGS) allows parallel sequencing of multiple genes at a very high depth of coverage. The need to analyze a variety of targets for diagnostic/prognostic/predictive purposes requires multi-gene characterization. Multi-gene panels are becoming standard approaches for the molecular analysis of solid lesions. We report a custom-designed 128 multi-gene panel engineered to cover the relevant targets in 22 oncogene/oncosuppressor genes for the analysis of the solid tumors most frequently subjected to routine genotyping. A total of 1695 solid tumors were analyzed for panel validation. The analytical sensitivity is 5%. Analytical validation: (i) Accuracy: sequencing results obtained using the multi-gene panel are concordant using two different NGS platforms and single-gene approach sequencing (100% of 83 cases); (ii) Precision: consistent results are obtained in the samples analyzed twice with the same platform (100% of 20 cases). Clinical validation: the frequency of mutations identified in different tumor types is consistent with the published literature. This custom-designed multi-gene panel allows to analyze with high sensitivity and throughput 22 oncogenes/oncosuppressor genes involved in diagnostic/prognostic/predictive characterization of central nervous system tumors, non-small-cell lung carcinomas, colorectal carcinomas, thyroid nodules, pancreatic lesions, melanoma, oral squamous carcinomas and gastrointestinal stromal tumors.

scholarly journals Next generation sequencing-based gene panel tests for the management of solid tumors

2018 ◽  
Vol 110 (1) ◽  
pp. 6-15 ◽  
Author(s):  
Masayuki Nagahashi ◽  
Yoshifumi Shimada ◽  
Hiroshi Ichikawa ◽  
Hitoshi Kameyama ◽  
Kazuaki Takabe ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Solid Tumors ◽  
Gene Panel ◽  
Next Generation ◽  
Generation Sequencing

Analysis of a large cohort of non-small cell lung cancers submitted for somatic variant analysis demonstrates that targeted next-generation sequencing is fit for purpose as a molecular diagnostic assay in routine practice

2018 ◽  
Vol 71 (11) ◽  
pp. 1001-1006 ◽  
Author(s):  
David Allan Moore ◽  
Kevin Balbi ◽  
Alexander Ingham ◽  
Hendrik-Tobias Arkenau ◽  
Philip Bennett
Keyword(s):  
Next Generation Sequencing ◽  
Single Gene ◽  
Small Cell ◽  
Molecular Diagnostic ◽  
Next Generation ◽  
Small Cell Lung ◽  
Targeted Next Generation Sequencing ◽  
Gene Testing ◽  
The Impact ◽  
Generation Sequencing

AimsTargeted next-generation sequencing (tNGS) is increasingly being adopted as an alternative to single gene testing in some centres. Our aim was to assess the overall fitness and utility of tNGS as a routine clinical test in non-small cell lung cancer (NSCLC).MethodsAll NSCLC cases submitted to a single laboratory for tNGS analysis over a 3-year period were included. Rejection/failure rates and turnaround times were calculated. For reportable cases, data relating to observed genetic changes likely to be driving tumour growth and/or contributing to therapeutic resistance were extracted. The impact of varied referral site practices (tissue processing and sample format submitted) on analytical outcomes was also considered.ResultsA total of 2796 cases were submitted, of which 217 (7.8%) were rejected and 131 (5.1%) failed. The median turnaround time was seven working days. Of 2448 reported cases, KRAS, EGFR or other recognised driver mutations were observed in 35%, 17% and 5.4%, respectively. Of the remaining cases, 3.5% demonstrated significant incidental evidence of gene amplification. In 15% of EGFR-driven cases, evidence of an EGFR tyrosine kinase inhibitor resistance mechanism was observed. Potential concerns around the provision of slides or precut ‘rolls’ only (cf, formalin fixed paraffin embedded (FFPE) tissue blocks) as standard practice by certain referral sites were identified.ConclusionsA tNGS panel approach is practically achievable, with acceptable success rates and turnaround times, in the context of a routine clinical service. Furthermore, it provides additional clinically and analytically relevant information, which is not available from single gene testing alone.

Experience with the targeted next-generation sequencing in the diagnosis of hereditary hypophosphatemic rickets

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Ihsan Turan ◽  
Sevcan Erdem ◽  
Leman Damla Kotan ◽  
Semine Ozdemir Dilek ◽  
Mehmet Tastan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Single Gene ◽  
Care Center ◽  
Tertiary Care ◽  
Gene Panel ◽  
Hypophosphatemic Rickets ◽  
Next Generation ◽  
Efficient Manner ◽  
Individual Gene ◽  
Generation Sequencing

Abstract Objectives Hereditary Hypophosphatemic Rickets (HHR) is a heterogeneous group of disorders characterized by hypophosphatemia. Although the X-linked dominant HHR is the most common form, the genetic etiology of HHR is variable. Recently, developed next-generation sequencing techniques may provide opportunities for making HHR diagnosis in a timely and efficient way. Methods We investigated clinical and genetic features for 18 consecutive probands and their 17 affected family members with HHR. All patient’s clinical and biochemical data were collected. We first analyzed a single gene with Next-generation sequencing if the patients have a strong clue for an individual gene. For the remaining cases, a Hypophosphatemic Rickets gene panel, including all known HHR genes by Next-generation sequencing, was employed. Results We were able to diagnosis all of the consecutive 35 patients in our tertiary care center. We detected nine novel and 10 previously described variants in PHEX (9; 50%), SLC34A3 (3; 17%), ENPP1 (3; 17%), SLC34A1 (1; 5%), CLCN5 (1; 5%), and DMP1 (1; 5%). Conclusions To delineate the etiology of HHR cases in a cost and time-efficient manner, we propose single gene analysis by next-generation sequencing if findings of patients indicate a strong clue for an individual gene. If that analysis is negative or for all other cases, a Next-generation Sequence gene panel, which includes all known HHR genes, should be employed.

scholarly journals Development, Implementation and Assessment of Molecular Diagnostics by Next Generation Sequencing in Personalized Treatment of Cancer: Experience of a Public Reference Healthcare Hospital

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1196 ◽  
Author(s):  
Simarro ◽  
Murria ◽  
Pérez-Simó ◽  
Llop ◽  
Mancheño ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Single Gene ◽  
Turnaround Time ◽  
Genetic Alterations ◽  
Molecular Diagnostic ◽  
Public Healthcare ◽  
Next Generation ◽  
Primary Resistance ◽  
Iso 15189 ◽  
Generation Sequencing

The establishment of precision medicine in cancer patients requires the study of several biomarkers. Single-gene testing approaches are limited by sample availability and turnaround time. Next generation sequencing (NGS) provides an alternative for detecting genetic alterations in several genes with low sample requirements. Here we show the implementation to routine diagnostics of a NGS assay under International Organization for Standardization (UNE-EN ISO 15189:2013) accreditation. For this purpose, 106 non-small cell lung cancer (NSCLC) and 102 metastatic colorectal cancer (mCRC) specimens were selected for NGS analysis with Oncomine Solid Tumor (ThermoFisher). In NSCLC the most prevalently mutated gene was TP53 (49%), followed by KRAS (31%) and EGFR (13%); in mCRC, TP53 (50%), KRAS (48%) and PIK3CA (16%) were the most frequently mutated genes. Moreover, NGS identified actionable genetic alterations in 58% of NSCLC patients, and 49% of mCRC patients did not harbor primary resistance mechanisms to anti-EGFR treatment. Validation with conventional approaches showed an overall agreement >90%. Turnaround time and cost analysis revealed that NGS implementation is feasible in the public healthcare context. Therefore, NGS is a multiplexed molecular diagnostic tool able to overcome the limitations of current molecular diagnosis in advanced cancer, allowing an improved and economically sustainable molecular profiling.

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.

scholarly journals Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Elton J. R. Vasconcelos ◽  
Chayan Roy ◽  
Joseph A. Geiger ◽  
Kristina M. Oney ◽  
Melody Koo ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Next Generation Sequencing ◽  
Complete Analysis ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Spotted Fever Group ◽  
Next Generation ◽  
Vector Borne ◽  
16 S Rrna ◽  
Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

scholarly journals Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

2019 ◽  
Vol 144 (1) ◽  
pp. 90-98 ◽  
Author(s):  
Robyn T. Sussman ◽  
Amanda R. Oran ◽  
Carmela Paolillo ◽  
David Lieberman ◽  
Jennifer J. D. Morrissette ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Solid Tumors ◽  
Gold Standard ◽  
University Hospital ◽  
Fusion Partner ◽  
Next Generation ◽  
Fixed Tissue ◽  
Fusion Transcripts ◽  
Genetic Abnormalities ◽  
Generation Sequencing

Context.— Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK. Objective.— To validate an anchored multiplex polymerase chain reaction–based panel for the detection of fusion transcripts in a university hospital–based clinical molecular diagnostics laboratory. Design.— We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffin-embedded specimens with as little as 10% tumor nuclei. Results.— Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant III (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific. Conclusions.— This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors.

37. Clinical validation of somatic alterations in solid tumors by next generation sequencing

2018 ◽  
Vol 226-227 ◽  
pp. 50
Author(s):  
Xia Li ◽  
Bryan Austin ◽  
Michael Donovan ◽  
Janet Orton ◽  
Guang Liu
Keyword(s):  
Next Generation Sequencing ◽  
Solid Tumors ◽  
Clinical Validation ◽  
Next Generation ◽  
Somatic Alterations ◽  
Generation Sequencing
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