scholarly journals Orthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay Discordance

2019 ◽  
pp. 1-9 ◽  
Author(s):  
Daniel Stetson ◽  
Ambar Ahmed ◽  
Xing Xu ◽  
Barrett R.B. Nuttall ◽  
Tristan J. Lubinski ◽  
...  
Keyword(s):  
Tumor Heterogeneity ◽  
False Negative ◽  
Variant Calling ◽  
Circulating Tumor Dna ◽  
Orthogonal Comparison ◽  
Novel Variants ◽  
Technical Factors ◽  
Next Generation Sequencing Ngs ◽  
Biologic Factors ◽  
Generation Sequencing

PURPOSE Discordance between plasma and tumor variant calling has been attributed primarily to tumor heterogeneity, whereas technical variables remain largely unexplored. MATERIALS AND METHODS To measure these variables, we tested four next-generation sequencing (NGS) gene panel assays for mutations in circulating tumor DNA (ctDNA) using replicate sets of 24 plasma samples and compared the results with matched tumor-normal tissue pairs. RESULTS Our orthogonal approach identified false-negative (FN) and false-positive (FP) variants with high confidence and revealed substantial variability among the ctDNA assays, with a range of sensitivity (38% to 89%) and positive predictive value (36% to 80%). Most discordance in our cross-vendor study was observed below 1% variant allele frequency. FP variants displayed mutational biases and tended to be novel variants not found in somatic databases. Of the 56 unique variants called by all four ctDNA assays, 41 (68%) resulted from technical discordance. CONCLUSION These findings suggest that most NGS assay discordance is a result of technical variations and, to a lesser extent, biologic factors such as clonal hematopoiesis of indeterminate potential and tumor heterogeneity.

scholarly journals Patient Derived Xenografts for Genome-Driven Therapy of Osteosarcoma

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 416
Author(s):  
Lorena Landuzzi ◽  
Maria Cristina Manara ◽  
Pier-Luigi Lollini ◽  
Katia Scotlandi
Keyword(s):  
Clinical Trials ◽  
Tumor Heterogeneity ◽  
Functional Studies ◽  
Ngs Data Analysis ◽  
The Many ◽  
Orthotopic Xenografts ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing ◽  
Somatic Copy Number Alterations

Osteosarcoma (OS) is a rare malignant primary tumor of mesenchymal origin affecting bone. It is characterized by a complex genotype, mainly due to the high frequency of chromothripsis, which leads to multiple somatic copy number alterations and structural rearrangements. Any effort to design genome-driven therapies must therefore consider such high inter- and intra-tumor heterogeneity. Therefore, many laboratories and international networks are developing and sharing OS patient-derived xenografts (OS PDX) to broaden the availability of models that reproduce OS complex clinical heterogeneity. OS PDXs, and new cell lines derived from PDXs, faithfully preserve tumor heterogeneity, genetic, and epigenetic features and are thus valuable tools for predicting drug responses. Here, we review recent achievements concerning OS PDXs, summarizing the methods used to obtain ectopic and orthotopic xenografts and to fully characterize these models. The availability of OS PDXs across the many international PDX platforms and their possible use in PDX clinical trials are also described. We recommend the coupling of next-generation sequencing (NGS) data analysis with functional studies in OS PDXs, as well as the setup of OS PDX clinical trials and co-clinical trials, to enhance the predictive power of experimental evidence and to accelerate the clinical translation of effective genome-guided therapies for this aggressive disease.

scholarly journals Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR

BioTechniques ◽  
2020 ◽  
Vol 68 (1) ◽  
pp. 48-51 ◽  
Author(s):  
Christopher R McEvoy ◽  
Timothy Semple ◽  
Bhargavi Yellapu ◽  
David Y Choong ◽  
Huiling Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Limiting Factor ◽  
Next Generation ◽  
Tumor Biopsy ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna ◽  
Generation Sequencing

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

scholarly journals Methylmalonic Acidemia with Novel MUT Gene Mutations

2017 ◽  
Vol 2017 ◽  
pp. 1-2
Author(s):  
Inusha Panigrahi ◽  
Savita Bhunwal ◽  
Harish Varma ◽  
Simranjeet Singh
Keyword(s):  
Sanger Sequencing ◽  
Gene Mutations ◽  
Methylmalonic Acidemia ◽  
Feeding Problems ◽  
Novel Variants ◽  
Mut Gene ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Poor Weight Gain

A 5-year-old boy presented with recurrent episodes of fever, feeding problems, lethargy, from the age of 11 months, and poor weight gain. He was admitted and evaluated for metabolic causes and diagnosed as having methylmalonic acidemia (MMA). He was treated with vit B12 and carnitine supplements and has been on follow-up for the last 3 years. Mutation analysis by next generation sequencing (NGS), supplemented with Sanger sequencing, revealed two novel variants in the MUT gene responsible for MMA in exon 5 and exon 3, respectively. Recently he developed dystonic movements including orofacial dyskinesia. With advent of NGS, judicious use of NGS with Sanger sequencing can help identify causative possibly pathogenic mutations.

scholarly journals A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiandong Zeng ◽  
Natalia T. Leach ◽  
Zhaoqing Zhou ◽  
Hui Zhu ◽  
Jean A. Smith ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Causative Mutation ◽  
Next Generation ◽  
Single Nucleotide ◽  
Highly Sensitive ◽  
Highly Sensitive Detection ◽  
Next Generation Sequencing Ngs ◽  
Reference Bias ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

scholarly journals The Saccharomyces cerevisiae W303-K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt

Open Biology ◽  
2012 ◽  
Vol 2 (8) ◽  
pp. 120093 ◽  
Author(s):  
Markus Ralser ◽  
Heiner Kuhl ◽  
Meryem Ralser ◽  
Martin Werber ◽  
Hans Lehrach ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Relationships ◽  
Model Organism ◽  
454 Sequencing ◽  
Variant Calling ◽  
Tiling Arrays ◽  
Genomic Studies ◽  
Cross Platform ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Saccharomyces cerevisiae strain W303 is a widely used model organism. However, little is known about its genetic origins, as it was created in the 1970s from crossing yeast strains of uncertain genealogy. To obtain insights into its ancestry and physiology, we sequenced the genome of its variant W303-K6001, a yeast model of ageing research. The combination of two next-generation sequencing (NGS) technologies (Illumina and Roche/454 sequencing) yielded an 11.8 Mb genome assembly at an N50 contig length of 262 kb. Although sequencing was substantially more precise and sensitive than whole-genome tiling arrays, both NGS platforms produced a number of false positives. At a 378× average coverage, only 74 per cent of called differences to the S288c reference genome were confirmed by both techniques. The consensus W303-K6001 genome differs in 8133 positions from S288c, predicting altered amino acid sequence in 799 proteins, including factors of ageing and stress resistance. The W303-K6001 (85.4%) genome is virtually identical (less than equal to 0.5 variations per kb) to S288c, and thus originates in the same ancestor. Non-S288c regions distribute unequally over the genome, with chromosome XVI the most (99.6%) and chromosome XI the least (54.5%) S288c-like. Several of these clusters are shared with Σ 1278B, another widely used S288c-related model, indicating that these strains share a second ancestor. Thus, the W303-K6001 genome pictures details of complex genetic relationships between the model strains that date back to the early days of experimental yeast genetics. Moreover, this study underlines the necessity of combining multiple NGS and genome-assembling techniques for achieving accurate variant calling in genomic studies.

scholarly journals Alectinib-Induced Pleural and Pericardial Effusions in ALK-Positive NSCLC

2021 ◽  
pp. 1323-1327
Author(s):  
Maiken Parm Ulhoi ◽  
Boe Sandahl Sorensen ◽  
Peter Meldgaard
Keyword(s):  
Progressive Disease ◽  
Heart Function ◽  
Circulating Tumor Dna ◽  
Driver Mutations ◽  
First Line ◽  
Alk Positive ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna ◽  
Generation Sequencing ◽  
Mild Toxicity

Alectinib is the first-line targeted treatment for advanced ALK-positive non-small-cell lung cancer. Although it has a relatively mild toxicity profile, adverse events (AEs) do occur. We present a case of alectinib-induced bilateral pleural effusions and pericardial effusion that has not previously been reported. The patient developed severe dyspnea 3 months after starting alectinib. He underwent thorough clinical examination including evaluations of heart function. The heart function was normal. There was no sign of pneumonitis or progressive disease on the CT scans. Cytology samples of the pleural fluid from multiple thoracocenteses were examined and showed no malignant cells. Next-generation sequencing (NGS) analysis of circulating tumor DNA from sequential blood samples was also carried out. NGS identified no known driver mutations associated with the effusions. Hence, the effusions were suspected to be an alectinib-induced AE. Alectinib was withdrawn, and the patient commenced brigatinib. The effusions subsequently regressed.

scholarly journals Differential Mutation Detection Capability Through Capture-Based Targeted Sequencing in Plasma Samples in Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Jian Gao ◽  
Lei Xi ◽  
Rentao Yu ◽  
Huailong Xu ◽  
Min Wu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Circulating Tumor Dna ◽  
Tumor Burden ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Tumor Protein P53 ◽  
Next Generation Sequencing Ngs ◽  
Coding Variants ◽  
G Protein Coupled ◽  
Generation Sequencing

Circulating tumor DNA (ctDNA) is a promising biomarker for accurate monitoring and less invasive assessment of tumor burden and treatment response. Here, targeted next-generation sequencing (NGS) with a designed gene panel of 176 cancer-relevant genes was used to assess mutations in 90 ctDNA samples from 90 patients with multiple types of liver disease and 10 healthy donor samples for control. Using our ctDNA detection panel, we identified mutations in 98.89% (89/90) of patient plasma biopsy samples, and 19 coding variants located in 10 cancer-related genes [ACVR2A, PCLO, TBCK, adhesion G protein-coupled receptor (ADGRV1), COL1A1, GABBR1, MUC16, MAGEC1, FASLG, and JAK1] were identified in 96.7% of patients (87/90). The 10 top mutated genes were tumor protein p53 (TP53), ACVR2A, ADGRV1, MUC16, TBCK, PCLO, COL11A1, titin (TTN), DNAH9, and GABBR1. TTN and TP53 and TTN and DNAH9 mutations tended to occur together in hepatocellular carcinoma samples. Most importantly, we found that most of those variants were insertions (frameshift insertions) and deletions (frameshift deletions and in-frame deletions), such as insertion variants in ACVR2A, PCLO, and TBCK; such mutations were detected in almost 95% of patients. Our study demonstrated that the targeted NGS-based ctDNA mutation profiling was a useful tool for hepatocellular carcinoma (HCC) monitoring and could potentially be used to guide treatment decisions in HCC.

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