Targeted Next-Generation Sequencing of Liquid Biopsy Samples from Patients with NSCLC

Liquid biopsy tests have become an integral part of the molecular diagnosis of patients with non-small cell lung cancer (NSCLC). We describe a new test panel that uses very low input (20 ng) of cell-free nucleic acids extracted from human plasma, which is designed to yield results in less than 72 h. In this study, we performed novel amplicon-based targeted next-generation sequencing with a semiconductor-based system, the Ion GeneStudio S5 Prime. The analytic performance of the assay was evaluated using contrived and retrospectively collected clinical specimens. The cumulative percent coefficient of variation for the new test process was very precise at 8.4% for inter-day, 4.0% for inter-operator and 3.4% for inter-instrument. We also observed significant agreement (95.7–100%) with an orthogonal, high-sensitivity droplet digital™ Polymerase Chain Reaction (ddPCR) test. This method offers a valuable supplement to assessing targeted mutations from blood while conserving specimens and maintaining sensitivity, with rapid turn-around times to actionable results.

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A Novel Method for Microsatellite Instability Detection by Liquid Biopsy Based on Next- Generation Sequencing

Current Bioinformatics ◽

10.2174/1574893615666200324133451 ◽

2020 ◽

Vol 15 ◽

Author(s):

Zheng Jiang ◽

Hui Liu ◽

Siwen Zhang ◽

Jia Liu ◽

Weitao Wang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Microsatellite Instability ◽

Liquid Biopsy ◽

Tumor Tissue ◽

High Sensitivity ◽

Next Generation ◽

Tissue Samples ◽

Next Generation Sequencing Technology ◽

Medication Selection ◽

Generation Sequencing

Background: Microsatellite instability (MSI) is a prognostic biomarker used to guide medication selection in multiple cancers, such as colorectal cancer. Traditional PCR with capillary electrophoresis and next-generation sequencing using paired tumor tissue and leukocyte samples are the main approaches for MSI detection due to their high sensitivity and specificity. Currently, patient tissue samples are obtained through puncture or surgery, which causes injury and risk of concurrent disease, further illustrating the need for MSI detection by liquid biopsy. Methods: We propose an analytic method using paired plasma/leukocyte samples and MSI detection using next-generation sequencing technology. Based on the theoretical progress of oncogenesis, we hypothesized that the microsatellite site length in plasma equals the combination of the distribution of tumor tissue and leukocytes. Thus, we defined a window-judgement method to identify whether biomarkers were stable. Results: Compared to traditional PCR as the standard, we evaluated three methods in 20 samples (MSI-H:3/MSS:17): peak shifting method using tissue vs. leukocytes, peak shifting method using plasma vs. leukocytes, and our method using plasma vs. leukocytes. Compared to traditional PCR, we observed a sensitivity of 100%, 0%, and 100%, and a specificity of 100.00%, 94.12%, and 88.24%, respectively. Conclusion: Our method has the advantage of possibly detecting MSI in a liquid biopsy and provides a novel direction for future studies to increase the specificity of the method.

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Comparison of broad-range polymerase chain reaction and metagenomic next-generation sequencing for the diagnosis of prosthetic joint infection

International Journal of Infectious Diseases ◽

10.1016/j.ijid.2020.03.055 ◽

2020 ◽

Vol 95 ◽

pp. 8-12 ◽

Cited By ~ 1

Author(s):

Chao-xin Wang ◽

Zida Huang ◽

Xinyu Fang ◽

Wenbo Li ◽

Bin Yang ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Next Generation Sequencing ◽

Prosthetic Joint Infection ◽

Joint Infection ◽

Next Generation ◽

Chain Reaction ◽

Prosthetic Joint ◽

Polymerase Chain ◽

Generation Sequencing

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Identification of an Emergent Pathogen, Bartonella vinsonii, Using Next-Generation Sequencing in a Patient With Culture-Negative Endocarditis

Journal of the Pediatric Infectious Diseases Society ◽

10.1093/jpids/piaa014 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rachel D Downey ◽

Susan M Russo ◽

Sarmistha B Hauger ◽

Donald K Murphey ◽

Grace Marx ◽

...

Keyword(s):

Next Generation Sequencing ◽

Immunohistochemical Staining ◽

Next Generation ◽

Chain Reaction ◽

Valve Tissue ◽

Free Dna ◽

Emergent Pathogen ◽

Polymerase Chain ◽

Culture Negative ◽

Generation Sequencing

Abstract Diagnosis and treatment of culture negative endocarditis remains a challenge. This report describes a rare cause of endocarditis in humans, Bartonella vinsonii, identified through next generation sequencing of plasma microbial cell-free DNA with confirmation of cardiac valve tissue infection through immunohistochemical staining and polymerase chain reaction.

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The long tail of molecular alterations in non-small cell lung cancer: a single-institution experience of next-generation sequencing in clinical molecular diagnostics

Journal of Clinical Pathology ◽

10.1136/jclinpath-2018-205032 ◽

2018 ◽

Vol 71 (9) ◽

pp. 767-773 ◽

Cited By ~ 6

Author(s):

Caterina Fumagalli ◽

Davide Vacirca ◽

Alessandra Rappa ◽

Antonio Passaro ◽

Juliana Guarize ◽

...

Keyword(s):

Next Generation Sequencing ◽

Molecular Diagnostics ◽

Small Cell ◽

Next Generation ◽

Small Cell Lung ◽

Single Institution ◽

Lung Cancers ◽

Targeted Next Generation Sequencing ◽

Molecular Alterations ◽

Generation Sequencing

BackgroundMolecular profiling of advanced non-small cell lung cancers (NSCLC) is essential to identify patients who may benefit from targeted treatments. In the last years, the number of potentially actionable molecular alterations has rapidly increased. Next-generation sequencing allows for the analysis of multiple genes simultaneously.AimsTo evaluate the feasibility and the throughput of next-generation sequencing in clinical molecular diagnostics of advanced NSCLC.MethodsA single-institution cohort of 535 non-squamous NSCLC was profiled using a next-generation sequencing panel targeting 22 actionable and cancer-related genes.Results441 non-squamous NSCLC (82.4%) harboured at least one gene alteration, including 340 cases (63.6%) with clinically relevant molecular aberrations. Mutations have been detected in all but one gene (FGFR1) of the panel. Recurrent alterations were observed in KRAS, TP53, EGFR, STK11 and MET genes, whereas the remaining genes were mutated in <5% of the cases. Concurrent mutations were detected in 183 tumours (34.2%), mostly impairing KRAS or EGFR in association with TP53 alterations.ConclusionsThe study highlights the feasibility of targeted next-generation sequencing in clinical setting. The majority of NSCLC harboured mutations in clinically relevant genes, thus identifying patients who might benefit from different targeted therapies.

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