scholarly journals PO-086 An efficient ion torrent™ next generation sequencing workflow for liquid biopsy research to assess cell-free total nucleic acid

2018 ◽  
Author(s):  
Y Li ◽  
K Lea ◽  
P Kshatriya ◽  
R Cao ◽  
J Gu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Liquid Biopsy ◽  
Ion Torrent ◽  
Total Nucleic Acid ◽  
Next Generation ◽  
Generation Sequencing

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.

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.

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

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.

scholarly journals Evaluation of Ion Torrent next-generation sequencing for thalassemia diagnosis

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052096777
Author(s):  
Peisong Chen ◽  
Xuegao Yu ◽  
Hao Huang ◽  
Wentao Zeng ◽  
Xiaohong He ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Ion Torrent ◽  
Next Generation ◽  
Large Deletions ◽  
Different Types ◽  
Variant Detection ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Accuracy And Repeatability ◽  
Generation Sequencing

Introduction To evaluate a next-generation sequencing (NGS) workflow in the screening and diagnosis of thalassemia. Methods In this prospective study, blood samples were obtained from people undergoing genetic screening for thalassemia at our centre in Guangzhou, China. Genomic DNA was polymerase chain reaction (PCR)-amplified and sequenced using the Ion Torrent system and results compared with traditional genetic analyses. Results Of the 359 subjects, 148 (41%) were confirmed to have thalassemia. Variant detection identified 35 different types including the most common. Identification of the mutational sites by NGS were consistent with those identified by Sanger sequencing and Gap-PCR. The sensitivity and specificities of the Ion Torrent NGS were 100%. In a separate test of 16 samples, results were consistent when repeated ten times. Conclusion Our NGS workflow based on the Ion Torrent sequencer was successful in the detection of large deletions and non-deletional defects in thalassemia with high accuracy and repeatability.

scholarly journals Metagenomic Next-Generation Sequencing of Bloodstream Microbial Cell-Free Nucleic Acid in Children With Suspected Sepsis in Pediatric Intensive Care Unit

2021 ◽  
Vol 11 ◽  
Author(s):  
Gangfeng Yan ◽  
Jing Liu ◽  
Weiming Chen ◽  
Yang Chen ◽  
Ye Cheng ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Pediatric Intensive Care Unit ◽  
Pediatric Intensive Care ◽  
Microbial Cell ◽  
Next Generation ◽  
Suspected Sepsis ◽  
Generation Sequencing

Bloodstream infection is a life-threatening complication in critically ill patients. Multi-drug resistant bacteria or fungi may increase the risk of invasive infections in hospitalized children and are difficult to treat in intensive care units. The purpose of this study was to use metagenomic next-generation sequencing (mNGS) to understand the bloodstream microbiomes of children with suspected sepsis in a pediatric intensive care unit (PICU). mNGS were performed on microbial cell-free nucleic acid from 34 children admitted to PICU, and potentially pathogenic microbes were identified. The associations of serological inflammation indicators, lymphocyte subpopulations, and other clinical phenotypes were also examined. mNGS of blood samples from children in PICU revealed potential eukaryotic microbial pathogens. The abundance of Pneumocystis jirovecii was positively correlated with a decrease in total white blood cell count and immunodeficiency. Hospital-acquired pneumonia patients showed a significant increase in blood bacterial species richness compared with community-acquired pneumonia children. The abundance of bloodstream bacteria was positively correlated with serum procalcitonin level. Microbial genome sequences from potential pathogens were detected in the bloodstream of children with suspected sepsis in PICU, suggesting the presence of bloodstream infections in these children.

scholarly journals Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6661 ◽  
Author(s):  
Arianna Nicolussi ◽  
Francesca Belardinilli ◽  
Yasaman Mahdavian ◽  
Valeria Colicchia ◽  
Sonia D’Inzeo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Germline Mutations ◽  
Ion Torrent ◽  
Next Generation ◽  
Training Set ◽  
Brca1 And Brca2 ◽  
Ion Torrent Pgm ◽  
Validation Set ◽  
Generation Sequencing

Background Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. Methods We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). Results By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. Discussion Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.

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