Use of two complementary new molecular techniques, next-generation sequencing and droplet digital PCR, for diagnosis of an F8 gene deletion and subsequent carrier analysis in a family with haemophilia A: A Case Report

Haemophilia ◽  
2018 ◽  
Vol 24 (6) ◽  
pp. e425-e427 ◽  
Author(s):  
Priyanka Gangodkar ◽  
Shatakshi Ranade ◽  
Siddharth Anand ◽  
Ashwini Bapat ◽  
Kavita Khatod ◽  
...  
Keyword(s):  
Case Report ◽  
Next Generation Sequencing ◽  
Gene Deletion ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Molecular Techniques ◽  
Haemophilia A ◽  
Next Generation ◽  
Carrier Analysis ◽  
Generation Sequencing

scholarly journals A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Bangchuan Hu ◽  
Yue Tao ◽  
Ziqiang Shao ◽  
Yang Zheng ◽  
Run Zhang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Blood Culture ◽  
Critically Ill ◽  
Critically Ill Patients ◽  
Pathogen Detection ◽  
Bloodstream Infections ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Next Generation ◽  
Generation Sequencing

Metagenomic next-generation sequencing (mNGS) and droplet digital PCR (ddPCR) have recently demonstrated a great potential for pathogen detection. However, few studies have been undertaken to compare these two nucleic acid detection methods for identifying pathogens in patients with bloodstream infections (BSIs). This prospective study was thus conducted to compare these two methods for diagnostic applications in a clinical setting for critically ill patients with suspected BSIs. Upon suspicion of BSIs, whole blood samples were simultaneously drawn for ddPCR covering 20 common isolated pathogens and four antimicrobial resistance (AMR) genes, mNGS, and blood culture. Then, a head-to-head comparison was performed between ddPCR and mNGS. A total of 60 episodes of suspected BSIs were investigated in 45 critically ill patients, and ddPCR was positive in 50 (83.3%), mNGS in 41 (68.3%, not including viruses), and blood culture in 10 (16.7%) episodes. Of the 10 positive blood cultures, nine were concordantly identified by both mNGS and ddPCR methods. The head-to-head comparison showed that ddPCR was more rapid (~4 h vs. ~2 days) and sensitive (88 vs. 53 detectable pathogens) than mNGS within the detection range of ddPCR, while mNGS detected a broader range of pathogens (126 vs. 88 detectable pathogens, including viruses) than ddPCR. In addition, a total of 17 AMR genes, including 14 blaKPC and 3 mecA genes, were exclusively identified by ddPCR. Based on their respective limitations and strengths, the ddPCR method is more useful for rapid detection of common isolated pathogens as well as AMR genes in critically ill patients with suspected BSI, whereas mNGS testing is more appropriate for the diagnosis of BSI where classic microbiological or molecular diagnostic approaches fail to identify causative pathogens.

scholarly journals Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

2018 ◽  
Vol 11 (5) ◽  
pp. 1220-1224 ◽  
Author(s):  
Christina Demuth ◽  
Karen-Lise Garm Spindler ◽  
Julia S. Johansen ◽  
Niels Pallisgaard ◽  
Dorte Nielsen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Droplet Digital Pcr ◽  
Next Generation ◽  
Kras Mutations ◽  
Tumor Dna ◽  
Generation Sequencing

scholarly journals Allele-Specific Droplet Digital PCR Combined with a Next-Generation Sequencing-Based Algorithm for Diagnostic Copy Number Analysis in Genes with High Homology: Proof of Concept Using Stereocilin

2018 ◽  
Vol 64 (4) ◽  
pp. 705-714 ◽  
Author(s):  
Sami S Amr ◽  
Elissa Murphy ◽  
Elizabeth Duffy ◽  
Rojeen Niazi ◽  
Jorune Balciuniene ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
High Homology ◽  
Next Generation ◽  
Copy Number Analysis ◽  
Confirmatory Testing ◽  
Allele Specific ◽  
Generation Sequencing

Abstract BACKGROUND Copy number variants (CNVs) can substantially contribute to the pathogenic variant spectrum in several disease genes. The detection of this type of variant is complicated in genes with high homology to other genomic sequences, yet such genomics regions are more likely to lead to CNVs, making it critical to address detection in these settings. METHODS We developed a copy number analysis approach for high homology genes/regions that consisted of next-generation sequencing (NGS)-based dosage analysis accompanied by allele-specific droplet digital PCR (ddPCR) confirmatory testing. We applied this approach to copy number analysis in STRC, a gene with 98.9% homology to a nonfunctional pseudogene, pSTRC, and characterized its accuracy in detecting different copy number states by use of known samples. RESULTS Using a cohort of 517 patients with hearing loss, we prospectively demonstrated the clinical utility of the approach, which contributed 30 of the 122 total positives (6%) to the diagnostic yield, increasing the overall yield from 17.6% to 23.6%. Positive STRC genotypes included homozygous (n = 15) or compound heterozygous (n = 8) deletions, or heterozygous deletions in trans with pathogenic sequence variants (n = 7). Finally, this approach limited ddPCR testing to cases with NGS copy number findings, thus markedly reducing the number of costly and laborious, albeit specific, ddPCR tests. CONCLUSIONS NGS-based CNV detection followed by allele-specific ddPCR confirmatory testing is a reliable and affordable approach for copy number analysis in medically relevant genes with homology issues.

scholarly journals Case Report: Next generation sequencing identifies a NAB2-STAT6 fusion in Glioblastoma

2016 ◽  
Vol 11 (1) ◽  
Author(s):  
Phedias Diamandis ◽  
Ruben Ferrer-Luna ◽  
Raymond Y. Huang ◽  
Rebecca D. Folkerth ◽  
Azra H. Ligon ◽  
...  
Keyword(s):  
Case Report ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Generation Sequencing
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