OC.04.5 EUS-FNA FOR KRAS MUTATIONAL ANALYSIS IN PANCREATIC LESIONS, PRELIMINARY RESULTS USING THREE DIFFERENT TECHNIQUES: SANGER SEQUENCING, NEXT GENERATION SEQUENCING AND REAL-TIME PCR

2012 ◽  
Vol 44 ◽  
pp. S65 ◽  
Author(s):  
M. Visani ◽  
D. De Biase ◽  
A.M. Polifemo ◽  
C. Fabbri ◽  
P. Baccarini ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Sanger Sequencing ◽  
Mutational Analysis ◽  
Next Generation ◽  
Preliminary Results ◽  
Generation Sequencing

scholarly journals Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuki Nagakubo ◽  
Yosuke Hirotsu ◽  
Kenji Amemiya ◽  
Toshio Oyama ◽  
Hitoshi Mochizuki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Sanger Sequencing ◽  
Colorectal Cancers ◽  
Next Generation ◽  
Braf Mutations ◽  
Generation Sequencing

Abstract Background Patients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed. Methods We applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples. Results In 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples. Conclusions Our results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy.

scholarly journals Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 461
Author(s):  
Madjid Morsli ◽  
Quentin Kerharo ◽  
Jeremy Delerce ◽  
Pierre-Hugues Roche ◽  
Lucas Troude ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Haemophilus Influenzae ◽  
Real Time ◽  
Real Time Pcr ◽  
Point Of Care ◽  
Next Generation ◽  
Oxford Nanopore ◽  
Sequence Encoding ◽  
Oxford Nanopore Technologies ◽  
Generation Sequencing

Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.

scholarly journals Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

2020 ◽  
Author(s):  
Dennis Back Holmgaard ◽  
Celine Barnadas ◽  
Seyed Hossein Mirbarati ◽  
Lee O’Brien Andersen ◽  
Henrik Vedel Nielsen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Relevance ◽  
Bacterial Species ◽  
Screening Method ◽  
Infectious Keratitis ◽  
Next Generation ◽  
Specificity And Sensitivity ◽  
Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

scholarly journals Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow

2018 ◽  
Author(s):  
A. Bal ◽  
M. Pichon ◽  
C. Picard ◽  
JS. Casalegno ◽  
M. Valette ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Viral Genome ◽  
Viral Infections ◽  
Rna Viruses ◽  
Next Generation ◽  
Dna And Rna ◽  
Generation Sequencing

AbstractBackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.MethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a notemplate-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).ResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6% to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).ConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

scholarly journals Legionella confirmation in cooling tower water Comparison of culture, real-time PCR and next generation sequencing

2018 ◽  
Vol 39 (2) ◽  
pp. 137-141 ◽  
Author(s):  
Maha Farhat ◽  
Raja Shaheed ◽  
Haidar Al-Ali ◽  
Abdullah Al-Ghamdi ◽  
Ghadeer Al-Hamaqi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Cooling Tower ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals IGH-FR1 and Leader Analysis By Next Generation Sequencing for the Detection of Clonality and Somatic Hypermutation in Lymphoid Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3868-3868
Author(s):  
Pier Paolo Piccaluga ◽  
Anna Gazzola ◽  
Ying Huang ◽  
Jeff Panganiban ◽  
Kasey Hutt ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Next Generation Sequencing ◽  
Diagnostic Accuracy ◽  
Predictive Value ◽  
Sanger Sequencing ◽  
Mutational Analysis ◽  
Somatic Hypermutation ◽  
Illumina Miseq ◽  
Next Generation ◽  
Generation Sequencing

Abstract The standard method to analyze clonality and somatic hypermutation of immunoglobulin genes in B-cell non Hodgkin lymphomas is a multiplex PCR followed by capillary electrophoresis and/or Sanger sequencing. Recently, Next Generation Sequencing (NGS) of immune receptor genes was demonstrated to be highly effective, with increased sensitivity for detecting sequences of interest. Based on that, it was proposed for the diagnosis and monitoring of lymphoid neoplasms. A phase 3 diagnostic accuracy study was designed to compare the value of the first commercially available kit for NGS of IGH/FR1 with the gold standard analysis. One hundred twenty samples were evaluated for IGH rearrangements with both traditional capillary electrophoresis (IdentiClone™ IGH Gene Clonality Assay by Invivoscribe Technologies) and NGS (LymphoTrackTM IGH Assay - MiSeq and LymphoTrack® IGH Somatic Hypermutation Assay - MiSeq by Invivoscribe Technologies on an Illumina MiSeq). The NGS clonality assay compared to conventional analysis had an overall diagnostic accuracy of 96% (63/66 cases), sensitivity (ST), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 95%, 100%, 100%, and 75%, respectively. Regarding mutational analysis, conventional detection and NGS gave comparable results. Notably, NGS was capable of analyzing all cases, including those failed by Sanger sequencing. In discrepant cases NGS results were confirmed by a different set of primers that provided coverage of the IGH Leader sequence. In conclusion NGS was effective for IGH analysis in lymphoid disease (even on FFPE samples). In order to apply them for routine diagnostics, NGS based approaches should be evaluated prospectively and cost-effectiveness assessments should be performed. Disclosures Huang: Invivoscribe: Employment. Panganiban:Invivoscribe: Employment. Hutt:Invivoscribe: Employment. Klass:Invivoscribe: Employment.

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