scholarly journals Leafhopper Viral Pathogens: Ultrastructure of salivary gland infection of Taastrup-like virus inPsammotettix alienusDahlbom; and a novel Rhabdovirus inHomalodisca vitripennis(Germar) Hemiptera: Cicadellidae

2018 ◽  
Author(s):  
Thorben Lundsgaard ◽  
Wayne B. Hunter ◽  
Scott Adkins
Keyword(s):  
Next Generation Sequencing ◽  
Plant Pathogens ◽  
Insect Pests ◽  
Economic Losses ◽  
Type I ◽  
Next Generation ◽  
Viral Pathogen ◽  
Thin Sections ◽  
Wide Range ◽  
Generation Sequencing

AbstractViruses that are pathogenic to insect pests can be exploited as biological control agents. Viruses that are pathogenic to beneficial insects and other arthropods, as in honey bees, silk worms, and shrimp, cause millions of dollars of losses to those industries. Current advances in next generation sequencing technologies along with molecular and cellular biology have produced a wealth of information about insect viruses and their potential applications. Leafhoppers cause economic losses as vectors of plant pathogens which significantly reduce the worlds’ food crops. Each year more viruses are discovered primarily through the use of next generation sequencing of the leafhopper hosts. The diversity of viruses from leafhoppers demonstrates a wide range of taxonomic members that includes genomes of DNA or RNA from families like: Reoviridae, Iridoviridae, Dicistroviridae, Iflaviridae, and others yet to be classified. Discussed is a recent viral pathogen isolated from the leafhopperPsammotettix alienus, name Taastrup Virus. Taastrup virus (TV) is a novel virus with a RNA genome, a Filovirus-like morphology, being tentatively placed within theMononegavirales. AdultPsammotettix alienusinfected with TV, showed the highest concentration of virions in salivary glands, consisting of a principal gland (type I-VI-cells) and an accessory gland. Examination of thin sections revealed enveloped particles, about 1300 nm long and 62 nm in diameter, located singly or in paracrystalline arrays in canaliculi of type III- and IV-cells. In gland cells with TV particles in canaliculi, granular masses up to 15 μm in diameter were present in the cytoplasm. These masses are believed to be viroplasms, the sites for viral replication. TV particles were observed at the connection between a canaliculus and the salivary duct system. A TV-like virus with strongly similar morphology was discovered in the ornamental plant,Liriope, near Fort Pierce, Florida, USA. When the virus was inoculated to a leafhopper cell culture, HvWH, made from the glassy-winged sharpshooter,Homalodisca vitripennis(Germar), the cells rapidly degraded with 100% mortality in 48 hours. These two instances are the only reported cases of this newly discovered viral pathogen of leafhoppers.

scholarly journals Frequency detection of BRAF V600E mutation in a cohort of pediatric langerhans cell histiocytosis patients by next-generation sequencing

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Shunqiao Feng ◽  
Lin Han ◽  
Mei Yue ◽  
Dixiao Zhong ◽  
Jing Cao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Langerhans Cell Histiocytosis ◽  
Mutation Screening ◽  
Langerhans Cell ◽  
Braf V600e ◽  
P Value ◽  
Type I ◽  
Next Generation ◽  
Mutation Status ◽  
Generation Sequencing

Abstract Background Langerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients. Results We retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence. Conclusions This is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

scholarly journals Next-Generation Sequencing Identification and Characterization of MicroRNAs in Dwarfed Citrus Trees Infected With Citrus Dwarfing Viroid in High-Density Plantings

2021 ◽  
Vol 12 ◽  
Author(s):  
Tyler Dang ◽  
Irene Lavagi-Craddock ◽  
Sohrab Bodaghi ◽  
Georgios Vidalakis
Keyword(s):  
Next Generation Sequencing ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
High Density ◽  
Poncirus Trifoliata ◽  
Next Generation ◽  
Developmental Processes ◽  
Wide Range ◽  
Generation Sequencing ◽  
Citrus Trees

Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.

scholarly journals No Metagenomic Evidence of Causative Viral Pathogens in Postencephalitic Parkinsonism Following Encephalitis Lethargica

2021 ◽  
Vol 9 (8) ◽  
pp. 1716
Author(s):  
Dániel Cadar ◽  
Kurt A. Jellinger ◽  
Peter Riederer ◽  
Sabrina Strobel ◽  
Camelia-Maria Monoranu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Neuronal Loss ◽  
Unknown Etiology ◽  
Next Generation ◽  
Encephalitis Lethargica ◽  
Tissue Samples ◽  
Viral Pathogen ◽  
Postencephalitic Parkinsonism ◽  
Generation Sequencing ◽  
Post Infectious

Postencephalitic parkinsonism (PEP) is a disease of unknown etiology and pathophysiology following encephalitis lethargica (EL), an acute-onset polioencephalitis of cryptic cause in the 1920s. PEP is a tauopathy with multisystem neuronal loss and gliosis, clinically characterized by bradykinesia, rigidity, rest tremor, and oculogyric crises. Though a viral cause of EL is likely, past polymerase chain reaction-based investigations in the etiology of both PEP and EL were negative. PEP might be caused directly by an unknown viral pathogen or the consequence of a post-infectious immunopathology. The development of metagenomic next-generation sequencing in conjunction with bioinformatic techniques has generated a broad-range tool for the detection of unknown pathogens in the recent past. Retrospective identification and characterization of pathogens responsible for past infectious diseases can be successfully performed with formalin-fixed paraffin-embedded (FFPE) tissue samples. In this study, we analyzed 24 FFPE brain samples from six patients with PEP by unbiased metagenomic next-generation sequencing. Our results show that no evidence for the presence of a specific or putative (novel) viral pathogen was found, suggesting a likely post-infectious immune-mediated etiology of PEP.

scholarly journals 355. A Novel Likelihood-Based Model to Estimate SARS-CoV-2 Viral Titer from Next-Generation Sequencing Data

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S281-S282
Author(s):  
Heather L Wells ◽  
Joseph Barrows ◽  
Mara Couto-Rodriguez ◽  
Xavier O Jirau Serrano ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Board Member ◽  
Clinical Specimen ◽  
Next Generation Sequencing Data ◽  
Viral Titer ◽  
Next Generation ◽  
Genome Coverage ◽  
Ct Value ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background The quantitative level of pathogens present in a host is a major driver of infectious disease (ID) state and outcome. However, the majority of ID diagnostics are qualitative. Next-generation sequencing (NGS) is an emerging ID diagnostics and research tool to provide insights, including tracking transmission, evolution, and identifying novel strains. Methods We built a novel likelihood-based computational method to leverage pathogen-specific genome-wide NGS data to detect SARS-CoV-2, profile genetic variants, and furthermore quantify levels of these pathogens. We used de-identified clinical specimens tested for SARS-CoV-2 using RT-PCR, SARS-CoV-2 NGS Assay (hybrid capture, Twist Bioscience), or ARTIC (amplicon-based) platform, and COVID-DX software. A training (n=87) and validation (n=22) set was selected to establish the strength of our quantification model. We fit non-uniform probabilistic error profiles to a deterministic sigmoidal equation that more realistically represents observed data and used likelihood maximized over several different read depths to improve accuracy over a wide range of values of viral load. Given the proportion of the genome covered at varying depths for a single sample as input data, our model estimated the Ct of that sample as the value that produces the maximum likelihood of generating the observed genome coverage data. Results The model fit on 87 SARS-CoV-2 NGS Assay training samples produced a good fit to the 22 validation samples, with a coefficient of correlation (r2) of ~0.8. The accuracy of the model was high (mean absolute % error of ~10%, meaning our model is able to predict the Ct value of each sample within a margin of ±10% on average). Because of the nature of the commonly used ARTIC protocol, we found that all quantitative signals in this data were lost during PCR amplification and the model is not applicable for quantification of samples captured this way. The ability to model quantification is a major advantage of the SARS-CoV-2 NGS assay protocol. The likelihood-based model to estimate SARS-CoV-2 viral titer Left Observed genome coverage (y-axis) plotted against Ct value (x-axis). The best-fitting logistic curve is demonstrated with a red line with shaded areas above and below representing the fitted error profile. RIGHT: Model-estimated Ct values (y-axis) compared to laboratory Ct values (x-axis) with grey bars representing estimated confidence intervals. The 1:1 diagonal is shown as a dotted line. Conclusion To our knowledge, this is the first model to incorporate sequence data mapped across the genome of a pathogen to quantify the level of that pathogen in a clinical specimen. This has implications in ID diagnostics, research, and metagenomics. Disclosures Heather L. Wells, MPH, Biotia, Inc. (Consultant) Joseph Barrows, MS, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Xavier O. Jirau Serrano, B.S., Biotia (Employee) Marilyne Debieu, PhD, Biotia (Employee) Karen Wessel, PhD, Labor Zotz/Klimas (Employee) Christopher Mason, PhD, Biotia (Board Member, Advisor or Review Panel member, Shareholder) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)

scholarly journals Unveiling Viruses Associated with Gastroenteritis Using a Metagenomics Approach

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1432
Author(s):  
Xavier Fernandez-Cassi ◽  
Sandra Martínez-Puchol ◽  
Marcelle Silva-Sales ◽  
Thais Cornejo ◽  
Rosa Bartolome ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Gastroenteritis ◽  
Clinical Analysis ◽  
Viral Metagenomics ◽  
Next Generation ◽  
Sequencing Data ◽  
Viral Pathogens ◽  
Viral Pathogen ◽  
Infectious Gastroenteritis ◽  
Generation Sequencing

Acute infectious gastroenteritis is an important illness worldwide, especially on children, with viruses accounting for approximately 70% of the acute cases. A high number of these cases have an unknown etiological agent and the rise of next generation sequencing technologies has opened new opportunities for viral pathogen detection and discovery. Viral metagenomics in routine clinical settings has the potential to identify unexpected or novel variants of viral pathogens that cause gastroenteritis. In this study, 124 samples from acute gastroenteritis patients from 2012–2014 previously tested negative for common gastroenteritis pathogens were pooled by age and analyzed by next generation sequencing (NGS) to elucidate unidentified viral infections. The most abundant sequences detected potentially associated to acute gastroenteritis were from Astroviridae and Caliciviridae families, with the detection of norovirus GIV and sapoviruses. Lower number of contigs associated to rotaviruses were detected. As expected, other viruses that may be associated to gastroenteritis but also produce persistent infections in the gut were identified including several Picornaviridae members (EV, parechoviruses, cardioviruses) and adenoviruses. According to the sequencing data, astroviruses, sapoviruses and NoV GIV should be added to the list of viral pathogens screened in routine clinical analysis.

Multicenter validation study for the certification of a CFTR gene scanning method using next generation sequencing technology

2018 ◽  
Vol 56 (7) ◽  
pp. 1046-1053 ◽  
Author(s):  
Anne Bergougnoux ◽  
Valeria D’Argenio ◽  
Stefanie Sollfrank ◽  
Fanny Verneau ◽  
Antonella Telese ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Genetic Analysis ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Sequencing Data ◽  
Scanning Method ◽  
Next Generation Sequencing Technology ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background: Many European laboratories offer molecular genetic analysis of the CFTR gene using a wide range of methods to identify mutations causative of cystic fibrosis (CF) and CFTR-related disorders (CFTR-RDs). Next-generation sequencing (NGS) strategies are widely used in diagnostic practice, and CE marking is now required for most in vitro diagnostic (IVD) tests in Europe. The aim of this multicenter study, which involved three European laboratories specialized in CF molecular analysis, was to evaluate the performance of Multiplicom’s CFTR MASTR Dx kit to obtain CE-IVD certification. Methods: A total of 164 samples, previously analyzed with well-established “reference” methods for the molecular diagnosis of the CFTR gene, were selected and re-sequenced using the Illumina MiSeq benchtop NGS platform. Sequencing data were analyzed using two different bioinformatic pipelines. Annotated variants were then compared to the previously obtained reference data. Results and conclusions: The analytical sensitivity, specificity and accuracy rates of the Multiplicom CFTR MASTR assay exceeded 99%. Because different types of CFTR mutations can be detected in a single workflow, the CFTR MASTR assay simplifies the overall process and is consequently well suited for routine diagnostics.

scholarly journals Genetic analyses of the NF1 gene in Turkish neurofibromatosis type I patients and definition of three novel variants

2017 ◽  
Vol 20 (1) ◽  
pp. 13-20 ◽  
Author(s):  
SD Ulusal ◽  
H Gürkan ◽  
E Atlı ◽  
SA Özal ◽  
M Çiftdemir ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Diagnosis ◽  
Neurofibromatosis Type ◽  
Type I ◽  
Neurofibromatosis Type I ◽  
Next Generation ◽  
Loss Of Function ◽  
Pathogenic Variants ◽  
Nf1 Gene ◽  
Generation Sequencing

Abstract Neurofibromatosis Type I (NF1) is a multi systemic autosomal dominant neurocutaneous disorder predisposing patients to have benign and/or malignant lesions predominantly of the skin, nervous system and bone. Loss of function mutations or deletions of the NF1 gene is responsible for NF1 disease. Involvement of various pathogenic variants, the size of the gene and presence of pseudogenes makes it difficult to analyze. We aimed to report the results of 2 years of multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) for genetic diagnosis of NF1 applied at our genetic diagnosis center. The MLPA, semiconductor sequencing and Sanger sequencing were performed in genomic DNA samples from 24 unrelated patients and their affected family members referred to our center suspected of having NF1. In total, three novel and 12 known pathogenic variants and a whole gene deletion were determined. We suggest that next generation sequencing is a practical tool for genetic analysis of NF1. Deletion/duplication analysis with MLPA may also be helpful for patients clinically diagnosed to carry NF1 but do not have a detectable mutation in NGS.

scholarly journals cDNA display coupled with next-generation sequencing for rapid activity-based screening: Comprehensive analysis of transglutaminase substrate preference

2021 ◽  
Author(s):  
Jasmina Damnjanović ◽  
Nana Odake ◽  
Jicheng Fan ◽  
Beixi Jia ◽  
Takaaki Kojima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Substrate Preference ◽  
Stable Complex ◽  
Next Generation ◽  
Library Size ◽  
Binary Model ◽  
Wide Range ◽  
Generation Sequencing ◽  
Selection Of

AbstractcDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, where a stable complex is formed suitable for a wide range of selection conditions. A great advantage of cDNA display is the ability to handle enormous library size (1012) in a microtube scale, in a matter of days. To harness its benefits, we aimed at developing a platform which combines the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing (NGS) for the selection of preferred substrate peptides of transglutaminase 2 (TG2), a protein cross-linking enzyme. After the optimization of the platform by the repeated screening of binary model libraries consisting of the substrate and non-substrate peptides at different ratios, screening and selection of combinatorial peptide library randomized at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by NGS and bioinformatics, revealing the comprehensive amino acid preference of the TG2 at targeted positions of the peptide backbone. This is the first report on the cDNA display/NGS screening system to yield comprehensive data on TG substrate preference. Although some issues remain to be solved, this platform can be applied to the selection of other TGs and easily adjusted for the selection of other peptide substrates and even larger biomolecules.

scholarly journals Assessment of Antibody Library Diversity through Next Generation Sequencing and Technical Error Compensation.

2016 ◽  
Author(s):  
Marco Fantini ◽  
Luca Pandolfini ◽  
Simonetta Lisi ◽  
Michele Chirichella ◽  
Ivan Arisi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Small Sample ◽  
Sequencing Error ◽  
Limited Information ◽  
Next Generation ◽  
Antibody Library ◽  
Illumina Platform ◽  
Wide Range ◽  
Antibody Libraries ◽  
Generation Sequencing

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its diversity, i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library diversity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Diversity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring diversity from such a small sample is, however, very rudimental and gives limited information about the real complexity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library diversity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the diversity, taking in consideration the sequencing error.

scholarly journals Next-generation sequencing in neuropathological diagnosis of infections of the nervous system

2016 ◽  
Author(s):  
Steven L. Salzberg ◽  
Florian Breitwieser ◽  
Anupama Kumar ◽  
Haiping Hao ◽  
Peter Burger ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Next Generation Sequencing ◽  
Large Scale ◽  
Infectious Agent ◽  
Next Generation ◽  
Dna And Rna ◽  
Wide Range ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Objective: To determine the feasibility of next-generation sequencing (NGS) microbiome approaches in the diagnosis of infectious disorders in brain or spinal cord biopsies in patients with suspected central nervous system (CNS) infections. Methods: In a prospective-pilot study, we applied NGS in combination with a new computational analysis pipeline to detect the presence of pathogenic microbes in brain or spinal cord biopsies from ten patients with neurological problems indicating possible infection but for whom conventional clinical and microbiology studies yielded negative or inconclusive results. Results: Direct DNA and RNA sequencing of brain tissue biopsies generated 8.3 million to 29.1 million sequence reads per sample, which successfully identified with high confidence the infectious agent in three patients, identified possible pathogens in two more, and helped to understand neuropathological processes in three others, demonstrating the power of large-scale unbiased sequencing as a novel diagnostic tool. Validation techniques confirmed the pathogens identified by NGS in each of the three positive cases. Clinical outcomes were consistent with the findings yielded by NGS on the presence or absence of an infectious pathogenic process in eight of ten cases, and were non-contributory in the remaining two. Conclusions: NGS-guided metagenomic studies of brain, spinal cord or meningeal biopsies offer the possibility for dramatic improvements in our ability to detect (or rule out) a wide range of CNS pathogens, with potential benefits in speed, sensitivity, and cost. NGS-based microbiome approaches present a major new opportunity to investigate the potential role of infectious pathogens in the pathogenesis of neuroinflammatory disorders.

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