Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco

Abstract Background: Reports have been surfacing surrounding CNS-associated symptoms in individuals affected by coronavirus disease 19 (COVID-19). Tourette syndrome is a neuropsychiatric disorder with usual onset in childhood. Gut microbiota can affect central physiology and function via the microbiota-gut-brain axis. The authors of this case report describe Tourette’s-like symptoms in a patient resulting from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection disrupting gut microbiota. Case Presentation: This case involves a 16-year-old female that developed acute onset Tourette’s-like symptoms along with neuropsychiatric symptoms after exposure to and infection from SARS-CoV-2. The patient had negative nasopharyngeal (NP) real-time reverse transcription-PCR (RT-PCR) tests for SARS-CoV-2 on five occasions from August of 2020 through June of 2021. The patient’s symptoms continued to worsen over the next six months until next-generation sequencing (NGS) revealed SARS-CoV-2 in her stool. Her treatment was adjusted as NGS revealed SARS-CoV-2 in her stool. Repair of the gastrointestinal microbiota, treatment with nutraceutical and pharmaceutical agents, as well as alterations in her surroundings resulted in dramatic improvement in the microbiome and a significant reduction of symptoms.Discussion: The use of (RT-PCR) testing to determine the presence or absence of SARS-CoV-2 may be inadequate and inaccurate for individuals that have been exposed to the virus. In addition, the impact of SARS-CoV-2 infection of the GI tract may cause significant havoc in the gut microbiota. Additional testing, eradication of infectious agents, as well as restoration of the gut microbiome are needed to effectively manage and treat this condition. The patient’s symptoms worsened over the next six months until next-generation sequencing (NGS) revealed SARS-CoV-2 in her stool and her treatment was adjusted. Treatment with nutraceuticals and alterations in her surroundings was followed by a more normal microbiome and a dramatic reduction in symptoms.

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Next-Generation Sequencing-Based Detection of Common Bean Viruses in Wild Plants from Tanzania and Their Mechanical Transmission to Common Bean Plants

Plant Disease ◽

10.1094/pdis-07-20-1420-re ◽

2021 ◽

Author(s):

Beatrice Mwaipopo ◽

Minna-Liisa Rajamäki ◽

Neema Ngowi ◽

Susan N’chimbi Msolla ◽

P Njau ◽

...

Keyword(s):

Next Generation Sequencing ◽

Common Bean ◽

Mosaic Virus ◽

Viral Disease ◽

Wild Plants ◽

Next Generation ◽

Rt Pcr ◽

Bean Plants ◽

Generation Sequencing ◽

Common Bean Plants

Viral diseases are a major threat for common bean production. In recent surveys, >15 different viruses belonging to 11 genera were shown to infect common bean (Phaseolus vulgaris L.) in Tanzania. Management of viruses requires an understanding of how they survive from one season to the next. In this study, we explored the possibility that alternative host plants have a central role in the survival of common bean viruses. We used next-generation sequencing (NGS) techniques to sequence virus-derived small interfering RNAs, together with conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect viruses in wild plants. Leaf samples for RNA extraction and NGS were collected from 1,430 wild plants around and within common bean fields in four agricultural zones in Tanzania. At least partial genome sequences of viruses potentially belonging to 25 genera were detected. The greatest virus diversity was detected in the eastern and northern zones, whereas wild plants in the Lake zone and especially in the southern highlands zone showed only a few viruses. RT-PCR analysis of all the collected plant samples confirmed the presence of yam bean mosaic virus and peanut mottle virus in wild legume plants. Of all viruses detected, only two viruses, cucumber mosaic virus and a novel bromovirus related to cowpea chlorotic mottle virus and brome mosaic virus, were mechanically transmitted from wild plants to common bean plants. The data generated in this study are crucial for development of viral disease management strategies and predicting crop viral disease outbreaks in different agricultural regions in Tanzania and beyond.

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Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

10.1101/2020.03.12.988246 ◽

2020 ◽

Cited By ~ 12

Author(s):

Chenyu Li ◽

David N. Debruyne ◽

Julia Spencer ◽

Vidushi Kapoor ◽

Lily Y. Liu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Multiplex Pcr ◽

High Sensitivity ◽

Detection Methods ◽

Next Generation ◽

Rt Pcr ◽

Copy Numbers ◽

Virus Rna ◽

Highly Sensitive ◽

Generation Sequencing

AbstractMany detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

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Chronic SARS-CoV-2 infection and viral evolution in a hypogammaglobulinaemic individual.

10.1101/2021.05.31.21257591 ◽

2021 ◽

Author(s):

Maia Kavanagh Williamson ◽

Fergus Hamilton ◽

Stephanie Hutchings ◽

Hannah M Pymont ◽

Mark Hackett ◽

...

Keyword(s):

Next Generation Sequencing ◽

Amino Acid ◽

Selective Pressure ◽

Viral Evolution ◽

Viral Escape ◽

Spike Protein ◽

Next Generation ◽

Rt Pcr ◽

The Face ◽

Generation Sequencing

There is widespread interest in the capacity for SARS-CoV-2 evolution in the face of selective pressures from host immunity, either naturally acquired post-exposure or from vaccine acquired immunity. Allied to this is the potential for long perm persistent infections within immune compromised individuals to allow a broader range of viral evolution in the face of sub-optimal immune driven selective pressure. Here we report on an immunocompromised individual who is hypogammaglobulinaemic and was persistently infected with SARS-CoV-2 for over 290 days, the longest persistent infection recorded in the literature to date. During this time, nine samples of viral nucleic acid were obtained and analysed by next-generation sequencing. Initially only a single mutation (L179I) was detected in the spike protein relative to the prototypic SARS-CoV-2 Wuhan-Hu-1 isolate, with no further changes identified at day 58. However, by day 155 the spike protein had acquired a further four amino acid changes, namely S255F, S477N, H655Y and D1620A and a two amino acid deletion (ΔH69/ΔV70). Infectious virus was cultured from a nasopharyngeal sample taken on day 155 and next-generation sequencing confirmed that the mutations in the virus mirrored those identified by sequencing of the corresponding swab sample. The isolated virus was susceptible to remdesivir in vitro, however a 17-day course of remdesivir started on day 213 had no effect on the viral RT-PCR cycle threshold (Ct) value. On day 265 the patient was treated with the combination of casirivimab and imdevimab. The patient experienced progressive resolution of all symptoms over the next 8 weeks and by day 311 the virus was no longer detectable by RT-PCR. The ΔH69/ΔV70 deletion in the N-terminus of the spike protein which arose in our patient is also present in the B.1.1.7 variant of concern and has been associated with viral escape mutagenesis after treatment of another immunocompromised patient with convalescent plasma. Our data confirms the significance of this deletion in immunocompromised patients but illustrates it can arise independently of passive antibody transfer, suggesting the deletion may be an enabling mutation that compensates for distant changes in the spike protein that arise under selective pressure.

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Validation of Saliva and Self-Administered Nasal Swabs for COVID-19 Testing

10.1101/2020.08.13.20173807 ◽

2020 ◽

Author(s):

Alvin Kuo Jing Teo ◽

Yukti Choudhury ◽

Iain Beehuat Tan ◽

Chae Yin Cher ◽

Shi Hao Chew ◽

...

Keyword(s):

Next Generation Sequencing ◽

Migrant Workers ◽

Phylogenetic Analyses ◽

Kappa Statistic ◽

Sample Type ◽

Next Generation ◽

Rt Pcr ◽

Male Adult ◽

Public Health Decision ◽

Generation Sequencing

Background Active cases of COVID-19 has primarily been diagnosed via RT-PCR of nasopharyngeal (NP) swabs. Saliva and self-administered nasal (SN) swabs can be collected safely without trained staff. We aimed to test the sensitivity of naso-oropharyngeal saliva and SN swabs compared to NP swabs in a large cohort of migrant workers in Singapore. Methods We recruited 200 male adult subjects: 45 with acute respiratory infection, 104 asymptomatic close contacts, and 51 confirmed COVID-19 cases. Each subject underwent NP swab, SN swab and saliva collection for RT-PCR testing at 1 to 3 timepoints. We additionally used a direct-from-sample amplicon-based next-generation sequencing (NGS) workflow to establish phylogeny. Results Of 200 subjects, 91 and 46 completed second and third rounds of testing, respectively. Of 337 sets of tests, there were 150 (44.5%) positive NP swabs, 127 (37.7%) positive SN swabs, and 209 (62.0%) positive saliva. Test concordance between different sample sites was good, with a kappa statistic of 0.616 for NP and SN swabs, and 0.537 for NP and saliva. In confirmed symptomatic COVID-19 subjects, the likelihood of a positive test from any sample fell beyond 14 days of symptom onset. NGS was conducted on 18 SN and saliva samples, with phylogenetic analyses demonstrating lineages for all samples tested were Clade O (GISAID nomenclature) and lineage B.6 (PANGOLIN nomenclature). Conclusion This study supports saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further delineates the role of oropharyngeal secretions in increasing the sensitivity of testing. However, SN swabs were inferior as an alternate sample type. Our study also provides evidence that a straightforward next-generation sequencing workflow can provide direct-from-sample phylogenetic analysis for public health decision-making.

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Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study

BMC Cancer ◽

10.1186/s12885-018-4736-4 ◽

2018 ◽

Vol 18 (1) ◽

Cited By ~ 6

Author(s):

Cecily P. Vaughn ◽

José Luis Costa ◽

Harriet E. Feilotter ◽

Rosella Petraroli ◽

Varun Bagai ◽

...

Keyword(s):

Next Generation Sequencing ◽

Research Study ◽

Simultaneous Detection ◽

Institutional Research ◽

Next Generation ◽

Rt Pcr ◽

Generation Sequencing

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Cost-effectiveness analysis comparing companion diagnostic tests for EGFR, ALK, and ROS1 versus next-generation sequencing (NGS) in advanced adenocarcinoma lung cancer patients

BMC Cancer ◽

10.1186/s12885-020-07240-2 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Luciene Schluckebier ◽

Rosangela Caetano ◽

Osvaldo Ulises Garay ◽

Giuliana T. Montenegro ◽

Marcelo Custodio ◽

...

Keyword(s):

Lung Cancer ◽

Cost Effectiveness ◽

Next Generation Sequencing ◽

Molecular Testing ◽

Next Generation ◽

Rt Pcr ◽

Mutational Status ◽

Life Years ◽

Sequential Tests ◽

Generation Sequencing

Abstract Background The treatment of choice for advanced non–small cell lung cancer is selected according to the presence of specific alterations. Patients should undergo molecular testing for relevant modifications and the mutational status of EGFR and translocation of ALK and ROS1 are commonly tested to offer the best intervention. In addition, the tests costs should also be taken in consideration. Therefore, this work was performed in order to evaluate the cost-effectiveness of a unique exam using NGS (next generation sequencing) versus other routinely used tests which involve RT-PCR and FISH. Methods The target population was NSCLC, adenocarcinoma, and candidates to first-line therapy. Two strategies were undertaken, strategy 1 corresponded to sequential tests with EGFR RT-PCR, then FISH for ALK and ROS1. Strategy 2 differed from 1 in that ALK and ROS1 translocation testing were performed simultaneously by FISH. Strategy 3 considered single test next-generation sequencing, a platform that includes EGFR, ALK and ROS1 genes. A decision tree analysis was used to model genetic testing options. From the test results, a microsimulation model was nested to estimate survival outcomes and costs of therapeutic options. Results The use of NGS added 24% extra true cases as well as extra costs attributed to the molecular testing. The ICER comparing NGS with sequential tests was US$ 3479.11/correct case detected. The NGS improved a slight gain in life years and QALYs. Conclusion Our results indicated that, although precise, the molecular diagnosis by NGS of patients with advanced stage NSCLC adenocarcinoma histology was not cost-effective in terms of quality-adjusted life years from the perspective of the Brazilian supplementary health system.

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