Facile method of curing toxicity in large viral genomes by high-throughput identification and removal of cryptic promoters

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

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Standard finishing categories for high-throughput sequencing of viral genomes

Revue Scientifique et Technique de l OIE ◽

10.20506/rst.35.1.2416 ◽

2016 ◽

Vol 35 (1) ◽

pp. 43-52 ◽

Cited By ~ 4

Author(s):

J.T. LADNER ◽

J.H. KUHN ◽

G. PALACIOS

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Viral Genomes

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Long-read viral metagenomics captures abundant and microdiverse viral populations and their niche-defining genomic islands

PeerJ ◽

10.7717/peerj.6800 ◽

2019 ◽

Vol 7 ◽

pp. e6800 ◽

Cited By ~ 35

Author(s):

Joanna Warwick-Dugdale ◽

Natalie Solonenko ◽

Karen Moore ◽

Lauren Chittick ◽

Ann C. Gregory ◽

...

Keyword(s):

High Throughput ◽

Low Cost ◽

Host Community ◽

Genomic Islands ◽

Viral Metagenomics ◽

Viral Gene ◽

Gene Markers ◽

Short Read ◽

Viral Genomes ◽

Long Read

Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and ‘universal’ gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The ‘VirION’ (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

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Use of High-Throughput Sequencing and Two RNA Input Methods to Identify Viruses Infecting Tomato Crops

Microorganisms ◽

10.3390/microorganisms9051043 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1043

Author(s):

Ayoub Maachi ◽

Covadonga Torre ◽

Raquel N. Sempere ◽

Yolanda Hernando ◽

Miguel A. Aranda ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Tomato Fruit ◽

Virus Detection ◽

Read Depth ◽

Cdna Libraries ◽

Virus Species ◽

Total Rna ◽

Viral Genomes ◽

First Time

We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection; all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species (Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.

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Next-Generation High-Throughput Functional Annotation of Microbial Genomes

mBio ◽

10.1128/mbio.01245-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 8

Author(s):

Ralph S. Baric ◽

Sean Crosson ◽

Blossom Damania ◽

Samuel I. Miller ◽

Eric J. Rubin

Keyword(s):

Functional Genomics ◽

High Throughput ◽

Cell Biology ◽

Microbial Pathogens ◽

Global Changes ◽

Next Generation ◽

Emerging Pathogens ◽

Viral Genomes ◽

Host Infection ◽

Diverse Data

ABSTRACT Host infection by microbial pathogens cues global changes in microbial and host cell biology that facilitate microbial replication and disease. The complete maps of thousands of bacterial and viral genomes have recently been defined; however, the rate at which physiological or biochemical functions have been assigned to genes has greatly lagged. The National Institute of Allergy and Infectious Diseases (NIAID) addressed this gap by creating functional genomics centers dedicated to developing high-throughput approaches to assign gene function. These centers require broad-based and collaborative research programs to generate and integrate diverse data to achieve a comprehensive understanding of microbial pathogenesis. High-throughput functional genomics can lead to new therapeutics and better understanding of the next generation of emerging pathogens by rapidly defining new general mechanisms by which organisms cause disease and replicate in host tissues and by facilitating the rate at which functional data reach the scientific community.

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Leveraging Novel Integrated Single-Cell Analyses to Define HIV-1 Latency Reversal

Viruses ◽

10.3390/v13071197 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1197

Author(s):

Suhui Zhao ◽

Athe Tsibris

Keyword(s):

Data Analysis ◽

Antiretroviral Therapy ◽

Single Cell ◽

Recent Work ◽

High Throughput ◽

Computational Approaches ◽

Integrative Data Analysis ◽

Viral Genomes ◽

Infection Models ◽

Hiv 1

While suppressive antiretroviral therapy can effectively limit HIV-1 replication and evolution, it leaves behind a residual pool of integrated viral genomes that persist in a state of reversible nonproductive infection, referred to as the HIV-1 reservoir. HIV-1 infection models were established to investigate HIV-1 latency and its reversal; recent work began to probe the dynamics of HIV-1 latency reversal at single-cell resolution. Signals that establish HIV-1 latency and govern its reactivation are complex and may not be completely resolved at the cellular and regulatory levels by the aggregated measurements of bulk cellular-sequencing methods. High-throughput single-cell technologies that characterize and quantify changes to the epigenome, transcriptome, and proteome continue to rapidly evolve. Combinations of single-cell techniques, in conjunction with novel computational approaches to analyze these data, were developed and provide an opportunity to improve the resolution of the heterogeneity that may exist in HIV-1 reactivation. In this review, we summarize the published single-cell HIV-1 transcriptomic work and explore how cutting-edge advances in single-cell techniques and integrative data-analysis tools may be leveraged to define the mechanisms that control the reversal of HIV-1 latency.

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Standards for Sequencing Viral Genomes in the Era of High-Throughput Sequencing

mBio ◽

10.1128/mbio.01360-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 59

Author(s):

Jason T. Ladner ◽

Brett Beitzel ◽

Patrick S. G. Chain ◽

Matthew G. Davenport ◽

Eric Donaldson ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Cost Benefit ◽

Genome Sequences ◽

Common Component ◽

Viral Genomes ◽

Genome Finishing ◽

Sequencing Technologies ◽

Trade Offs ◽

Sequencing Platforms

ABSTRACT Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques.

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High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples

10.1101/2021.02.09.430478 ◽

2021 ◽

Author(s):

Adrian A. Pater ◽

Michael S. Bosmeny ◽

Mansi Parasrampuria ◽

Seth B. Eddington ◽

Katy N. Ovington ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Rna Extraction ◽

Nanopore Sequencing ◽

The Novel ◽

Viral Genomes ◽

Primer Sets ◽

Novel Coronavirus ◽

Sequencing Platforms ◽

Effective Public Health

ABSTRACTIn late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking movement and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using online visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable ‘how-to’ reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and next-generation nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.

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High-throughput SARS-CoV-2 and host genome sequencing from single nasopharyngeal swabs

10.1101/2020.07.27.20163147 ◽

2020 ◽

Author(s):

John E Gorzynski ◽

Hannah N De Jong ◽

David Amar ◽

Chris R Hughes ◽

Alexander Ioannidis ◽

...

Keyword(s):

Genome Sequencing ◽

High Throughput ◽

Genomic Data ◽

High Fidelity ◽

Whole Genome ◽

Data Generation ◽

Viral Genomes ◽

Human Transcriptome ◽

Low Pass ◽

Rapid Generation

During COVID19 and other viral pandemics, rapid generation of host and pathogen genomic data is critical to tracking infection and informing therapies. There is an urgent need for efficient approaches to this data generation at scale. We have developed a scalable, high throughput approach to generate high fidelity low pass whole genome and HLA sequencing, viral genomes, and representation of human transcriptome from single nasopharyngeal swabs of COVID19 patients.

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