Metagenomic Studies of Viruses in Weeds and Wild Plants: A Powerful Approach to Characterise Variable Virus Communities

High throughput sequencing (HTS) has revolutionised virus detection and discovery, allowing for the untargeted characterisation of whole viromes. Viral metagenomics studies have demonstrated the ubiquity of virus infection – often in the absence of disease symptoms – and tend to discover many novel viruses, highlighting the small fraction of virus biodiversity described to date. The majority of the studies using high-throughput sequencing to characterise plant viromes have focused on economically important crops, and only a small number of studies have considered weeds and wild plants. Characterising the viromes of wild plants is highly relevant, as these plants can affect disease dynamics in crops, often by acting as viral reservoirs. Moreover, the viruses in unmanaged systems may also have important effects on wild plant populations and communities. Here, we review metagenomic studies on weeds and wild plants to show the benefits and limitations of this approach and identify knowledge gaps. We consider key genomics developments that are likely to benefit the field in the near future. Although only a small number of HTS studies have been performed on weeds and wild plants, these studies have already discovered many novel viruses, demonstrated unexpected trends in virus distributions, and highlighted the potential of metagenomics as an approach.

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Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽

10.1094/pdis-05-21-0949-re ◽

2021 ◽

Author(s):

Dan Edward Veloso Villamor ◽

Karen E Keller ◽

Robert Martin ◽

Ioannis Emmanouil Tzanetakis

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Wide Spectrum ◽

Virus Detection ◽

Rt Pcr ◽

Host Interactions ◽

Growing Seasons ◽

Berry Crops ◽

Sampling Times ◽

Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

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Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽

10.3390/v10100566 ◽

2018 ◽

Vol 10 (10) ◽

pp. 566 ◽

Cited By ~ 9

Author(s):

Siemon Ng ◽

Cassandra Braxton ◽

Marc Eloit ◽

Szi Feng ◽

Romain Fragnoud ◽

...

Keyword(s):

Sample Preparation ◽

Nucleic Acids ◽

High Throughput ◽

High Throughput Sequencing ◽

Virus Detection ◽

Extraction Methods ◽

Control Measures ◽

Acid Extraction ◽

Library Preparation ◽

Sample Processing

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

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Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species

Applied and Environmental Microbiology ◽

10.1128/aem.03538-15 ◽

2016 ◽

Vol 82 (6) ◽

pp. 1966-1975 ◽

Cited By ~ 15

Author(s):

Christelle Lacroix ◽

Kurra Renner ◽

Ellen Cole ◽

Eric W. Seabloom ◽

Elizabeth T. Borer ◽

...

Keyword(s):

Plant Species ◽

Disease Risk ◽

Virus Detection ◽

Plant Viruses ◽

Wild Plants ◽

Wild Plant ◽

Content Type ◽

Wild Plant Species ◽

Methodological Guidelines ◽

Wild Hosts

ABSTRACTEcological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally importantBarley yellow dwarf virusPAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts.

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High Throughput Sequencing For Plant Virus Detection and Discovery

Phytopathology ◽

10.1094/phyto-07-18-0257-rvw ◽

2019 ◽

Vol 109 (5) ◽

pp. 716-725 ◽

Cited By ~ 44

Author(s):

D. E. V. Villamor ◽

T. Ho ◽

M. Al Rwahnih ◽

R. R. Martin ◽

I. E. Tzanetakis

Keyword(s):

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing ◽

Virus Detection ◽

Agricultural Crops ◽

Virus Discovery ◽

Plant Virus Detection ◽

Virus Biology ◽

Disease Causality

Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists’ utility in their research.

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Advanced Virus Detection Technologies Interest Group (AVDTIG): Efforts on High Throughput Sequencing (HTS) for Virus Detection

PDA Journal of Pharmaceutical Science and Technology ◽

10.5731/pdajpst.2016.007161 ◽

2016 ◽

Vol 70 (6) ◽

pp. 591-595 ◽

Cited By ~ 12

Author(s):

A. S. Khan ◽

D. A. Vacante ◽

J.-P. Cassart ◽

S. H. S. Ng ◽

C. Lambert ◽

...

Keyword(s):

Interest Group ◽

High Throughput ◽

High Throughput Sequencing ◽

Virus Detection ◽

Detection Technologies

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Small RNA Expression Profiling by High-Throughput Sequencing: Implications of Enzymatic Manipulation

Journal of Nucleic Acids ◽

10.1155/2012/360358 ◽

2012 ◽

Vol 2012 ◽

pp. 1-15 ◽

Cited By ~ 20

Author(s):

Fanglei Zhuang ◽

Ryan T. Fuchs ◽

G. Brett Robb

Keyword(s):

High Throughput ◽

Expression Profiling ◽

Messenger Rna ◽

High Throughput Sequencing ◽

Biological Processes ◽

Cellular Processes ◽

Disease States ◽

Powerful Approach ◽

Sequencing Library ◽

Rna Expression Profiling

Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments.

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Virus detection in high-throughput sequencing data without a reference genome of the host

Infection Genetics and Evolution ◽

10.1016/j.meegid.2018.09.026 ◽

2018 ◽

Vol 66 ◽

pp. 180-187 ◽

Cited By ~ 3

Author(s):

Jochen Kruppa ◽

Wendy K. Jo ◽

Erhard van der Vries ◽

Martin Ludlow ◽

Albert Osterhaus ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Reference Genome ◽

Virus Detection ◽

Sequencing Data ◽

High Throughput Sequencing Data

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Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies

Phytopathology ◽

10.1094/phyto-02-18-0067-r ◽

2019 ◽

Vol 109 (3) ◽

pp. 488-497 ◽

Cited By ~ 29

Author(s):

Sebastien Massart ◽

Michela Chiumenti ◽

Kris De Jonghe ◽

Rachel Glover ◽

Annelies Haegeman ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

High Throughput Sequencing ◽

Performance Test ◽

Virus Detection ◽

Performance Testing ◽

Plant Viruses ◽

Reference Sequence ◽

Bioinformatics Pipeline ◽

Double Blind

Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

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Broad-Range Virus Detection and Discovery Using Microfluidic PCR Coupled with High-throughput Sequencing

10.1101/2020.06.10.145052 ◽

2020 ◽

Author(s):

Ying Tao ◽

Clinton R. Paden ◽

Krista Queen ◽

Jing Zhang ◽

Eishita Tyagi ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Virus Detection ◽

Viral Pathogens ◽

Large Numbers ◽

Straightforward Method ◽

Sequence Identification ◽

Pcr Assays ◽

Taxonomic Groups ◽

Next Generation Sequencing Ngs

AbstractThere is a need for a comprehensive and sensitive method to test for a broad range of viral pathogens in samples without any identifiable pathogen detected. Real-time PCR assays are sensitive and rapid, but their specificity limits their utility in detecting divergent agents. Shotgun high-throughput sequencing methods provide unbiased sequence identification, however, they have limited sensitivity and require complex analyses. In order to meet the need for a sensitive, high-throughput virus detection and discovery platform with good sensitivity, we combine two existing technologies, broadly-reactive consensus-degenerate pan-viral group PCR and the MiSeq sequencer (Illumina), using the Access Array (Fluidigm), a commercially-available microfluidic PCR system. Pan-viral group primers target conserved regions of virus taxonomic groups and can amplify known and potentially novel species. The Access Array employs dozens of these assays in parallel, which are then sequenced all at once on the MiSeq. In this study, we run a respiratory panel of pan-viral group PCR assays using AA-PCR-Seq. We validate the panel on a collection of representative human and animal samples, comparing it to qPCR and shotgun next-generation sequencing (NGS). AA-PCR-Seq provides a robust, straightforward method for screening large numbers of samples for virus detection and discovery.

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