Current impact and future directions of high throughput sequencing in plant virus diagnostics

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT’s feasibility as a valuable component to the diagnostician’s toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.

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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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How Do We Define the Genomic Landscape?

Blood ◽

10.1182/blood.v124.21.sci-13.sci-13 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-13-SCI-13

Author(s):

Sandeep S. Dave

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Conflicts Of Interest ◽

Genomic Analysis ◽

Future Directions ◽

Tissue Samples ◽

Genomic Landscape ◽

Fresh Frozen ◽

Definition Of ◽

Formalin Fixed

High throughput sequencing is a revolutionary technology for the definition of the genomic features of tumors. This talk will provide a review of the relevant methodologies for non-experts in the field. The presentation will include a discussion of how high throughput sequencing is performed, its relative strengths and weaknesses, and how it is applicable to formalin-fixed and fresh/frozen tissue samples. The talk will also describe future directions in the genomic analysis of tumors. Disclosures No relevant conflicts of interest to declare.

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Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics

Viruses ◽

10.3390/v13061130 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1130

Author(s):

Nourolah Soltani ◽

Kristian A. Stevens ◽

Vicki Klaassen ◽

Min-Sook Hwang ◽

Deborah A. Golino ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Plant Viruses ◽

Molecular Diagnostic ◽

Virus Species ◽

Certification Programs ◽

Performance Specifications ◽

Virus Diagnostics ◽

Diagnostic Research ◽

And Performance

Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.

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High-Throughput Sequencing Facilitates Characterization of a “Forgotten” Plant Virus: The Case of a Henbane Mosaic Virus Infecting Tomato

Frontiers in Microbiology ◽

10.3389/fmicb.2018.02739 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Anja Pecman ◽

Denis Kutnjak ◽

Nataša Mehle ◽

Magda Tušek Žnidarič ◽

Ion Gutiérrez-Aguirre ◽

...

Keyword(s):

Mosaic Virus ◽

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing

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Comparative study on three viral enrichment approaches based on RNA extraction for plant virus/viroid detection using high-throughput sequencing

PLoS ONE ◽

10.1371/journal.pone.0237951 ◽

2020 ◽

Vol 15 (8) ◽

pp. e0237951

Author(s):

Yahya Zakaria Abdou Gaafar ◽

Heiko Ziebell

Keyword(s):

Comparative Study ◽

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing ◽

Rna Extraction

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Novel Insights for Biosurveillance of Bat-Borne Viruses

Proceedings ◽

10.3390/proceedings2020050047 ◽

2020 ◽

Vol 50 (1) ◽

pp. 47

Author(s):

Adrian C. Paskey ◽

Justin H. J. Ng ◽

Gregory K. Rice ◽

Wan Ni Chia ◽

Casandra W. Philipson ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Temporal Patterns ◽

Population Level ◽

The Body ◽

Population Persistence ◽

Target Enrichment ◽

Species Barrier ◽

Future Directions ◽

New Knowledge

Bats are rich reservoirs of viruses, including several high consequence zoonoses. In this study, high throughput sequencing was used to characterize the virome through a longitudinal study of a captive colony of lesser dawn bats, species Eonycteris spelaea, in Singapore. This study utilized viral RNA extracted from swabs of four body sites per bat per timepoint. Swabs of the exterior of the bat (head and body) were used to evaluate virus populations and demonstrate utility as a sample site for future surveillance to extrapolate population-level infection. Through unbiased shotgun and target-enrichment sequencing, we identified both the known and previously unknown viruses of zoonotic relevance and defined the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. We observed the population persistence of three zoonotic-related viral families that are known to be associated with spillover from bats to humans: Paramyxoviridae, Reoviridae, and Coronaviridae. To our knowledge, this is the first study that combines probe-based viral enrichment with high-throughput sequencing or that creates a viral profile from multiple swab sites on individual bats and their cohorts. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Noninvasive surveillance methods that target the body of bats not only detect viruses shed within the colony but can also represent viral populations dispersed throughout the entire colony. New knowledge of persistent viral families should inform future directions for the biosurveillance of viruses that have the potential to cross the species barrier from bats to humans or other amplifying hosts.

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Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets

Journal of Clinical Microbiology ◽

10.1128/jcm.00466-19 ◽

2019 ◽

Vol 57 (8) ◽

Cited By ~ 11

Author(s):

Annika Brinkmann ◽

Andreas Andrusch ◽

Ariane Belka ◽

Claudia Wylezich ◽

Dirk Höper ◽

...

Keyword(s):

Proficiency Testing ◽

High Throughput ◽

In Silico ◽

High Throughput Sequencing ◽

Bioinformatics Analysis ◽

Sequence Data ◽

Correct Identification ◽

Illumina Hiseq ◽

Data Set ◽

Virus Diagnostics

ABSTRACT Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants’ analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

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Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence

Pathogens ◽

10.3390/pathogens10091174 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1174

Author(s):

Yahya Z. A. Gaafar ◽

Marcel Westenberg ◽

Marleen Botermans ◽

Krizbai László ◽

Kris De Jonghe ◽

...

Keyword(s):

Sample Preparation ◽

High Throughput ◽

Ribosomal Rna ◽

Diagnostic Tool ◽

Plant Virus ◽

High Throughput Sequencing ◽

Interlaboratory Comparison ◽

Virus Detection ◽

Plant Viruses ◽

Comparison Study

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data

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A Primer on the Analysis of High-Throughput Sequencing Data for Detection of Plant Viruses

Microorganisms ◽

10.3390/microorganisms9040841 ◽

2021 ◽

Vol 9 (4) ◽

pp. 841

Author(s):

Denis Kutnjak ◽

Lucie Tamisier ◽

Ian Adams ◽

Neil Boonham ◽

Thierry Candresse ◽

...

Keyword(s):

Data Analysis ◽

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing ◽

Plant Viruses ◽

Acid Extraction ◽

Sequencing Data ◽

Bioinformatic Tools ◽

Advantages And Disadvantages ◽

Plant Virus Detection

High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

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