Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

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Applications of Next Generation High Throughput Sequencing Technologies in Characterization, Discovery and Molecular Interaction of Plant Viruses

Indian Journal of Virology ◽

10.1007/s13337-013-0133-4 ◽

2013 ◽

Vol 24 (2) ◽

pp. 157-165 ◽

Cited By ~ 26

Author(s):

K. Prabha ◽

V. K. Baranwal ◽

R. K. Jain

Keyword(s):

High Throughput ◽

Molecular Interaction ◽

High Throughput Sequencing ◽

Plant Viruses ◽

Next Generation ◽

Sequencing Technologies

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GrigoraSNPs: Optimized HTS DNA Forensic SNP Analysis

10.1101/173716 ◽

2017 ◽

Cited By ~ 3

Author(s):

Darrell O. Ricke ◽

Anna Shcherbina ◽

Adam Michaleas ◽

Philip Fremont-Smith

Keyword(s):

High Throughput ◽

Tandem Repeats ◽

High Throughput Sequencing ◽

Dna Analysis ◽

Sequence Data ◽

Snp Analysis ◽

Analysis Pipeline ◽

Sequencing Technologies ◽

High Throughput Dna Sequencing

AbstractHigh throughput DNA sequencing technologies enable improved characterization of forensic DNA samples enabling greater insights into DNA contributor(s). Current DNA forensics techniques rely upon allele sizing of short tandem repeats by capillary electrophoresis. High throughput sequencing enables forensic sample characterizations for large numbers of single nucleotide polymorphism loci. The slowest computational component of the DNA forensics analysis pipeline is the characterization of raw sequence data. This paper optimizes the SNP calling module of the DNA analysis pipeline with runtime results that scale linearly with the number of HTS sequences (patent pending)[1]. GrigoraSNPs can analyze 100 million reads in less than 5 minutes using 3 threads on a 4.0 GHz Intel i7-6700K laptop CPU.

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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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Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico

Phytopathology ◽

10.1094/phyto-07-19-0253-fi ◽

2020 ◽

Vol 110 (1) ◽

pp. 106-120 ◽

Cited By ~ 1

Author(s):

Avijit Roy ◽

Andrew L. Stone ◽

Gabriel Otero-Colina ◽

Gang Wei ◽

Ronald H. Brlansky ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sensu Stricto ◽

Genome Segment ◽

Rt Pcr ◽

Sequence Comparisons ◽

Orchid Fleck Virus ◽

Reverse Transcription Pcr ◽

Sequencing Technologies ◽

Negative Sense

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit–specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit–specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

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Assessing genotyping errors in mammalian museum study skins using high-throughput genotyping-by-sequencing

Conservation Genetics Resources ◽

10.1007/s12686-021-01213-8 ◽

2021 ◽

Author(s):

Stella C. Yuan ◽

Eric Malekos ◽

Melissa T. R. Hawkins

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Massively Parallel Sequencing ◽

Massively Parallel ◽

Museum Specimens ◽

Museum Specimen ◽

Genotyping Errors ◽

Allelic Dropout ◽

Parallel Sequencing ◽

Sequencing Technologies

AbstractThe use of museum specimens held in natural history repositories for population and conservation genetic research is increasing in tandem with the use of massively parallel sequencing technologies. Short Tandem Repeats (STRs), or microsatellite loci, are commonly used genetic markers in wildlife and population genetic studies. However, they traditionally suffered from a host of issues including length homoplasy, high costs, low throughput, and difficulties in reproducibility across laboratories. Massively parallel sequencing technologies can address these problems, but the incorporation of museum specimen derived DNA suffers from significant fragmentation and exogenous DNA contamination. Combatting these issues requires extra measures of stringency in the lab and during data analysis, yet there have not been any high-throughput sequencing studies evaluating microsatellite allelic dropout from museum specimen extracted DNA. In this study, we evaluate genotyping errors derived from mammalian museum skin DNA extracts for previously characterized microsatellites across PCR replicates utilizing high-throughput sequencing. We found it useful to classify samples based on DNA concentration, which determined the rate by which genotypes were accurately recovered. Longer microsatellites performed worse in all museum specimens. Allelic dropout rates across loci were dependent on sample quantity, with high concentration museum specimens performing as well and recovering quality metrics nearly as high as the frozen tissue sample. Based on our results, we provide a set of best practices for quality assurance and incorporation of reliable genotypes from museum specimens.

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