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

Viruses ◽  
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.

scholarly journals High-Throughput Sequencing Facilitates Discovery of New Plant Viruses in Poland

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 820
Author(s):  
Julia Minicka ◽  
Aleksandra Zarzyńska-Nowak ◽  
Daria Budzyńska ◽  
Natasza Borodynko-Filas ◽  
Beata Hasiów-Jaroszewska
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Plant Viruses ◽  
Yellow Mosaic Virus ◽  
Virus Species ◽  
Phylogenetic Groups ◽  
Accurate Identification ◽  
Detection And Identification ◽  
New Finding ◽  
Recent Occurrence

Viruses cause epidemics on all major crops of agronomic importance, and a timely and accurate identification is essential for control. High throughput sequencing (HTS) is a technology that allows the identification of all viruses without prior knowledge on the targeted pathogens. In this paper, we used HTS technique for the detection and identification of different viral species occurring in single and mixed infections in plants in Poland. We analysed various host plants representing different families. Within the 20 tested samples, we identified a total of 13 different virus species, including those whose presence has not been reported in Poland before: clover yellow mosaic virus (ClYMV) and melandrium yellow fleck virus (MYFV). Due to this new finding, the obtained sequences were compared with others retrieved from GenBank. In addition, cucurbit aphid-borne yellows virus (CABYV) was also detected, and due to the recent occurrence of this virus in Poland, a phylogenetic analysis of these new isolates was performed. The analysis revealed that CABYV population is highly diverse and the Polish isolates of CABYV belong to two different phylogenetic groups. Our results showed that HTS-based technology is a valuable diagnostic tool for the identification of different virus species originating from variable hosts, and can provide rapid information about the spectrum of plant viruses previously not detected in a region.

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

2014 ◽  
Vol 188 ◽  
pp. 90-96 ◽  
Author(s):  
Sebastien Massart ◽  
Antonio Olmos ◽  
Haissam Jijakli ◽  
Thierry Candresse
Keyword(s):  
High Throughput ◽  
Plant Virus ◽  
High Throughput Sequencing ◽  
Future Directions ◽  
Virus Diagnostics ◽  
Current Impact

scholarly journals A high-throughput amplicon sequencing approach for population-wide species diversity and composition survey

2020 ◽  
Author(s):  
WT Tay ◽  
LN Court ◽  
S Macfadyen ◽  
F Jacomb ◽  
S Vyskočilová ◽  
...  
Keyword(s):  
Species Diversity ◽  
High Throughput ◽  
Plant Viruses ◽  
Small Sample ◽  
Sub Saharan Africa ◽  
Molecular Diagnostic ◽  
Sampling Effort ◽  
Ecological Knowledge ◽  
Pest Species ◽  
Test Species

AbstractManagement of agricultural pests requires an understanding of pest species diversity, their interactions with beneficial insects and spatial-temporal patterns of pest abundance. Invasive and agriculturally important insect pests can build up very high populations, especially in cropping landscapes. Traditionally, sampling effort for species identification involves small sample sizes and is labour intensive. Here, we describe a high throughput sequencing (HTS) PCR amplicon method and associated analytical workflow for a rapid, intensive, high-volume survey of pest species compositions. We demonstrate our method using the Bemisia pest cryptic species complex as examples. The whiteflies Bemisia including the ‘tabaci’ species are agriculturally important capable of vectoring diverse plant viruses that cause diseases and crop losses. We use our HTS amplicon platform to simultaneously process high volumes of whitefly individuals, with efficiency to detect rare (i.e., 1%) test-species and beneficial hymenopteran parasitoid species. Field-testing our HTS amplicon method across the Tanzania, Uganda and Malawi cassava cultivation landscapes, we identified the sub-Saharan Africa 1 Bemisia putative species as the dominant pest species, with other cryptic Bemisia species being detected at various abundances. We also provide evidence that Bemisia species compositions can be affected by sampling techniques that target either nymphs or adults. Our method is particularly suitable to molecular diagnostic surveys of cryptic insect species with high population densities. Our approach can be adopted to understand species biodiversity across landscapes, with broad implications for improving trans-boundary biosecurity preparedness, thus contributing to molecular ecological knowledge and the development of control strategies for high-density, cryptic, pest-species complexes.

scholarly journals Assessment of Common Soybean-Infecting Viruses in Ohio, USA, Through Multi-site Sampling and High-Throughput Sequencing

2016 ◽  
Vol 17 (2) ◽  
pp. 133-140 ◽  
Author(s):  
Junping Han ◽  
Leslie L. Domier ◽  
Bryan J. Cassone ◽  
Anne Dorrance ◽  
Feng Qu
Keyword(s):  
Mosaic Virus ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Disease ◽  
Soybean Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Plant Viruses ◽  
Yellow Mosaic Virus ◽  
Tobacco Streak Virus ◽  
Streak Virus

Multi-site sampling was conducted during 2011 and 2012 to assess the scope of virus disease problems of soybean in Ohio, USA. A total of 259 samples were collected from 80 soybean fields distributed in 42 Ohio counties, accounting for more than 90% of major soybean-growing counties in Ohio. A high-throughput RNA-Seq approach was adopted to identify all viruses in the samples that share sufficient sequence similarities with known plant viruses. To minimize sequencing costs, total RNA extracted from up to 20 samples were first pooled to make up regional pools, resulting in eight regional pools per year in both 2011 and 2012. These regional pools were further pooled into two yearly master pools of RNA, and sequenced using the Illumina's HiSeq2000 platform. Bioinformatic analyses of sequence reads led to the identification of signature sequences of nine different viruses. The originating locations of these viruses were then mapped with PCR or RT-PCR. This study confirmed the widespread distribution of Bean pod mottle virus, Soybean vein necrosis virus, Tobacco ringspot virus, and Tobacco streak virus in Ohio. It additionally revealed occasional association of Alfalfa mosaic virus, Bean yellow mosaic virus, Clover yellow vein virus, Soybean mosaic virus, and Soybean Putnam virus with Ohio soybean. This is the first statewide survey of soybean viruses in Ohio, and provides the much-needed baseline information for management of virus diseases of soybean. Accepted for publication 20 May 2016. Published 10 June 2016.

scholarly journals Double-Stranded RNA High-Throughput Sequencing Reveals a New Cytorhabdovirus in a Bean Golden Mosaic Virus-Resistant Common Bean Transgenic Line

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 90 ◽  
Author(s):  
Dione M. T. Alves-Freitas ◽  
Bruna Pinheiro-Lima ◽  
Josias C. Faria ◽  
Cristiano Lacorte ◽  
Simone G. Ribeiro ◽  
...  
Keyword(s):  
Phaseolus Vulgaris ◽  
Common Bean ◽  
Mosaic Virus ◽  
High Throughput ◽  
Transgenic Line ◽  
High Throughput Sequencing ◽  
Open Reading Frames ◽  
Virus Species ◽  
Homologous Proteins ◽  
Bean Golden Mosaic Virus

Using double-strand RNA (dsRNA) high-throughput sequencing, we identified five RNA viruses in a bean golden mosaic virus (BGMV)-resistant common bean transgenic line with symptoms of viral infection. Four of the identified viruses had already been described as infecting common bean (cowpea mild mottle virus, bean rugose mosaic virus, Phaseolus vulgaris alphaendornavirus 1, and Phaseolus vulgaris alphaendornavirus 2) and one is a putative new plant rhabdovirus (genus Cytorhabdovirus), tentatively named bean-associated cytorhabdovirus (BaCV). The BaCV genome presented all five open reading frames (ORFs) found in most rhabdoviruses: nucleoprotein (N) (ORF1) (451 amino acids, aa), phosphoprotein (P) (ORF2) (445 aa), matrix (M) (ORF4) (287 aa), glycoprotein (G) (ORF5) (520 aa), and an RNA-dependent RNA polymerase (L) (ORF6) (114 aa), as well as a putative movement protein (P3) (ORF3) (189 aa) and the hypothetical small protein P4. The predicted BaCV proteins were compared to homologous proteins from the closest cytorhabdoviruses, and a low level of sequence identity (15–39%) was observed. The phylogenetic analysis shows that BaCV clustered with yerba mate chlorosis-associated virus (YmCaV) and rice stripe mosaic virus (RSMV). Overall, our results provide strong evidence that BaCV is indeed a new virus species in the genus Cytorhabdovirus (family Rhabdoviridae), the first rhabdovirus to be identified infecting common bean.

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

2019 ◽  
Vol 109 (3) ◽  
pp. 488-497 ◽  
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.

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

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 436 ◽  
Author(s):  
Varvara Maliogka ◽  
Angelantonio Minafra ◽  
Pasquale Saldarelli ◽  
Ana Ruiz-García ◽  
Miroslav Glasa ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Infected Plant ◽  
Fruit Trees ◽  
Plant Viruses ◽  
Fruit Tree ◽  
Advantages And Disadvantages ◽  
Sequencing Technologies ◽  
Recent Advances

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.

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

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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