PVDP: A portable open source pipeline for detection of plant viruses in RNAseq data. A case study on potato viruses in Antioquia (Colombia)

Gutiérrez, Pablo; Rivillas, Ary; Tejada, Daniel; Giraldo, Susana; Restrepo, Andrea; Ospina, M María Carolina; Cadavid, S Prieto; García, Yuliana Gallo

doi:10.1016/j.pmpp.2021.101604

Cited by 19 publications

(15 citation statements)

References 35 publications

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“…However, we started losing sensitivity before we could eliminate false positives. PVDP reports useful parameters such as evenness, read dispersion, and certainty score to help identify false positives [ 32 ]. Nevertheless, because the reads resulting from index misassignment showed even distribution across the virus or viroid genomes to which they matched, these measurements did not assist in flagging false positives.…”

Section: Discussionmentioning

confidence: 99%

“…However, their sensitivity also makes direct read annotation methods more prone to detect false positives (e.g., cross-talk, detection of endogenous viruses). Therefore, they can be used as first-pass screening tools to identify viral sequences and guide downstream bioinformatics procedures such as reference-guided assembly, as pointed out by Gutierrez et al [ 32 ]. In comparison, de novo assembly-based approaches enable assembling complete viral genomes and thus provide a better resolution in complex cases such as multiple related viruses co-infecting the same plant, discriminating recombination events, detecting sequence divergence, or identifying new viral species.…”

Section: Discussionmentioning

confidence: 99%

“…Four bioinformatics approaches were executed on the ribodepleted RNA-Seq samples to detect viruses and viroids. We trialed two methods that use direct annotation on quality trimmed reads without any assembly: the plant virus detection pipeline (PVDP) [ 32 ] and Kodoja [ 33 ]. These workflows were run on default parameters using the pre-computed database kodojaDB_v1.0 and the PlantVirusesDB_0420v4_masked.fa for Kodoja and PVDP, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis

Gauthier

Lelwala

Elliott³

et al. 2022

Biology

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Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis

Gauthier

Lelwala

Elliott³

et al. 2022

Biology

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“…Additionally, complete chloroplast genome sequences from the same genera of host plants held at PEQ were retrieved from NCBI nucleotide database [ 40 ] in GenBank format and will be referred to as ‘Chloroplast dataset’ from here on. The GenBank-formatted sequences were converted into FASTA format with annotated taxonomic information using a custom script by Gutiérrez et al [ 43 ]. In the regulated dataset, the unknown base percentages of each sequence were determined using the Seqtk version 1.3 [ 44 ], and sequences with >5% N content were removed along with host plant sequences and sequences >20 Kb, except for complete chloroplast genomes that ranged from 100 to 200 kbp.…”

Section: Methodsmentioning

confidence: 99%

Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants

Lelwala

LeBlanc

Gauthier

et al. 2022

Viruses

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High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21–22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ.

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“…sequences was performed with the Plant Virus Detection Platform, PVDP (Gutiérrez et al, 2021). Viral sequences were assembled de novo with rnaSPAdes (Bushmanova et al, 2019).…”

Section: Bioinformatic Analyses Preliminary Detection Of Viralmentioning

confidence: 99%

Natural infection of purple passion fruit (Passiflora edulis f. edulis) by a novel member of the family Tymoviridae in Colombia

Cardona¹,

Restrepo²,

Higuita³

et al. 2022

Self Cite

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Purple passion fruit is one of the most important fruit exports of Colombia, but its productivity is being compromised by the emergence of several viral diseases. High-throughput sequencing (HTS) surveys of viruses in purple passion fruit fields in the province of Antioquia suggested infection by a new member of the family Tymoviridae. In this work, we characterize the complete genome sequence of this virus, tentatively named purple passionfruit leaf deformation virus (PpLDV), and evaluate its distribution in Antioquia. PpLDV was assembled at high coverage in four datasets from different regions. The 6.1 kb genome of PpLDV encodes a single polyprotein with domains characteristic of the family Tymoviridae, contains a marafibox-like promoter and the 3'-UTR can fold into a tRNA-like secondary structure with a valine anti-codon. Phylogenetic analysis of the polyprotein revealed that PpLDV is a distinct member of the family Tymoviridae, more closely related to the genus Tymovirus and the unclassified Poinsettia mosaic virus (PnMV). The presence of PpLDV was confirmed by RT-qPCR and RT-PCR in samples from commercial purple passion fruit fields, plantlets and seed sprouts collected in Antioquia using primers designed in this study.

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PVDP: A portable open source pipeline for detection of plant viruses in RNAseq data. A case study on potato viruses in Antioquia (Colombia)

Cited by 19 publications

References 35 publications

Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis

Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis

Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants

Natural infection of purple passion fruit (Passiflora edulis f. edulis) by a novel member of the family Tymoviridae in Colombia

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