Highly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from Hops
Implementing high-throughput sequencing (HTS) in virus detection has recently become widespread. Bioinformatic expertise, programming skills, and access to high-capacity computers are essential for HTS data analysis. The E-Probe Diagnostic Nucleic Acid Analysis, a bioinformatic detection system that...
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The American Phytopathological Society
2025-06-01
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| Series: | PhytoFrontiers |
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| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-24-0106-FI |
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| author | Ali Pasha Andres S. Espindola Heiko Ziebell Francisco M. Ochoa‐Corona |
| author_facet | Ali Pasha Andres S. Espindola Heiko Ziebell Francisco M. Ochoa‐Corona |
| author_sort | Ali Pasha |
| collection | DOAJ |
| description | Implementing high-throughput sequencing (HTS) in virus detection has recently become widespread. Bioinformatic expertise, programming skills, and access to high-capacity computers are essential for HTS data analysis. The E-Probe Diagnostic Nucleic Acid Analysis, a bioinformatic detection system that addresses these HTS constraints and performs searches on non-normalized HTS data, was adopted. The E-Probe Diagnostic Nucleic Acid Analysis theoretical framework was later updated for cross-platform accessibility via a web browser and renamed Microbe Finder (MiFi). In this study, the MiFi platform was used to design electronic probes (e-probes) for two viroids and three viruses that infect the hop plant (Humulus lupulus L.). These e-probes underwent a rigorous curation process followed by in silico validation to determine their theoretical limit of detection. Interestingly, curated 40-nt e-probes of all targeted pathogens in this study proved to have a better combination of sensitivity and specificity for their target genomes than other e-probe lengths. Additionally, an in vitro limit of detection analysis was performed by constructing an artificial positive control plasmid that contained all curated e-probes. The stock plasmid was serially diluted before sequencing to validate the detection limit. The curated and validated e-probes successfully detected the targeted pathogens when applied to HTS datasets previously analyzed using other bioinformatics tools. The e-probes developed in this study allow the direct detection of targeted hop viruses and viroids in unassembled HTS datasets. [Figure: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license. |
| format | Article |
| id | doaj-art-9aedc1f4402d4bdabe75693fa1882120 |
| institution | OA Journals |
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| language | English |
| publishDate | 2025-06-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | PhytoFrontiers |
| spelling | doaj-art-9aedc1f4402d4bdabe75693fa18821202025-08-20T02:38:01ZengThe American Phytopathological SocietyPhytoFrontiers2690-54422025-06-015216517310.1094/PHYTOFR-09-24-0106-FIHighly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from HopsAli Pasha0Andres S. Espindola1Heiko Ziebell2Francisco M. Ochoa‐Corona3Julius Kühn Institute (JKI)-Federal Research Center for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, GermanyInstitute for Biosecurity and Microbial Forensics (IBMF), Oklahoma State University, Stillwater, OK, U.S.A.Julius Kühn Institute (JKI)-Federal Research Center for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, GermanyInstitute for Biosecurity and Microbial Forensics (IBMF), Oklahoma State University, Stillwater, OK, U.S.A.Implementing high-throughput sequencing (HTS) in virus detection has recently become widespread. Bioinformatic expertise, programming skills, and access to high-capacity computers are essential for HTS data analysis. The E-Probe Diagnostic Nucleic Acid Analysis, a bioinformatic detection system that addresses these HTS constraints and performs searches on non-normalized HTS data, was adopted. The E-Probe Diagnostic Nucleic Acid Analysis theoretical framework was later updated for cross-platform accessibility via a web browser and renamed Microbe Finder (MiFi). In this study, the MiFi platform was used to design electronic probes (e-probes) for two viroids and three viruses that infect the hop plant (Humulus lupulus L.). These e-probes underwent a rigorous curation process followed by in silico validation to determine their theoretical limit of detection. Interestingly, curated 40-nt e-probes of all targeted pathogens in this study proved to have a better combination of sensitivity and specificity for their target genomes than other e-probe lengths. Additionally, an in vitro limit of detection analysis was performed by constructing an artificial positive control plasmid that contained all curated e-probes. The stock plasmid was serially diluted before sequencing to validate the detection limit. The curated and validated e-probes successfully detected the targeted pathogens when applied to HTS datasets previously analyzed using other bioinformatics tools. The e-probes developed in this study allow the direct detection of targeted hop viruses and viroids in unassembled HTS datasets. [Figure: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-24-0106-FIbioinformaticsdetectionEDNA-MiFie-probehopHTS |
| spellingShingle | Ali Pasha Andres S. Espindola Heiko Ziebell Francisco M. Ochoa‐Corona Highly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from Hops PhytoFrontiers bioinformatics detection EDNA-MiFi e-probe hop HTS |
| title | Highly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from Hops |
| title_full | Highly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from Hops |
| title_fullStr | Highly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from Hops |
| title_full_unstemmed | Highly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from Hops |
| title_short | Highly Curated and Reliable E-Probes for Detection of Viral Pathogens in Unassembled High-Throughput Sequencing Datasets from Hops |
| title_sort | highly curated and reliable e probes for detection of viral pathogens in unassembled high throughput sequencing datasets from hops |
| topic | bioinformatics detection EDNA-MiFi e-probe hop HTS |
| url | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-09-24-0106-FI |
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