Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets
High-throughput sequencing (HTS) technology is applied to plant virus diagnosis using virus discovery pipelines, and direct detection of plant viruses from unassembled HTS is needed. The e-probe diagnostic nucleic acid assay Microbe Finder (EDNA-MiFi) is a pipeline for pathogen detection for unassem...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
The American Phytopathological Society
2025-06-01
|
| Series: | PhytoFrontiers |
| Subjects: | |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-11-24-0121-FI |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850109696802816000 |
|---|---|
| author | Lizbeth Peña-Zúñiga Andres Espindola Darren Hagen Akhtar Ali Francisco Ochoa-Corona |
| author_facet | Lizbeth Peña-Zúñiga Andres Espindola Darren Hagen Akhtar Ali Francisco Ochoa-Corona |
| author_sort | Lizbeth Peña-Zúñiga |
| collection | DOAJ |
| description | High-throughput sequencing (HTS) technology is applied to plant virus diagnosis using virus discovery pipelines, and direct detection of plant viruses from unassembled HTS is needed. The e-probe diagnostic nucleic acid assay Microbe Finder (EDNA-MiFi) is a pipeline for pathogen detection for unassembled HTS datasets. The application of EDNA-MiFi as a diagnostic tool was demonstrated in detecting plant pathogens of different taxa. This study demonstrates that the limit of detection of EDNA-MiFi for sensitive virus detection and quantification can be calculated. A synthetic construct mimicking the Arabis mosaic virus (ArMV) RNA2 was synthesized in vitro. E-probes for the ArMV-RNA2 were designed de novo and curated for target specificity. The new and specific e-probes of ArMV RNA2 were 20 and 60 nt long and were queried against 28 HTS unassembled spiked genome libraries obtained by spiking a serially diluted gradient of the synthetic construct carrying the ArMV RNA2 with purified genomic DNA from Vitis vinifera L. The experiment generated 28 HTS spiked datasets simulating a range of virus abundance levels, such as stages of virus infection. The limit of detection of EDNA-MiFi using the synthetic ArMV-RNA2 construct in vitro and e-probes 20 nt long varies depending on the E value, with 0.0000001 ng (14.1 copies) at an E value of 1, which is comparable to the qPCR limit of detection. Other reliable detections were at 0.001 ng at an E value of 1e−1 and 0.01 at an E value of 1e−2. EDNA-MiFi rapidly queries the spiked genome libraries with sets of predetermined plant virus e-probes, allowing for rapid and sensitive detection without requiring genome sequence assembly. [Figure: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license. |
| format | Article |
| id | doaj-art-75d5424b357344218950e7bb3617536a |
| institution | OA Journals |
| issn | 2690-5442 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | PhytoFrontiers |
| spelling | doaj-art-75d5424b357344218950e7bb3617536a2025-08-20T02:38:01ZengThe American Phytopathological SocietyPhytoFrontiers2690-54422025-06-015226427110.1094/PHYTOFR-11-24-0121-FIAssessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing DatasetsLizbeth Peña-Zúñiga0Andres Espindola1Darren Hagen2Akhtar Ali3Francisco Ochoa-Corona4Institute of Biosecurity and Microbial Forensics, Department Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, U.S.A.Institute of Biosecurity and Microbial Forensics, Department Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, U.S.A.Department of Animal and Food Sciences, Oklahoma State University, Stillwater, OK, U.S.A.Department of Biological Science, University of Tulsa, Tulsa, OK, U.S.A.Institute of Biosecurity and Microbial Forensics, Department Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, U.S.A.High-throughput sequencing (HTS) technology is applied to plant virus diagnosis using virus discovery pipelines, and direct detection of plant viruses from unassembled HTS is needed. The e-probe diagnostic nucleic acid assay Microbe Finder (EDNA-MiFi) is a pipeline for pathogen detection for unassembled HTS datasets. The application of EDNA-MiFi as a diagnostic tool was demonstrated in detecting plant pathogens of different taxa. This study demonstrates that the limit of detection of EDNA-MiFi for sensitive virus detection and quantification can be calculated. A synthetic construct mimicking the Arabis mosaic virus (ArMV) RNA2 was synthesized in vitro. E-probes for the ArMV-RNA2 were designed de novo and curated for target specificity. The new and specific e-probes of ArMV RNA2 were 20 and 60 nt long and were queried against 28 HTS unassembled spiked genome libraries obtained by spiking a serially diluted gradient of the synthetic construct carrying the ArMV RNA2 with purified genomic DNA from Vitis vinifera L. The experiment generated 28 HTS spiked datasets simulating a range of virus abundance levels, such as stages of virus infection. The limit of detection of EDNA-MiFi using the synthetic ArMV-RNA2 construct in vitro and e-probes 20 nt long varies depending on the E value, with 0.0000001 ng (14.1 copies) at an E value of 1, which is comparable to the qPCR limit of detection. Other reliable detections were at 0.001 ng at an E value of 1e−1 and 0.01 at an E value of 1e−2. EDNA-MiFi rapidly queries the spiked genome libraries with sets of predetermined plant virus e-probes, allowing for rapid and sensitive detection without requiring genome sequence assembly. [Figure: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-11-24-0121-FIArMVEDNA-MiFiHTS diagnosticsHTS limit of detectionHTS sensitivity |
| spellingShingle | Lizbeth Peña-Zúñiga Andres Espindola Darren Hagen Akhtar Ali Francisco Ochoa-Corona Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets PhytoFrontiers ArMV EDNA-MiFi HTS diagnostics HTS limit of detection HTS sensitivity |
| title | Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets |
| title_full | Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets |
| title_fullStr | Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets |
| title_full_unstemmed | Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets |
| title_short | Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets |
| title_sort | assessment of viral limit of detection in spiked unassembled high throughput sequencing datasets |
| topic | ArMV EDNA-MiFi HTS diagnostics HTS limit of detection HTS sensitivity |
| url | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-11-24-0121-FI |
| work_keys_str_mv | AT lizbethpenazuniga assessmentofvirallimitofdetectioninspikedunassembledhighthroughputsequencingdatasets AT andresespindola assessmentofvirallimitofdetectioninspikedunassembledhighthroughputsequencingdatasets AT darrenhagen assessmentofvirallimitofdetectioninspikedunassembledhighthroughputsequencingdatasets AT akhtarali assessmentofvirallimitofdetectioninspikedunassembledhighthroughputsequencingdatasets AT franciscoochoacorona assessmentofvirallimitofdetectioninspikedunassembledhighthroughputsequencingdatasets |