High-throughput sequencing for the diagnostic of plant pathologies and identification of pests: recommendations and challenges
Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests
Recommendation: posted 12 September 2022, validated 07 October 2022
Schumpp, O. (2022) High-throughput sequencing for the diagnostic of plant pathologies and identification of pests: recommendations and challenges. Peer Community In Infections, 100002. https://doi.org/10.24072/pci.infections.100002
High-throughput sequencing (HTS) has revealed an incredible diversity of microorganisms in ecosystems and is also changing the monitoring of macroorganism biodiversity (Deiner et al. 2017; Piper et al. 2019).
The diagnostic of plant pathogens and the identification of pests is gradually integrating the use of these techniques, but there are still obstacles. Most of them are related to the reliability of these analyses, which have long been considered insufficient because of their dependence on a succession of sophisticated operations involving parameters that are sometimes difficult to adapt to complex matrices or certain diagnostic contexts. The need to validate HTS approaches is gradually being highlighted in recent work but remains poorly documented (Bester et al. 2022).
In this paper, a large community of experts presents and discusses the key steps for optimal control of HTS performance and reliability in a diagnostic context (Massart et al. 2022). It also addresses the issue of costs. The article provides recommendations that closely combine the quality control requirements commonly used in conventional diagnostics with newer or HTS-specific control elements and concepts that are not yet widely used. It discusses the value of these for the use of the various techniques currently covered by the terms "High Throughput Sequencing" in diagnostic activities. The elements presented are intended to limit false positive or false negative results but will also optimise the interpretation of contentious results close to the limits of analytical sensitivity or unexpected results, both of which appear to be frequent when using HTS.
Furthermore, the need for risk analysis, verification and validation of methods is well illustrated with numerous examples for each of the steps considered crucial to ensure reliable use of HTS. The clear contextualisation of the proposals made by the authors complements and clarifies the need for user expertise according to the experimental objectives. Some unanswered questions that will require further development and validation are also presented.
This article should benefit a large audience including researchers with some level of expertise in HTS but unfamiliar with the recent concepts of controls common in the diagnostic world as well as scientists with strong diagnostic expertise but less at ease with the numerous and complex procedures associated with HTS.
Bester R, Steyn C, Breytenbach JHJ, de Bruyn R, Cook G, Maree HJ (2022) Reproducibility and Sensitivity of High-Throughput Sequencing (HTS)-Based Detection of Citrus Tristeza Virus and Three Citrus Viroids. Plants, 11, 1939. https://doi.org/10.3390/plants11151939
Deiner K, Bik HM, Mächler E, Seymour M, Lacoursière-Roussel A, Altermatt F, Creer S, Bista I, Lodge DM, de Vere N, Pfrender ME, Bernatchez L (2017) Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology, 26, 5872–5895. https://doi.org/10.1111/mec.14350
Massart, S et al. (2022) Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests. Zenodo, 6637519, ver. 3 peer-reviewed and recommended by Peer Community in Infections. https://doi.org/10.5281/zenodo.6637519
Piper AM, Batovska J, Cogan NOI, Weiss J, Cunningham JP, Rodoni BC, Blacket MJ (2019) Prospects and challenges of implementing DNA metabarcoding for high-throughput insect surveillance. GigaScience, 8, giz092. https://doi.org/10.1093/gigascience/giz092
The recommender in charge of the evaluation of the article and the reviewers declared that they have no conflict of interest (as defined in the code of conduct of PCI) with the authors or with the content of the article. The authors declared that they comply with the PCI rule of having no financial conflicts of interest in relation to the content of the article.
Evaluation round #1
DOI or URL of the preprint: https://doi.org/10.5281/zenodo.6637518
Version of the preprint: 1
Author's Reply, 06 Sep 2022
Decision by Olivier Schumpp, posted 06 Aug 2022
Thank you for submitting your manuscript to PCI Infection. I particularly enjoyed reading it. The manuscript was evaluated by 2 reviewers and both found it to be of great interest and high quality. However, both raised a number of points which I invite you to address before a final decision is made.
For my side, I have two additional minor comments:
- L1091: ... "and Massart S." (?)
- Diagram of figures 1 and 3: should there not be an exit arrow at the end of the verification and/or validation steps?
I look forward to receiving your revised version.