Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests

Abstract

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health.

High-throughput sequencing application in the detection and discovery of viruses associated with the regulated citrus leprosis disease complex

“…Initially we thought that there was an early-stage contamination or cross-contamination when multiplexing several samples in a single sequencing experiment. To prevent the contamination, we followed the guidelines for reliable use of HTS ( Lebas et al., 2022 ; Massart et al., 2022 ) and repeated the RNA extraction followed by library preparation in a separate laboratory set up. The new dual indexed libraries were constructed in Molecular Plant Pathology Laboratory (MPPL), Beltsville, MD instead of PPCDL, MD using new kits and reagents.…”

High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing

“…There is also a risk of cross-contamination among the samples within a sequencing run. The analytical sensitivity will depend on the contamination threshold fixed for the run ( Massart et al., 2022 ). Indeed, unexpected viral background amplifications were observed but in irrelevant number of reads that, in general, fell below our threshold of 500 and 100 reads to consider a sample as a true positive for a specific virus or viroid on NextSeq and MiSeq runs, respectively ( Figure 1 and Figure 3 ).…”

Long term anthropic management and associated loss of plant diversity deeply impact virome richness and composition ofPoaceaecommunities

“…To control proper extraction of RNA and DNA viruses (and to deal with cross-samples contaminations, a series of blank (water) and alien controls were used in each batch of sequenced samples. An alien control, which have been recommended in recent guidelines (51), corresponds to a plant sample infected by an virus (called alien virus) that cannot infect the studied plant samples. For this study, potato leaf roll virus (genus Polerovirus, RNA virus ) and banana streak virus (genus Badnavirus , DNA virus) were selected.…”