Abstract
High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.
Highlights
Plant quarantine agencies worldwide have shown interest in adopting High-throughput sequencing (HTS) as a diagnostic tool [1,2,3]
When plant RNA viruses are in low concentration, their detection becomes challenging with any molecular technique, including HTS
Primers needed for TArget-SPecific Reverse Transcript (TASPERT) can be developed for any pathogen, but most importantly for RNA viruses, which do not have a known generalized barcode to allow enrichment
Summary
Plant quarantine agencies worldwide have shown interest in adopting HTS as a diagnostic tool [1,2,3]. The advent of new HTS platforms such as MinION from Oxford Nanopore Technologies (ONT) has driven down the cost of sequencing, contributing to portability and the capacity to stop sequencing when the diagnostic target is found [4]. These are valued characteristics that make MinION an attractive choice for diagnostics. MinION has been used to detect multiple human and animal viruses worldwide [5,6], including a proof of concept for plant virus diagnostics [7] Competing technologies such as Illumina offer short reads with decreased error rates, yet the equipment is costly and lacks the portability feature. When plant RNA viruses are in low concentration, their detection becomes challenging with any molecular technique, including HTS
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