Abstract
Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic platform that accurately detects both known and unknown virus species, as well as unreported variants, is playing a pivotal role across plant germplasm quarantine programs. Here we propose the addition of high-throughput sequencing (HTS) from total RNA to the routine quarantine diagnostic workflow of sugarcane viruses. We evaluated the impact of sequencing depth needed for the HTS-based identification of seven regulated sugarcane RNA/DNA viruses across two different growing seasons (spring and fall). Our HTS analysis revealed that viral normalized read counts (RPKM) was up to 23-times higher in spring than in the fall season for six out of the seven viruses. Random read subsampling analyses suggested that the minimum number of reads required for reliable detection of RNA viruses was 0.5 million, with a viral genome coverage of at least 92%. Using an HTS-based total RNA metagenomics approach, we identified all targeted viruses independent of the time of the year, highlighting that higher sequencing depth is needed for the identification of DNA viruses.
Highlights
We evaluated the use of high-throughput sequencing (HTS) as a diagnostic tool for the virus detection of seven sugarcane viruses of regulatory relevance for the U.S
In order to correct for differences associated with sequencing depth and the length of viral genome, normalized read counts were calculated as reads per kilobase of the reference sequence per million reads (RPKM) values by taking into consideration (a) the length of the viral reference genome, (b) the number of reads mapped to each genome, and (c) the total number of reads per sample [43]
Total number of reads obtained per sample; Abbreviations: sugarcane yellow leaf virus-Brazil (SCYLV-BRA); sugarcane mosaic virus (SCMV); sugarcane yellow leaf virus-China (SCYLV-CHN); sugarcane striate mosaic associated virus (SCSMaV); fiji disease virus (FDV); sugarcane streak Egypt virus (SCSEV); sugarcane white streak virus (SCWSV); sugarcane streak mosaic virus (SCSMV); 3 Total number of unique reads mapped to viral reference sequences; 4 RPKM: Reads per kilobase of transcript per million mapped reads; 5 Percentage of viral reads from total number of reads obtained per sample; 6 Percentage identity between de novo-assembled viral genome and the best-hit viral genome
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Current diagnostic workflows include conventional indexing using indicator plants, serological tests (ELISAs), PCR-based techniques, and electron microscopy These techniques are well established and are utilized by plant quarantine and disease diagnostic programs throughout the world. For many years, these assays have been successfully implemented and validated to identify an array of well-characterized quarantine pathogens within the context of quarantine standards because of their specificity to target pathogens and the ability to detect systemic pathogens. We applied similar parameters under which conventional diagnostics tests, such as PCR-based tests, are validated in the PGQP This validation included comparing the seasonal sampling effect (spring and fall) while adding the sequencing depth needed for the detection on seven sugarcane viruses. A better understanding of the seasonal virus accumulation pattern and the incorporation of HTS analysis into existing biological indexing methods will improve the current quarantine framework for improved pathogen detection
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