Comparison of Nanopore Sequencing Protocols and Real-Time Analysis for Phytopathogen Diagnostics

Abstract

Next-generation sequencing technologies have aided molecular detection and diagnosis of plant pathogens. Short read sequencing technologies can be expensive, time consuming, and lack portability. The Oxford Nanopore Technologies (ONT) MinION sequencer offers plant diagnosticians an alternative for whole-genome and transcriptome sequencing with the advantages of portability, rapid results and access to sequences generated, and relatively low cost. Although this company offers multiple sequencing products, most previous articles focused on comparing this technology with other sequencing technology (Sanger, Illumina) and not comparing ONT product performance. In this project, we explored the potential of using ONT’s kits without modifications for the sequencing of plant pathogens. We compared the performance of DNA library preparation protocols (1D versus 1D2 versus Rapid) and RNA library preparation protocols (cDNA PCR Sequencing versus Direct RNA) on the MinION sequencer and examined different data analysis approaches for two well-characterized plant pathogens of agricultural concern, Ralstonia solanacearum and plum pox virus. For bacterial DNA sequencing, the Rapid Sequencing kit had the lowest throughput but offered the shortest sample-to-sequence turnaround time. The cDNA PCR Sequencing kit was better suited for plant RNA viruses than the Direct RNA. Whole-genome assemblies using all tested kits provided accurate identification of the target pathogen when pure cultures or infected plant material was used. This technology offers a shorter sample-to-sequence timeline than previous technologies for both DNA and RNA samples. Data analysis can still be time- and resource-consuming, but we found that 1 h of data generation was sufficient for accurate identification of the target pathogens, which can alleviate computing resources because a full 48-h run of sequencing data is not required. With the decreasing costs of flow cells, MinION is now an additional tool to be considered by plant diagnosticians for whole-genome and transcriptome-based diagnostics.

Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics

“…However, the results of the latter study showed that the PCR-cDNA sequencing kit could produce longer reads and 400 times more reads than the direct RNA sequencing kit. In addition, the mean quality score and mean read length were higher for the PCR-cDNA sequencing kit compared to the direct RNA sequencing kit [37].…”

“…Nanopore sequencing technology has lower throughput and high error rates compared with second-generation sequencing technologies. However several features, such as the small size of the sequencer, ease of library preparation, low sequencing cost and short run times, make it an interesting tool for the surveillance of viruses and other pathogens [37]. In recent years, ONT has improved the read quality of nanopore sequencing by changing the chemical reagents used in library preparation kits and flow cells and developing improved algorithms for basecalling.…”

“…For instance, a field diagnosis system based on nanopore sequencing has been developed to rapidly (3 hours) detect Cassava mosaic begomoviruses in cassava plants [40]. In another study, a comparison between RNA and PCR-cDNA nanopore sequencing kits for plum pox virus detection in tobacco samples indicated that nanopore technology could effectively identify plant viruses [37]. However, the results of the latter study showed that the PCR-cDNA sequencing kit could produce longer reads and 400 times more reads than the direct RNA sequencing kit.…”

Metagenomic Studies of Viruses in Weeds and Wild Plants: A Powerful Approach to Characterise Variable Virus Communities

“…Due to the small genome size of plant viruses, ranging from 4 to 30 kb [75], long read sequencing can capture entire genome segments in a single read. For plant viruses, long read sequencing has been used on geminiviruses [76], tombusviridae [77], and several potyviruses including Wheat streak mosaic virus [78], Plum pox virus [79] and Yam mild mosaic virus [80]. However, the host plants in these studies were all crop plants, with the exception of the geminiviruses found in Medicago arborea, a shrub in the Fabaceae family [76].…”

Nanopore Metagenomics Sequencing for Rapid Diagnosis and Characterization of Lily Viruses

“…Oxford Nanopore Technologies (ONT) is a compact, portable device for real-time single-molecule DNA and RNA sequencing that directly detects changes in the electric currents, which are generated when DNA and RNA molecules pass through nanopore proteins embedded in the device’s polymer membrane, thereby enabling the detection of biological molecules ( Deamer et al, 2016 ; Eid et al, 2009 ; Phannareth et al, 2020 ). These electrical signals are converted into nucleotide sequences by base calling, which is a computational algorithm ( Theuns et al, 2018 ).…”