Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens

Abstract

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases. IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.

Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

“…Another genome-based approach to resolve species complexes in prokaryotes is Percentage of Conserved Proteins (POCP) analysis (Qin et al 2014;Martinez-Romero and Ormeño-Orrillo 2019;Peix et al 2019;Wittouck et al 2019;Rensink et al 2020), a method that has now also been implemented in fungi (Wibberg et al 2020). These strategies are still impractical for broad exploration of fungal diversity, as the accurate analysis of fungal genomes is a time-consuming process and sampling remains sparse, although high quality genomes requiring fewer analytical resources may soon become available with improved third generation sequencing techniques, such as PacBio Sequel and Oxford Nanopore Technologies (Tedersoo et al 2018b;Loit et al 2019;Stadler et al 2020;Wibberg et al 2020). For difficult species complexes, sequencing of restriction siteassociated DNA markers (RADSeq) is another emerging approach in fungal taxonomy (Grewe et al 2017(Grewe et al , 2018Salas-Lizana and Oono 2018).…”

Perspectives from Ten Years of Protist Studies by High‐Throughput Metabarcoding

“…portability and real‐time sequencing) that may make it an alternative in the near future, but due to its high error rate it is currently not best‐suited to assess the diversity of complex microbial samples (Loit et al. ).…”

Comparative sequencing data analysis of Ion Torrent and MinION sequencing platforms using a clinical diagnostic haematology panel

“…Currently, as per the available scientific data, MinION has an excellent advantage in metagenomics workflow not only due to its portability but also because of its higher accuracy in identifying dominant pathogenic organisms. In a comparative study of two long read sequencers, PacBio Sequel and ONT MinION by Loit et al regarding identifying plant pathogens, MinION was noted to be more accurate and faster than the former 12 . In another study, MinION was compared with the very popular MiSeq sequencing platform by Illumina Inc by Lindberg et al in sequencing of single and mixed source human mitochondrial genome 13 .…”