Format

Send to

Choose Destination
Appl Environ Microbiol. 2019 Aug 23. pii: AEM.01368-19. doi: 10.1128/AEM.01368-19. [Epub ahead of print]

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

Author information

1
Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Fr.R. Kreutzwaldi, 5, 51006 Tartu, Estonia.
2
Institute of Forestry and Rural Engineering, Estonian University of Life Sciences, Fr.R. Kreutzwaldi, 5, 51006 Tartu, Estonia.
3
Department of Ecology, Swedish University of Agricultural Sciences, Ulls väg 16, 75651 Uppsala, Sweden.
4
Institute of Ecology and Earth Sciences, University of Tartu, 14a Ravila, 50411 Tartu, Estonia.
5
Zoological Institute, Technische Universität Braunschweig, Mendelssohnstrasse 4, 38106 Braunschweig, Germany.
6
Institute of Ecology and Earth Sciences, University of Tartu, 14a Ravila, 50411 Tartu, Estonia leho.tedersoo@ut.ee.
7
Natural History Museum, University of Tartu, 14a Ravila, 50411 Tartu, Estonia.

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 performance of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences) in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae spp.) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to the 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 two and half hours. 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 improves 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.

PMID:
31444199
DOI:
10.1128/AEM.01368-19

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center