Dataset Information

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.

SUBMITTER: Loit K

PROVIDER: S-EPMC6803294 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Loit Kaire K Adamson Kalev K Bahram Mohammad M Puusepp Rasmus R Anslan Sten S Kiiker Riinu R Drenkhan Rein R Tedersoo Leho L

Applied and environmental microbiology 20191016 21

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 smal ...[more]

PMID: 31444199

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Project description:BackgroundSequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.ResultsHere, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies' MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.ConclusionsOur pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

Project description:Illumina MiSeq is the current standard for characterizing microbial communities in soil. The newer alternative, Oxford Nanopore Technologies MinION sequencer, is quickly gaining popularity because of the low initial cost and longer sequence reads. However, the accuracy of MinION, per base, is much lower than MiSeq (95% versus 99.9%). The effects of this difference in base-calling accuracy on taxonomic and diversity estimates remains unclear. We compared the effects of platform, primers, and bioinformatics on mock community and agricultural soil samples using short MiSeq, and short and full-length MinION 16S rRNA amplicon sequencing. For all three methods, we found that taxonomic assignments of the mock community at both the genus and species level matched expectations with minimal deviation (genus: 80.9-90.5%; species: 70.9-85.2% Bray-Curtis similarity); however, the short MiSeq with error correction (DADA2) resulted in the correct estimate of mock community species richness and much lower alpha diversity for soils. Several filtering strategies were tested to improve these estimates with varying results. The sequencing platform also had a significant influence on the relative abundances of taxa with MiSeq resulting in significantly higher abundances Actinobacteria, Chloroflexi, and Gemmatimonadetes and lower abundances of Acidobacteria, Bacteroides, Firmicutes, Proteobacteria, and Verrucomicrobia compared to the MinION platform. When comparing agricultural soils from two different sites (Fort Collins, CO and Pendleton, OR), methods varied in the taxa identified as significantly different between sites. At all taxonomic levels, the full-length MinION method had the highest similarity to the short MiSeq method with DADA2 correction with 73.2%, 69.3%, 74.1%, 79.3%, 79.4%, and 82.28% of the taxa at the phyla, class, order, family, genus, and species levels, respectively, showing similar patterns in differences between the sites. In summary, although both platforms appear suitable for 16S rRNA microbial community composition, biases for different taxa may make the comparison between studies problematic; and even with a single study (i.e., comparing sites or treatments), the sequencing platform can influence the differentially abundant taxa identified.

Dataset Information

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

Publications

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

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