Diagnostic detection of aquatic pathogens using real-time next generation sequencing
CSIRO Australian Animal Health Laboratory
Current diagnostic programs generally rely on highly -specific assays for pathogen detection. While these techniques are invaluable, they are one dimensional and do not provide detailed information critical to a disease investigation. These gaps include the inability to detect unknown pathogens and potential variants of know pathogens and provide no additional genomic or transcriptomic data. Moreover, samples must be shipped to trained personnel in a laboratory, further delaying the time to diagnosis. The MinION, on the other hand, can theoretically detect any pathogen and can potentially be deployed to the field. Moreover, the MinION can rapidly generate full-length genomes, allowing for epidemiological tracking of viral or bacterial strains in near real-time. Such rapid data, which cannot be obtained as quickly using existing methods, are vital if the intention is to intervene in an outbreak and reduce impacts on the productivity and profitability of aquaculture facilities. For example, a rapid, early diagnosis may allow mitigating actions to be taken on-farm, such as the diversion of intake water, movement restrictions of stock and the isolation of infected ponds. These qualities make the MinION an attractive complimentary platform to fill several gaps in the data obtained during disease outbreak investigations, or routine diagnostics, and potentially for use in the field. However, results from the misuse or lack of understanding of the technology could also have adverse regulatory implications for aquaculture industries. For example, without appropriate guidelines, an inexperienced diagnostician may misinterpret a distant DNA match in a pathogen database as a significant result, this may create unwanted attention to industry and potential stock destruction or changes to disease status that are unjustified. Thus, it is critical that the MinION is evaluated at the Australian Animal Health Laboratory, and guidelines and procedures are developed for accurate diagnostic evaluations. The activities detailed in this application will establish the feasibility of using the MinION for diagnostic applications, and ensure that the data is reliably generated and interpreted appropriately.
1. Evaluate if MinION data meets or exceeds the data obtained using established laboratory-based NGS platforms. Objectives (1) and (2) align with Methods section (1).The first objective of this project is to demonstrate if the MinION can obtain quality genome assemblies of known pathogens, such as WSSV, AHPND, OsHV-1 and HaHV that have been created using existing NGS technology. Moreover, determine if the MinION is capable of producing a diagnostic result more rapidly and with greater confidence than traditional techniques. STOP/GO POINT: If MinION data does not produce reliable genome assemblies, no improvement in genome quality, or is significantly more laborious to set-up/run or analyse than existing NGS technologies, do not proceed with objective 2.
2. Evaluate the performance of the MinION using existing diagnostic extraction techniques and produce robust methods and protocols for sample preparation, sequencing and data analysis. This objective will optimise MinION protocols for sample pre-processing, optimal sequencing conditions, and data post-processing. We will then evaluate the MinION data produced from a range of aquatic organisms against data produced using traditional techniques from the same samples. STOP/GO POINT: If after these optimisations, the MinION cannot detect pathogens as reliably as traditional techniques, do not proceed with objective 3.
3. Compare the applicability of MinION to standard molecular assays for identification of pathogens in diagnostic samples. Objective (3) is aligned with Methods section (2).In this objective, diagnostic samples will be tested using existing diagnostics tools (qPCR, cPCR) and MinION sequencing. Analysis between the methods will be detailed, including time to result, pathogen identity and genomic information. This objective will not only provide an insight into real-time sequencing for diagnostics, but in addition the feasibility of MinION technology for field application in the future.