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The development of tools for real-time detection of antimicrobial resistant (AMR) pathogens is a priority topic of the One Health EJP. For real-time analysis to be achievable on-site (away from the laboratory setting), robust culture independent detection methods, employing minimal equipment are required. Metagenomic sequencing using short-read data has provided insight and detailed compositions of a variety of microbial communities, as well as for the detection of potential pathogens and AMR or virulence genes. In addition to the currently bulky nature of short-read technologies and the difficulty to perform this analysis on site, an important limitation is their inability to reliably associate the genetic context of individual genes to bacteria (including pathogens) within a community. The FARMED project aims to address these issues by using the Oxford Nanopore Technologies (ONT) MinION, comparing to the current gold standard short-read technology, to evaluate its capability for diagnostic use on a range of sample matrices, particularly on-site at/near point of sample collection. This is enabled by the portability of the ONT technology, allowing on-site analysis, in contrast to short read sequencing. An additional advantage of using ONT sequencing or long-read metagenomic sequencing, is that the local genetic context of AMR genes can be derived, and as such, the presence of the AMR genes can be attributed to specific species or plasmids, within the bacterial community. This technology will enable the identification of a plethora of bacterial species and linkage of AMR genes to particular species. However, the successful application of on-site microbial detection/monitoring is influenced by various factors such as resource-limited working environment, sample collection and importantly the quality of the input DNA for sequencing. In addition, the availability of on-site sequencing and subsequent data analysis needs to be taken into account. This deliverable will review the scientific literature on existing DNA extraction methods and determine which have the potential/are suitable for rapid on-site metagenomic analysis. For on-site DNA extraction, it is essential that the implemented methods use minimal transportable equipment. Furthermore, the method(s) need to be free of hazardous chemicals for field personnel and the working environment, as well as components that require sub-zero transport conditions. We will also discuss the requirements of DNA for long-read sequencing and suitability of the different methods. We consider methods suitable for on-site DNA extraction from different ‘simple’ and ‘complex’ sample matrices, as each has different considerations. Finally, we will deliver recommendations for the FARMED consortium to be tested using ONT sequencing.

The development of tools for real-time detection of antimicrobial resistant (AMR) pathogens is a priority topic of the One Health EJP. For real-time analysis to be achievable on-site (away from the laboratory setting), robust culture independent detection methods, employing minimal equipment are required. Metagenomic sequencing using short-read data has provided insight and detailed compositions of a variety of microbial communities, as well as for the detection of potential pathogens and AMR or virulence genes. In addition to the currently bulky nature of short-read technologies and the difficulty to perform this analysis on site, an important limitation is their inability to reliably associate the genetic context of individual genes to bacteria (including pathogens) within a community. The FARMED project aims to address these issues by using the Oxford Nanopore Technologies (ONT) MinION, comparing to the current gold standard short-read technology, to evaluate its capability for diagnostic use on a range of sample matrices, particularly on-site at/near point of sample collection. This is enabled by the portability of the ONT technology, allowing on-site analysis, in contrast to short read sequencing. An additional advantage of using ONT sequencing or long-read metagenomic sequencing, is that the local genetic context of AMR genes can be derived, and as such, the presence of the AMR genes can be attributed to specific species or plasmids, within the bacterial community. This technology will enable the identification of a plethora of bacterial species and linkage of AMR genes to particular species. However, the successful application of on-site microbial detection/monitoring is influenced by various factors such as resource-limited working environment, sample collection and importantly the quality of the input DNA for sequencing. In addition, the availability of on-site sequencing and subsequent data analysis needs to be taken into account. This deliverable will review the scientific literature on existing DNA extraction methods and determine which have the potential/are suitable for rapid on-site metagenomic analysis. For on-site DNA extraction, it is essential that the implemented methods use minimal transportable equipment. Furthermore, the method(s) need to be free of hazardous chemicals for field personnel and the working environment, as well as components that require sub-zero transport conditions. We will also discuss the requirements of DNA for long-read sequencing and suitability of the different methods. We consider methods suitable for on-site DNA extraction from different ‘simple’ and ‘complex’ sample matrices, as each has different considerations. Finally, we will deliver recommendations for the FARMED consortium to be tested using ONT sequencing.