Simple Summary Metagenomic sequencing, as an emerging detection technology, has the capability to non-specifically detect various bacteria and viruses. However, a series of issues have limited its application in animal clinical diagnostics. In response to these challenges, this study focused on clinical samples from cats and pigs and optimized sequencing and analysis methods. The results demonstrated that the optimized nanopore metagenomic sequencing method could effectively detect bacteria and viruses in the samples while reducing interference from host genes. When combined with real-time cloud-based and local analysis, it enabled the quick determination of when sequencing should be terminated and facilitated the assembly of relatively complete viral genomes. This method is characterized by its low equipment requirements, short sequencing time, and cost-effectiveness, making it suitable for deployment in small- to medium-sized animal clinical testing laboratories. Abstract Metagenomic sequencing is a valuable tool for non-specifically detecting various microorganisms in samples, offering unique advantages for detecting emerging pathogens, fastidious or uncultivable pathogens, and mixed infections. It has recently been applied to clinically detect pathogenic microorganisms in animals; however, the high proportion of host genes, expensive sequencing equipment, and the complexity of sequencing and data analysis methods have limited its clinical utility. In this study, a combination of tissue homogenization and nuclease digestion was employed to remove host genes from pig and cat samples; DNA and RNA were then extracted and subjected to nonselective PCR amplification to simultaneously detect DNA and RNA pathogen genomes using R9.4.1 or R10.4.1 flow cells on the MinION platform. Real-time pathogen detection was conducted using EPI2M WIMP, and viral genome assembly was performed using NanoFilt, minimap2, samtools, and ivar. Pathogens in five clinical samples (serum, nasopharyngeal swab, feces, or ascites) from cats and four clinical samples (lung or small intestine tissue) from pigs were examined by metagenomic sequencing, and the results were consistent with those obtained by PCR and bacterial culture. Additionally, we detected four viruses and three bacteria that may be associated with diseases. A comparison of results before and after host gene removal in three samples showed a 9–50% reduction in host genes. We also compared the assembly efficiency of six virus genomes and found that data volumes ranging from 3.3 to 98.3 MB were sufficient to assemble >90% of the viral genomes. In summary, this study utilized optimized nanopore metagenomic sequencing and analysis methods to reduce host genes, decrease the required data volume for sequencing analysis, and enable real-time detection to determine when to stop sequencing. The streamlined sequencing and analysis process overcomes barriers to the veterinary clinical application of metagenomic sequencing and provides a reference for clinical implementation.
【저자키워드】 nanopore sequencing, metagenomic sequencing, host gene removal, veterinary pathogenic microorganisms,