Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species

摘要

Despite considerable advancements in the characterization of genetic sequence data afforded by next-generation sequencing (NGS) technologies, significant limitations in applying them to the veterinary diagnostic setting remain. NGS can fail to detect low-copy-number pathogens in complex biological samples due to low pathogen/host nucleic acid ratios. For example, if 10% of mammalian cells (approximately 3 × 109 bp per cell) in a blood sample are infected with a pathogen with a 10-kb genome, only 0.000033% of sequences in an NGS output file will be of pathogen origin. Additionally, pathogens isolated from clinical samples often have poor or only partially characterized genomes, can exist as genetically diverse populations within a single individual, or may be present in low abundance due to latency or stage of infection. This combination of small genome size, low relative copy number, and heterogeneous genomic content complicates pathogen detection from complex samples in the context of NGS diagnostic capabilities. For these reasons, virus isolation or culture methods for bacteria and fungi are often required prior to NGS to increase the proportion of on-target reads from clinical samples. These limitations prevent the adoption of NGS as a standard workflow in veterinary diagnostic laboratories.