Optimized approaches to profile low biomass DNA and RNA virome in the upper respiratory tract of beef cattle

Abstract Background Viruses play a key role in cattle respiratory tract (RT) health, yet, profiling the upper respiratory tract (URT) virome (DNA and RNA viruses) from deep nasopharyngeal swabs (DNS) remains challenging due to their low biomass, lack of well-defined enrichment approaches, and a high proportion of unclassified viral sequences. This study aimed to optimize DNS preservation, nucleic acid extraction, and bioinformatics pipelines to enhance URT virome characterization in beef calves. Results Three DNS preservation methods were tested (RNAlater, brain heart infusion (BHI) medium/glycerol at -80 °C, and BHI medium/glycerol followed by RNAlater). Three nucleic acid extraction kits (QIAamp Fast DNA Stool Mini Kit, GeneJET Genomic DNA Purification Kit, and mirVana™ miRNA Isolation Kit) were evaluated to compare total nucleic acid yields. Viral DNA and RNA were extracted separately using the NucleoSpin Virus Kit, which was optimized for viral nucleic acid recovery. The DNS samples that preserved in RNAlater and extracted using the NucleoSpin Virus kit generated highest viral DNA (1.35 ± 0.12 µg) and RNA (1.57 ± 0.09 µg) (mean ± SEM) yields for metagenomics sequencing. Metagenomic sequencing and viral taxonomic assignment using Kraken2 and Kaiju, respectively showed that Kaiju allowed maximum exact matches for viral taxonomy assignment and provided high-resolution outputs for the profiling of cattle URT viromes. Conclusion The use of an optimized nucleic acid extraction method and Kaiju-based classification enhanced viral nucleic acid recovery and taxonomic classification from low-biomass DNS samples. The findings highlighted the importance of effective storage conditions and extraction methods in maximizing viral nucleic acid yield, while demonstrating that Kaiju provided more reliable viral taxonomic assignments than Kraken2. These advancements provide a framework for more accurate assessment of the URT virome in cattle and other farm animals, enabling deeper investigation into the roles of viruses in RT health.