Abstract
Foodborne diseases are major public health problems globally. Metagenomics has emerged as a widely used tool for pathogen screening. In this study, we conducted an updated Tn5 transposase-assisted RNA/DNA hybrid co-tagmentation (TRACE) library construction approach. To address the detection of prevalent known foodborne viruses and the discovery of unknown pathogens, we employed both specific primers and oligo-T primers during reverse transcription. The method was validated using clinical samples confirmed by RT-qPCR and compared with standard RNA-seq library construction methods. The mapping-based approach enabled the retrieval of nearly complete genomes (>95%) for the majority of virus genome segments (86 out of 88, 97.73%), with a mean coverage depth of 21,494.53× (ranging from 77.94× to 55,688.58×). Co-infection phenomena involving prevalent genotypes of Norovirus with Astrovirus and Human betaherpesvirus 6B were observed in two samples. The updated TRACE-seq exhibited superior performance in viral reads percentages compared to standard RNA-seq library preparation methods. This updated method has expanded its target pathogens beyond solely Norovirus to include other prevalent foodborne viruses. The feasibility and potential effectiveness of this approach were then evaluated as an alternative method for surveilling foodborne viruses, thus paving the way for further exploration into whole-genome sequencing of viruses.