Targeted metagenomic sequencing with spiked primers for enrichment of viruses in wastewater for pathogen surveillance

ABSTRACT Background Wastewater surveillance offers an underutilized opportunity to identify high-risk viral pathogens that pose public health risks. Although metagenomic approaches have been increasingly adopted for human wastewater surveillance, little attention has been given to its application in rural agricultural settings. Untargeted metagenomic sequencing of wastewater poses considerable technical challenges for viral detection due to fragmented genomes and low viral abundance. While existing enrichment methods partially address these challenges, high costs and proprietary protocols limit adoption in resource-constrained settings. Objective We developed a fully open-source primer design algorithm ( Open MSSPE Design ) and evaluated Metagenomic Sequencing with Spiked Primer Enrichment (MSSPE) 1 , as a practical strategy for metagenomic surveillance of swine slurry and farm wastewater samples collected from rural agricultural settings. This non-invasive MSSPE strategy addresses the challenge of detecting extremely low-abundance viral targets amid samples dominated by a background of bacterial and eukaryotic nucleic acids. Methodology We generated fifteen primer sets targeting high-priority DNA and RNA viral pathogens affecting human and animal health in Southeast Asia using our open-source primer design algorithm. Twenty-five wastewater and swine slurry samples from smallholder farms in northern Thailand underwent parallel library preparation—untargeted mNGS and MSSPE—for direct comparison. Libraries were sequenced on Illumina platforms and analyzed using Chan Zuckerberg ID (CZ ID) 2 . Rarefaction analysis assessed performance at sequencing depths of 100,000–1.5 million reads per sample. Results We detected multiple high-risk DNA and RNA viruses in wastewater samples from smallholder farm operations. MSSPE achieved substantial viral enrichment across nine pathogenic DNA and RNA viruses, with a median two-fold enrichment of reads per million, with variability across targets (IQR: 1.01–3.44x) and a nearly 10% median increase in breadth of genome coverage (IQR: 4.54–11.84%), while retaining sensitivity for untargeted pathogens. MSSPE also increased the odds of detecting targeted viruses (OR 1.35, CI 1.14–1.60), with the greatest advantage at shallow sequencing depths where MSSPE required fewer reads to identify targeted viral taxa relative to mNGS. Conclusions MSSPE demonstrated the ability to enrich shallow-depth sequencing (<2M reads per sample) sufficiently to detect priority viruses without substantially increasing library preparation time or cost. This open-source workflow supports cost-effective metagenomic viral surveillance for resource-constrained settings, providing a non-invasive method for detecting low-abundance viral targets in high-background sample types at rural agricultural interfaces with elevated risk of zoonotic spillover.