Enriched Long-Read Sequencing of Co-circulating Viruses in Complex Samples

At present, no single workflow is available for quick and accurate identification and analysis of genomes of various viruses present together in a field or clinical sample, particularly when followed by long-read sequencing. Our work addressed this limitation by combining targeted enrichment with long-read, real-time sequencing. Using a panel of probes targeting 16,069 complete viral genomes, we validated this workflow (termed TELSVirus) on complex sample matrices collected from pigs and compared its performance to traditional methods including real-time reverse transcription polymerase chain reaction and shotgun metagenomics. Using serial dilutions of samples with known viral status, we observed that TELSVirus generated viral reads for dilutions up to 10⁻⁹. TELSVirus was able to detect viral targets when shotgun metagenomic long- and short-read datasets did not and when rRT-PCR results were undetermined. Finally, we performed TELSVirus on 144 oral fluid samples collected in the field, which are highly complex and diverse samples used for viral surveillance in swine. We identified a high prevalence of relatively understudied viruses, often found co-circulating with better-characterized viruses. In many cases, TELSVirus generated ultra-deep genome coverage, allowing for further genomic epidemiological investigations, although bioinformatic methods need further development to work robustly with TELSVirus data. Our results support using TELSVirus for rapid detection and genomic characterization of multiple low-abundance viruses from single samples using long-read sequencing.