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

Area of Science:

• Virology
• Genomics
• Bioinformatics

Background:

• Current workflows lack efficiency for simultaneous identification and analysis of diverse viral genomes in complex samples, especially with long-read sequencing.
• Accurate viral identification is crucial for diagnostics, surveillance, and understanding viral ecosystems.

Purpose of the Study:

• To develop and validate a novel workflow, TELSVirus, for rapid, accurate, and comprehensive identification and genomic analysis of multiple viruses in single samples.
• To assess the performance of TELSVirus compared to traditional methods like real-time reverse transcription polymerase chain reaction (rRT-PCR) and shotgun metagenomics.

Main Methods:

• Developed TELSVirus by combining targeted enrichment with long-read, real-time sequencing using a probe panel for 16,069 viral genomes.
• Validated the workflow on complex swine sample matrices, including serial dilutions, and compared results with rRT-PCR and shotgun metagenomics.
• Applied TELSVirus to 144 field-collected swine oral fluid samples for viral surveillance.

Main Results:

• TELSVirus successfully generated viral reads for dilutions up to 10⁻⁹, demonstrating high sensitivity.
• The workflow detected viral targets missed by shotgun metagenomics and when rRT-PCR results were undetermined.
• Analysis of swine oral fluids revealed high prevalence of understudied co-circulating viruses, with TELSVirus providing ultra-deep genome coverage.

Conclusions:

• TELSVirus offers a significant advancement for rapid detection and genomic characterization of multiple low-abundance viruses from single samples using long-read sequencing.
• The workflow shows promise for viral surveillance and epidemiological investigations, though bioinformatic analysis methods require further development.
• TELSVirus enhances the ability to study complex viral communities and discover novel or understudied viruses.