A nanopore-based next-generation sequencing workflow for comprehensive adventitious virus testing

Abstract Traditional adventitious virus testing of biological drugs has missed the presence of viral contaminants in the past. Next-generation sequencing (NGS) has emerged as a promising additional tool with unprecedented capability for breadth of detection. This study introduces a Nanopore-based NGS workflow for detection of all classes of replicating viruses in banked cells employed in medicinal biotechnology (e.g., production cell lines, cell lines employed for adventitious agent testing [AAT], allogeneic cell therapies). Starting with isolation of total cellular RNA, cDNA library preparation was designed to detect also viruses which do not poly-adenylate their transcripts while retaining strand-specific information. The newly generated bioinformatic analysis pipeline provides versatility with respect to the use of databases for host sequence depletion (publicly available, customized) and convenience features (direct comparison of sample to run-specific negative control, implemented visualization of sample read alignment to virus database entries). Via deliberate infection of typical biotechnological cell lines, the workflow was found to specifically identify diverse classes of viruses. Preliminary results are available within 26 h, a feature particularly useful for emergency situations requiring fast decision-making, e.g., to investigate potentially positive signals in traditional AAT.