2nd Edition of Cell & Gene Therapy World Conference 2026

Abstract

Next-generation sequencing (NGS) is emerging as a powerful tool for detecting adventitious viral agents in biologics, aligning with recommendations from ICH Q5A(R2). However, the performance of RNA-seq based detection assays is highly dependent on upstream sample preparation steps. In this study, we systematically evaluated five RNA extraction methods and seven RNA-seq library preparation protocols using multiple sample matrices such as U937, HEK293, and primary human leukocyte with model virus panels (ATCC MSA 2008, BEI NR-59622). Total RNA was extracted, and libraries were constructed using different commercially available kits or a combination of kits. Libraries were sequenced on Illumina NovaSeq 6000, and metagenomic analysis was performed to assess viral read counts. Our results demonstrate significant variability in viral RNA recovery depending on extraction chemistry, and virus and sample matrix type. Tri Reagent-based methods excelled for certain viruses, while magnetic bead-based extraction yielded the highest total RNA in most matrices tested. Among RNA-seq library prep methods, rRNA depletion post library preparation combined with dual indexing improved viral signal detection and minimized artifacts such as index hopping. Finally, to improve the accuracy of viral detection and minimize false positives, we applied a customized bioinformatics pipeline designed to prefilter host-derived reads before viral mapping. This approach was essential, as initial analyses without host read removal revealed a hepatitis E virus (HEV) signal in unspiked control samples. Spiked viruses- Reo, FeLV, RSV, HEV and ZIKV were detected from the RNAseq data, though we observed differences in read numbers aligning to different viral genomes with the tested library preparation methods. Low-level viral signals detected in control samples using untargeted bioinformatics pipelines further emphasize the need for defined detection thresholds and confirmatory strategies. Our findings highlight critical analytical variables that influence NGS assay performance and provide practical guidance for optimizing workflow components to support regulatory adoption of NGS for adventitious agent detection in cell and gene therapy products.