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Background Next-generation sequencing (NGS) has become an essential tool in the development and quality control of cell therapy products for cancer treatment. As advanced therapies expand into clinical oncology, there is growing regulatory and scientific emphasis on molecular approaches to ensure product identity and viral safety. NGS enables high-resolution genomic analysis, supporting rigorous assessment of plasmid and viral vector (VV) sequence fidelity and detection of adventitious viruses (AVD). Recent updates to international guidance, including ICH Q5A(R2), reflect FDA’s push to reduce animal-based biosafety testing in favor of molecular methods. 1 Meanwhile, industry interest in NGS has grown substantially, driven by its sensitivity, throughput, and support for complex characterization. This work introduces NGS-based methods aimed at improving the quality control and viral safety of oncology cell therapy products (figure 1).Methods Plasmid identity testing was performed using spike-in mutant plasmids mixed with wild-type controls at predefined ratios. Variant frequency accuracy and sensitivity were assessed post DNA-seq. For VV identity testing, RNA extracted from viral supernatant was converted to cDNA and sequenced. Reads were aligned to a reference vector genome to evaluate sequence fidelity and mapping coverage. AVD detection was conducted by spiking FDA-recommended DNA and RNA viruses into human cell lines, followed by a single DNA/RNA-seq run to assess detection breadth ( figure 2).Results The plasmid assay reliably detected known sequence variants with a sensitivity as low as 1%, demonstrating precise detection of low-frequency events. The VV identity workflow achieved high mapping efficiency, with up to 60% of sequencing reads aligning to the reference vector genome, significantly improving genome coverage and sequence resolution. For AVD, both DNA and RNA viruses were successfully identified in a single sequencing run, confirming the workflow’s capacity for broad and simultaneous detection of diverse viral classes.Conclusions We developed and validated fit-for-purpose NGS workflows that support precise molecular characterization and biosafety assessment of cell therapy products. These methods demonstrate strong sensitivity, broad detection capability, and alignment with evolving regulatory expectations. Beyond technical merit, integrating NGS into quality frameworks is critical to enabling the safe, consistent manufacturing of oncology cell therapies. As these therapies advance toward wider clinical use, reliable molecular tools like NGS are essential to ensure both scientific rigor and patient safety. Industry adoption is accelerating, underscoring NGS’s growing role in next-generation cancer treatment development.Reference ICH Q5A(R2): Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. International Council for Harmonisation, 2023.Abstract 155 Figure 1NGS applications supporting identity and biosafety testing in cell therapy workflowsAbstract 155 Figure 2NGS workflow