Seeing the invisible: How RNA extraction and library prep shape viral read recovery in RNA-seq
- 1Tumor Vaccine and Biotechnology Branch, Division of Cellular Therapy 2, Office of Cellular Therapy and Human Tissue, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
- 2Division of Bioinformatics and Biostatistics, Office of Research, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
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 and data analysis. In this study, we systematically evaluated five RNA extraction methods and seven RNA-seq library preparation protocols across multiple sample matrices spiked with model viruses. Our results demonstrate variability in viral RNA recovery depending on extraction chemistry, 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 construction combined with dual indexing improved viral signal detection and minimized artifacts such as index hopping. Finally, we implemented a custom bioinformatics pipeline to enhance sensitivity and minimize host-background interference. These findings highlight critical analytical variables that influence NGS assay performance and provide practical guidance for optimizing workflow components to support the use of NGS for adventitious agent detection in cell and gene therapy product development.
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BibTeX
@article{afroj2026seeing,
title = {{Seeing the invisible: How RNA extraction and library prep shape viral read recovery in RNA-seq}},
author = {Sayma Afroj and Binsheng Gong and Aaron Scholl and Bingjie Li and Joshua Xu and Sandip De},
journal = {Molecular therapy. Advances},
year = {2026},
month = oct,
doi = {10.1016/j.omta.2026.201858},
url = {https://europepmc.org/article/PMC/PMC13718637},
note = {CC BY},
}
RIS
TY - JOUR
TI - Seeing the invisible: How RNA extraction and library prep shape viral read recovery in RNA-seq
AU - Sayma Afroj
AU - Binsheng Gong
AU - Aaron Scholl
AU - Bingjie Li
AU - Joshua Xu
AU - Sandip De
PY - 2026
DA - 2026/10/06
JO - Molecular therapy. Advances
DO - 10.1016/j.omta.2026.201858
UR - https://europepmc.org/article/PMC/PMC13718637
AB - 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 and data analysis. In this study, we systematically evaluated five RNA extraction methods and seven RNA-seq library preparation protocols across multiple sample matrices spiked with model viruses. Our results demonstrate variability in viral RNA recovery depending on extraction chemistry, 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 construction combined with dual indexing improved viral signal detection and minimized artifacts such as index hopping. Finally, we implemented a custom bioinformatics pipeline to enhance sensitivity and minimize host-background interference. These findings highlight critical analytical variables that influence NGS assay performance and provide practical guidance for optimizing workflow components to support the use of NGS for adventitious agent detection in cell and gene therapy product development.
SN - 3117-387X
ER -