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Seven laboratories evaluated short-read high-throughput sequencing (HTS) for sensitivity and breadth of adventitious virus detection using viruses with distinct physicochemical and genome properties. These five viruses are currently designated as CBER NGS Virus Reagents and include: Epstein-Barr virus (EBV; or human herpes virus 4), feline leukemia virus (FeLV), respiratory syncytial virus (RSV), mammalian orthoreovirus type 1 (Reo1), and porcine circovirus type 1 (PCV1). To evaluate adventitious virus detection in a biological material with a high production virus titer and low cellular background, the 5 viruses were mixed and different copies of the viral genomes spiked into 1 \u2013 5 \u00d7 10<jats:sup>9<\/jats:sup> genome copies per mL (GC\/mL) of purified adenovirus 5. Independent protocols were used by each laboratory for the entire HTS workflow. All laboratories detected 10<jats:sup>4<\/jats:sup> GC\/mL of the five viruses by both targeted and non-targeted bioinformatic analyses. Additionally, the limit of detection of squirrel monkey retrovirus and porcine endogenous retrovirus, which pre-existed in EBV and PCV1 virus stocks, respectively, was evaluated. The five laboratories that tested 10<jats:sup>3<\/jats:sup> GC\/mL, detected all 5 viruses with the targeted analysis, and Reo1 and EBV with the non-targeted analysis. It was noted that some laboratories achieved a better sensitivity for detection of the five viruses (\u2009\u226410<jats:sup>2<\/jats:sup>\u2009GC\/mL). This study presents an approach for HTS validation for viral safety testing of vaccines and other biologics using a panel of reference viruses. 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