title: Copy Number Heterogeneity of JC Virus Standards.
abstract: Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to obtain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and the Exact v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T antigen region. Intriguingly, several of the JCV standards sequenced in this study with large T antigen deletions were cultured in cell lines immortalized using simian virus 40 (SV40) T antigen, suggesting the possibility of transcomplementation in cell culture. Using a cutoff 5% allele fraction for junctional reads, 7 different rearrangements were present in the JC virus sequences present in the WHO standard across multiple library preparations and sequencing runs. Neither the copy number differences nor the rearrangements were observed in a clinical sample with a high copy number of JCV or a plasmid control. These results were also confirmed by the quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR), and Sanger sequencing of multiple rearrangements. In summary, targeting different regions of the same international standard can result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA agrening@uw.edu.
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Greninger
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Allen C
authors.initials: AC
authors.lastname: Bateman
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Ederlyn E
authors.initials: EE
authors.lastname: Atienza
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Sharon
authors.initials: S
authors.lastname: Wendt
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Negar
authors.initials: N
authors.lastname: Makhsous
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.|Fred Hutchinson Cancer Research Institute, Seattle, Washington, USA.
authors.forename: Keith R
authors.initials: KR
authors.lastname: Jerome
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Cook
mesh_terms: D018628:Gene Dosage; D006801:Humans; D007577:JC Virus / Q000302:isolation & purification*; D027601:Polyomavirus Infections / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D017422:Sequence Analysis, DNA; D014412:Tumor Virus Infections / Q000821:virology*; D019562:Viral Load / Q000379:methods / Q000592:standards*
keywords: BKV; JC virus; T antigen; clinical standards; deep sequencing; polyomavirus; qPCR; simian virus 40