RT Journal Article SR Electronic T1 Standard Issue: Copy number heterogeneity of JC virus standards discovered through next-generation sequencing JF bioRxiv FD Cold Spring Harbor Laboratory SP 085415 DO 10.1101/085415 A1 Alexander L. Greninger A1 Allen C. Bateman A1 Ederlyn E. Atienza A1 Sharon Wendt A1 Keith R. Jerome A1 Linda Cook YR 2016 UL http://biorxiv.org/content/early/2016/11/03/085415.abstract AB 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 gain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and ExactTM 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. No such variation was seen for a clinical sample with high copy-number of JCV nor a plasmid control. Intriguingly, several of the JCV standards sequenced in this study with large T-antigen deletions were cultured in cell lines immortalized using SV40 T-antigen, suggesting the possibility of trans-complementation in cell culture. Using a cut-off of 2% variant allele fraction for junctional reads to define the presence of a strain, 11 different strains were present in the WHO standard. In summary, targeting of different regions of the same international standard could result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology.