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Copy number heterogeneity of JC virus standards.

Copy number heterogeneity of JC virus standards.
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Greninger AL, Bateman AC, Atienza EE, Wendt S, Makhsous N, Jerome KR, Cook L,


Greninger AL, Bateman AC, Atienza EE, Wendt S, Makhsous N, Jerome KR, Cook L, (click to view)

Greninger AL, Bateman AC, Atienza EE, Wendt S, Makhsous N, Jerome KR, Cook L,

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Journal of clinical microbiology 2016 12 14() pii

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 gain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and 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 SV40 T-antigen, suggesting the possibility of trans-complementation in cell culture. Using a cut-off of 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 high copy-number of JCV or a plasmid control. These results were also confirmed by qPCR, ddPCR, and Sanger sequencing of multiple rearrangements. 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.

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