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Standard Issue: Copy number heterogeneity of JC virus standards discovered through next-generation sequencing

View ORCID ProfileAlexander L. Greninger, Allen C. Bateman, Ederlyn E. Atienza, Sharon Wendt, Keith R. Jerome, Linda Cook
doi: https://doi.org/10.1101/085415
Alexander L. Greninger
1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Allen C. Bateman
1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Ederlyn E. Atienza
1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Sharon Wendt
1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Keith R. Jerome
1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
2Fred Hutchinson Cancer Research Institute, Seattle, WA, USA
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Linda Cook
1Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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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 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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted November 03, 2016.
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Standard Issue: Copy number heterogeneity of JC virus standards discovered through next-generation sequencing
Alexander L. Greninger, Allen C. Bateman, Ederlyn E. Atienza, Sharon Wendt, Keith R. Jerome, Linda Cook
bioRxiv 085415; doi: https://doi.org/10.1101/085415
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Standard Issue: Copy number heterogeneity of JC virus standards discovered through next-generation sequencing
Alexander L. Greninger, Allen C. Bateman, Ederlyn E. Atienza, Sharon Wendt, Keith R. Jerome, Linda Cook
bioRxiv 085415; doi: https://doi.org/10.1101/085415

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