Detection of cytomegalovirus drug resistance mutations by next-Generation sequencing

Malaya K. Sahoo, Martina I. Lefterova, Fumiko Yamamoto, Jesse J. Waggoner, Sunwen Chou, Susan P. Holmes, Matthew W. Anderson, Benjamin A. Pinsky

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients.However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54.We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument.Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants.The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance.Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run.This led to the identification of seven resistance mutations, three of which were present in20% of the sequenced population.Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations

Original languageEnglish (US)
Pages (from-to)3700-3710
Number of pages11
JournalJournal of Clinical Microbiology
Volume51
Issue number11
DOIs
StatePublished - Nov 2013

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Cytomegalovirus
Drug Resistance
Mutation
Antiviral Agents
Plasmids
Play Therapy
Aptitude
Microfluidics
DNA-Directed DNA Polymerase
Hematopoietic Stem Cells
Viral Load
Libraries
Phosphotransferases
Clone Cells
Transplants
Polymerase Chain Reaction
Population
Genes

ASJC Scopus subject areas

  • Microbiology (medical)

Cite this

Sahoo, M. K., Lefterova, M. I., Yamamoto, F., Waggoner, J. J., Chou, S., Holmes, S. P., ... Pinsky, B. A. (2013). Detection of cytomegalovirus drug resistance mutations by next-Generation sequencing. Journal of Clinical Microbiology, 51(11), 3700-3710. https://doi.org/10.1128/JCM.01605-13

Detection of cytomegalovirus drug resistance mutations by next-Generation sequencing. / Sahoo, Malaya K.; Lefterova, Martina I.; Yamamoto, Fumiko; Waggoner, Jesse J.; Chou, Sunwen; Holmes, Susan P.; Anderson, Matthew W.; Pinsky, Benjamin A.

In: Journal of Clinical Microbiology, Vol. 51, No. 11, 11.2013, p. 3700-3710.

Research output: Contribution to journalArticle

Sahoo, MK, Lefterova, MI, Yamamoto, F, Waggoner, JJ, Chou, S, Holmes, SP, Anderson, MW & Pinsky, BA 2013, 'Detection of cytomegalovirus drug resistance mutations by next-Generation sequencing', Journal of Clinical Microbiology, vol. 51, no. 11, pp. 3700-3710. https://doi.org/10.1128/JCM.01605-13
Sahoo, Malaya K. ; Lefterova, Martina I. ; Yamamoto, Fumiko ; Waggoner, Jesse J. ; Chou, Sunwen ; Holmes, Susan P. ; Anderson, Matthew W. ; Pinsky, Benjamin A. / Detection of cytomegalovirus drug resistance mutations by next-Generation sequencing. In: Journal of Clinical Microbiology. 2013 ; Vol. 51, No. 11. pp. 3700-3710.
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