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False-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis

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Abstract
A viral metagenomic approach using virion enrichment, random amplification and next-generation sequencing was used to investigate an undiagnosed cluster of dairy cattle presenting with high persistent fever, unresponsive to anti-microbial and anti-inflammatory treatment, diarrhoea and redness of nose and teat. Serum and whole blood samples were taken in the predicted hyperviraemic state of an animal that a few days later presented with these clinical signs. Bioinformatics analysis of the resulting data from the DNA virus identification workflow (a total of 32 757 sequences with average read length 335 bases) initially demonstrated the presence of parvovirus-like sequences in the tested blood sample. Thorough follow-up using specific real-time RT-PCR assays targeting the detected sequence fragments confirmed the presence of these sequences in the original sample as well as in a sample of an additional animal, but a contamination with an identical genetic signature in negative extraction controls was demonstrated. Further investigation using an alternative extraction method identified a contamination of the originally used Qiagen extraction columns with parvovirus-like nucleic acids or virus particles. Although we did not find any relevant virus that could be associated with the disease, these observations clearly illustrate the importance of using a proper control strategy and follow-up diagnostic tests in any viral metagenomic study.
Keywords
contamination, parvovirus, VETERINARY-MEDICINE, ORIGIN, SERONEGATIVE HEPATITIS, GENERATION SEQUENCING TECHNOLOGIES, PARVOVIRUS, next-generation sequencing, viral metagenomics, IDENTIFICATION, SAMPLES, CHINESE PATIENTS, VIRAL METAGENOMICS, TOOL

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Chicago
Rosseel, Toon, Bart Pardon, Kris De Clercq, Orkun Ozhelvaci, and Steven Van Borm. 2014. “False-positive Results in Metagenomic Virus Discovery: a Strong Case for Follow-up Diagnosis.” Transboundary and Emerging Diseases 61 (4): 293–299.
APA
Rosseel, T., Pardon, B., De Clercq, K., Ozhelvaci, O., & Van Borm, S. (2014). False-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis. TRANSBOUNDARY AND EMERGING DISEASES, 61(4), 293–299.
Vancouver
1.
Rosseel T, Pardon B, De Clercq K, Ozhelvaci O, Van Borm S. False-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis. TRANSBOUNDARY AND EMERGING DISEASES. 2014;61(4):293–9.
MLA
Rosseel, Toon, Bart Pardon, Kris De Clercq, et al. “False-positive Results in Metagenomic Virus Discovery: a Strong Case for Follow-up Diagnosis.” TRANSBOUNDARY AND EMERGING DISEASES 61.4 (2014): 293–299. Print.
@article{4411643,
  abstract     = {A viral metagenomic approach using virion enrichment, random amplification and next-generation sequencing was used to investigate an undiagnosed cluster of dairy cattle presenting with high persistent fever, unresponsive to anti-microbial and anti-inflammatory treatment, diarrhoea and redness of nose and teat. Serum and whole blood samples were taken in the predicted hyperviraemic state of an animal that a few days later presented with these clinical signs. Bioinformatics analysis of the resulting data from the DNA virus identification workflow (a total of 32 757 sequences with average read length 335 bases) initially demonstrated the presence of parvovirus-like sequences in the tested blood sample. Thorough follow-up using specific real-time RT-PCR assays targeting the detected sequence fragments confirmed the presence of these sequences in the original sample as well as in a sample of an additional animal, but a contamination with an identical genetic signature in negative extraction controls was demonstrated. Further investigation using an alternative extraction method identified a contamination of the originally used Qiagen extraction columns with parvovirus-like nucleic acids or virus particles. Although we did not find any relevant virus that could be associated with the disease, these observations clearly illustrate the importance of using a proper control strategy and follow-up diagnostic tests in any viral metagenomic study.},
  author       = {Rosseel, Toon and Pardon, Bart and De Clercq, Kris and Ozhelvaci, Orkun and Van Borm, Steven},
  issn         = {1865-1674},
  journal      = {TRANSBOUNDARY AND EMERGING DISEASES},
  keyword      = {contamination,parvovirus,VETERINARY-MEDICINE,ORIGIN,SERONEGATIVE HEPATITIS,GENERATION SEQUENCING TECHNOLOGIES,PARVOVIRUS,next-generation sequencing,viral metagenomics,IDENTIFICATION,SAMPLES,CHINESE PATIENTS,VIRAL METAGENOMICS,TOOL},
  language     = {eng},
  number       = {4},
  pages        = {293--299},
  title        = {False-positive results in metagenomic virus discovery: a strong case for follow-up diagnosis},
  url          = {http://dx.doi.org/10.1111/tbed.12251},
  volume       = {61},
  year         = {2014},
}

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