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Letter

Traces of ATCV-1 associated with laboratory component contamination

View ORCID ProfileKristín Rós Kjartansdóttir, Jens Friis-Nielsen, Maria Asplund, Sarah Mollerup, Tobias Mourier, Randi Holm Jensen, View ORCID ProfileThomas Arn Hansen, Alba Rey-Iglesia, Stine Raith Richter, David E. Alquezar-Planas, Pernille V. S. Olsen, Lasse Vinner, Helena Fridholm, Thomas Sicheritz-Pontén, Lars Peter Nielsen, Søren Brunak, Eske Willerslev, View ORCID ProfileJose M. G. Izarzugaza, and Anders Johannes Hansen
PNAS March 3, 2015 112 (9) E925-E926; first published February 5, 2015; https://doi.org/10.1073/pnas.1423756112
Kristín Rós Kjartansdóttir
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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  • ORCID record for Kristín Rós Kjartansdóttir
Jens Friis-Nielsen
bCenter for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; and
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Maria Asplund
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Sarah Mollerup
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Tobias Mourier
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Randi Holm Jensen
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Thomas Arn Hansen
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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  • ORCID record for Thomas Arn Hansen
Alba Rey-Iglesia
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Stine Raith Richter
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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David E. Alquezar-Planas
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Pernille V. S. Olsen
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Lasse Vinner
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Helena Fridholm
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Thomas Sicheritz-Pontén
bCenter for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; and
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Lars Peter Nielsen
cDepartment of Autoimmunology and Biomarkers, Statens Serum Institut, DK-2300 Copenhagen S, Denmark
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Søren Brunak
bCenter for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; and
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Eske Willerslev
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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Jose M. G. Izarzugaza
bCenter for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; and
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Anders Johannes Hansen
aCentre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen, Denmark;
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  • For correspondence: ajhansen@snm.ku.dk

This Letter has a Reply and related content. Please see:

  • Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice - October 27, 2014
  • Reply to Kjartansdóttir et al.: Chlorovirus ATCV-1 findings not explained by contamination - February 05, 2015
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Yolken et al. (1) claim detection of Acanthocystis turfacea chlorella virus 1 (ATCV-1, gi119953744) in the normal human oropharyngeal viral flora and associate it with altered cognitive function. However, the reported presence of a freshwater algae virus, previously not known to infect other species, was based on a few sequence reads homologous to ATCV-1 identified with BLASTn. These reads span relatively few bases (97–698 bp) per sample, dispersed over a minor fraction (0.03–0.24%) of the 288 kb ATCV-1 genome.

In an unrelated sequencing experiment involving 289 human specimens, we detected traces of ATCV-1 in 16 samples of 7 different types (including skin, colon, bone marrow, and breast) and a nontemplate control. The coverage (0.01–0.05%) and length of homologous regions (50–222 bp) are comparable to those obtained by Yolken et al. (1). In the absence of full coverage of the genome of the ATCV-1 virus, we hypothesize that the scattered presence of homology in so diverse sample types—including a negative control—derived from low-level laboratory contamination. The traces of ATCV-1 identified in our samples appear in close vicinity, suggesting they do not correspond to sporadic artifacts (Fig. 1).

Fig. 1.
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Fig. 1.

Schematic view of ATCV-1-like reads identified in the 16 different samples mapping to the ATCV-1 genome (EF101928.1). The outer circle represents the linear double-stranded DNA genome of ATCV-1. The radial gray lines depict where, in the genome of ATCV-1, there is a matching read from any sample, and the black regions show the sample-specific positions of all BLASTn hits. The inner gray concentric circles serve as a visual guide for each sample.

Nucleic acid contamination from laboratory reagents and kits have previously been documented; for example, NIH-CQV/PHV in silica membranes (2), Legionella DNA in Qiagen DNA extraction kits (3), and murine DNA in extraction columns (4). Consequently, we correlated the presence of ATCV-1 in our samples with metadata from our laboratory procedures. Two laboratory components, Promega RQ1 RNase-Free DNase and Qiagen RNeasy MinElute cleanup, correlated significantly with the positive identification of ATCV-1 in a one-tailed Fisher’s exact test (P values 3.16 × 10−4 and 1.61 × 10−6, respectively) (Table 1).

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Table 1.

Association between the detection of ATCV-1 in our sample treatments and metadata describing our laboratory procedures

In several samples harboring traces of ATCV-1 reads, we concomitantly identified other algae viral species nonhomologous to ATCV-1. These findings support the presence of low-level algae viral sequence contamination.

To confirm their metagenomic findings, Yolken et al. (1) performed quantitative PCR screening. This type of analysis is prone to the same types of artifacts as the metagenomic sequencing, and the results reported by Yolken et al. could similarly originate from low-level contamination.

The discovery of novel viruses or their characterization in new hosts demands thorough investigation and validation beyond the identification of a few sequencing reads (5). Furthermore, the use of well-defined controls and critical awareness of the possible sources of contamination, either common or rare, introduced by the experimental reagents are a prerequisite. Based on our investigation of 289 human samples, we are confident that the presence of ATCV-1 sequences reported by Yolken et al. (1) originates from low-level laboratory contamination and, consequently, are rebutting the reported presence of ATCV-1 in human oropharynx.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: ajhansen{at}snm.ku.dk.
  • Author contributions: K.R.K., J.F.-N., S.M., T.M., L.V., H.F., L.P.N., S.B., E.W., J.M.G.I., and A.J.H. designed research; K.R.K., S.M., R.H.J., T.A.H., A.R.-I., S.R.R., D.E.A.-P., P.V.S.O., L.V., and H.F. performed research; K.R.K., J.F.-N., M.A., S.M., T.M., T.S.-P., L.P.N., S.B., E.W., J.M.G.I., and A.J.H. analyzed data; and K.R.K., J.F.-N., M.A., S.M., T.M., L.V., J.M.G.I., and A.J.H. wrote the paper.

  • The authors declare no conflict of interest.

View Abstract

References

  1. ↵
    1. Yolken RH, et al.
    (2014) Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice. Proc Natl Acad Sci USA 111(45):16106–16111
    .
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Naccache SN, et al.
    (2013) The perils of pathogen discovery: Origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns. J Virol 87(22):11966–11977
    .
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Evans GE, et al.
    (2003) Contamination of Qiagen DNA extraction kits with Legionella DNA. J Clin Microbiol 41(7):3452–3453
    .
    OpenUrlFREE Full Text
  4. ↵
    1. Erlwein O, et al.
    (2011) DNA extraction columns contaminated with murine sequences. PLoS ONE 6(8):e23484
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    OpenUrlCrossRefPubMed
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    1. Dalton-Griffin L,
    2. Kellam P
    (2009) Infectious causes of cancer and their detection. J Biol 8(7):67
    .
    OpenUrlCrossRefPubMed
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ATCV-1 associated with laboratory contamination
Kristín Rós Kjartansdóttir, Jens Friis-Nielsen, Maria Asplund, Sarah Mollerup, Tobias Mourier, Randi Holm Jensen, Thomas Arn Hansen, Alba Rey-Iglesia, Stine Raith Richter, David E. Alquezar-Planas, Pernille V. S. Olsen, Lasse Vinner, Helena Fridholm, Thomas Sicheritz-Pontén, Lars Peter Nielsen, Søren Brunak, Eske Willerslev, Jose M. G. Izarzugaza, Anders Johannes Hansen
Proceedings of the National Academy of Sciences Mar 2015, 112 (9) E925-E926; DOI: 10.1073/pnas.1423756112

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ATCV-1 associated with laboratory contamination
Kristín Rós Kjartansdóttir, Jens Friis-Nielsen, Maria Asplund, Sarah Mollerup, Tobias Mourier, Randi Holm Jensen, Thomas Arn Hansen, Alba Rey-Iglesia, Stine Raith Richter, David E. Alquezar-Planas, Pernille V. S. Olsen, Lasse Vinner, Helena Fridholm, Thomas Sicheritz-Pontén, Lars Peter Nielsen, Søren Brunak, Eske Willerslev, Jose M. G. Izarzugaza, Anders Johannes Hansen
Proceedings of the National Academy of Sciences Mar 2015, 112 (9) E925-E926; DOI: 10.1073/pnas.1423756112
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