Originally published as Genetics Published Articles Ahead of Print on November 19, 2005.

Genetics, Vol. 172, 733-741, February 2006, Copyright © 2006
doi:10.1534/genetics.105.049718

Assessing the Fidelity of Ancient DNA Sequences Amplified From Nuclear Genes

Jonas Binladen^*, Carsten Wiuf, M. Thomas P. Gilbert, Michael Bunce, Ross Barnett^**, Greger Larson^**, Alex D. Greenwood^,, James Haile^**, Simon Y. W. Ho^**, Anders J. Hansen^* and Eske Willerslev^*,1

^* Ancient DNA and Evolution Group, Centre for Ancient Genetics, Niels Bohr Institute and Biological Institute, University of Copenhagen, Copenhagen DK-2100, Denmark, Bioinformatics Research Center, University of Aarhus, Aarhus DK-8000, Denmark, Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, Department of Anthropology, McMaster University, Hamilton, Ontario L8S 4L9, Canada, ^** Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom, Department of Vertebrate Zoology, American Museum of Natural History, New York, New York 10024 and Institute of Molecular Virology, GSF-National Research Center for Environment and Health, 85764 Neuherberg, Germany

¹ Corresponding author: Ancient DNA and Evolution Group, Centre for Ancient Genetics, Niels Bohr Institute and Biological Institute, University of Copenhagen, Juliane Maries vej 30, DK-2100, Denmark.
E-mail: ewillerslev{at}gfy.ku.dk

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine guanine and thymine cytosine) and type 2 transitions (cytosine thymine and guanine adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences.

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