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Nucleic Acids Research Advance Access published online on August 18, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl483
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genomics

Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis

M. Thomas P. Gilbert1,*, Jonas Binladen1, Webb Miller2, Carsten Wiuf3,4, Eske Willerslev1, Hendrik Poinar5, John E. Carlson6, James H. Leebens-Mack7 and Stephan C. Schuster2

1 Center for Ancient Genetics, Niels Bohr Institute and Biological Institutes, The University of Copenhagen, Juliane Maries vej 30 DK-2100 Copenhagen Ø, Denmark 2 Center for Comparative Genomics and Bioinformatics, Penn State University 310 Wartik Lab, University Park, PA 16802, USA 3 Bioinformatics Research Center, University of Aarhus Aarhus DK-8000, Denmark 4 Molecular Diagnostic Laboratory, Aarhus University Hospital DK-8200, Denmark 5 McMaster Ancient DNA Center, Department of Anthropology and Pathology & Molecular Medicine, McMaster University, Hamilton Ontario L82 4L9, Canada 6 School of Forest Resources, Penn State University 323 Forest Resources Building, University Park, PA16802, USA 7 Department of Plant Biology, University of Georgia Athens, Georgia 30602-7271

*To whom correspondence should be addressed. Tel: +45 35 32 05 87; Fax: +45 35 36 53 57; Email: mtpgilbert{at}gmail.com

Received May 25, 2006. Revised June 21, 2006. Accepted June 23, 2006.

Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error. Furthermore, considerable disagreement and speculation exists on which specific damage events underlie observed miscoding lesions. The root of the problem is that it has previously been difficult to assemble sufficient data to test the hypotheses, and near-impossible to accurately determine the specific strand of origin of observed damage events. With the advent emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin, and subsequently interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390 965 bp modern chloroplast and 131 474 bp ancient woolly mammoth GS20 sequence data we conclusively demonstrate that Type 2 (C->T/G->A) miscoding lesions represent the overwhelming majority of damage derived miscoding lesions. In addition, we show that an as yet unidentified G->A modification, not the conventionally argued cytosine->uracil deamination, underpins the majority of Type 2 damage.


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