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Improving the performance of true single molecule sequencing for ancient DNA

Aurelien Ginolhac1, Julia Vilstrup1, Jesper Stenderup1, Morten Rasmussen1, Mathias Stiller2, Beth Shapiro2, Grant Zazula3, Duane Froese4, Kathleen E Steinmann56, John F Thompson57, Khaled AS AL-Rasheid8, Thomas MP Gilbert1, Eske Willerslev1 and Ludovic Orlando1*

Author Affiliations

1 Centre for GeoGenetics, Natural History Museum of Denmark, Copenhagen University, 5-7 Øster Voldgade, København, 1350, Denmark

2 Department of Biology, The Pennsylvania State University, 326 Mueller Lab, University Park, PA, 16802, USA

3 Department of Tourism and Culture, Government of Yukon, Yukon, Palaeontology Program, PO Box 2703 L2A, Whitehorse, Yukon Territory Y1A, 2C6, Canada

4 Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, Alberta T6G 2E3, Canada

5 Applications, Methods and Collaborations, Helicos BioSciences, One Kendall Square Bldg 200LL, Cambridge, MA, 02139, USA

6 Present address: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, 021423, USA

7 Present address: NABsys Inc, 60 Clifford Street, Providence, RI, 0290, USA

8 Zoology Department, College of Science King Saud University, P.O. Box, 2455, Riyadh, 11451, Saudi Arabia

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BMC Genomics 2012, 13:177  doi:10.1186/1471-2164-13-177

Published: 10 May 2012



Second-generation sequencing technologies have revolutionized our ability to recover genetic information from the past, allowing the characterization of the first complete genomes from past individuals and extinct species. Recently, third generation Helicos sequencing platforms, which perform true Single-Molecule DNA Sequencing (tSMS), have shown great potential for sequencing DNA molecules from Pleistocene fossils. Here, we aim at improving even further the performance of tSMS for ancient DNA by testing two novel tSMS template preparation methods for Pleistocene bone fossils, namely oligonucleotide spiking and treatment with DNA phosphatase.


We found that a significantly larger fraction of the horse genome could be covered following oligonucleotide spiking however not reproducibly and at the cost of extra post-sequencing filtering procedures and skewed %GC content. In contrast, we showed that treating ancient DNA extracts with DNA phosphatase improved the amount of endogenous sequence information recovered per sequencing channel by up to 3.3-fold, while still providing molecular signatures of endogenous ancient DNA damage, including cytosine deamination and fragmentation by depurination. Additionally, we confirmed the existence of molecular preservation niches in large bone crystals from which DNA could be preferentially extracted.


We propose DNA phosphatase treatment as a mechanism to increase sequence coverage of ancient genomes when using Helicos tSMS as a sequencing platform. Together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, this simple preparation procedure can improve overall Helicos tSMS performance when damaged DNA templates are targeted.

Ancient DNA; True Single Molecule DNA Sequencing; Next-Generation Sequencing