Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments
Contributed by Svante Pääbo, August 6, 2013 (sent for review July 11, 2013)
Significance
Outside of permafrost, no contiguous DNA sequences have been generated from material older than ∼120,000 y. By improving our ability to sequence very short DNA fragments, we have recovered the mitochondrial genome sequence of a >300,000-y-old cave bear from Sima de los Huesos, a Spanish cave site that is famous for its rich collection of Middle Pleistocene human fossils. This finding demonstrates that DNA can survive for hundreds of thousands of years outside of permafrost and opens the prospect of making more samples from this time period accessible to genetic studies.
Abstract
Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.
Data Availability
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. KF437625).
Acknowledgments
We thank Qiaomei Fu, Ignacio Martínez, and Bence Viola for helpful discussions; Ayinuer Aximu, Barbara Höber, and Barbara Höffner for performing the sequencing runs; Janet Kelso, Gabriel Renaud, and Udo Stenzel for bioinformatic support; Christoph Kantzke for help in the laboratory; and Torsten Blass for help with R. This work was supported by the Max Planck Society and Ministerio de Ciencia e Innovacion Grant CGL2009-12703-C03-03.
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Data Availability
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. KF437625).
Submission history
Published online: September 9, 2013
Published in issue: September 24, 2013
Acknowledgments
We thank Qiaomei Fu, Ignacio Martínez, and Bence Viola for helpful discussions; Ayinuer Aximu, Barbara Höber, and Barbara Höffner for performing the sequencing runs; Janet Kelso, Gabriel Renaud, and Udo Stenzel for bioinformatic support; Christoph Kantzke for help in the laboratory; and Torsten Blass for help with R. This work was supported by the Max Planck Society and Ministerio de Ciencia e Innovacion Grant CGL2009-12703-C03-03.
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Competing Interests
The authors declare no conflict of interest.
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Cite this article
Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments, Proc. Natl. Acad. Sci. U.S.A.
110 (39) 15758-15763,
https://doi.org/10.1073/pnas.1314445110
(2013).
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