Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

Jesse Dabney
Michael Knapp
Isabelle Glocke

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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. Trace amounts of DNA can occasionally survive the decomposition of organic matter for long periods of time after the death of an organism. However, the retrieval of these ancient DNA molecules is severely impeded by their small size. DNA fragmentation is at least partly driven by depurination (1, 2), a continually occurring process. It is thus predicted that the degree of DNA fragmentation increases with sample age. This correlation has, in fact, been established in a recent study that analyzed samples of different ages from the same archeological sites (3), but the correlation vanishes in comparisons across different sites (4). The important role of environmental conditions, especially temperature, in DNA preservation is well recognized and reflected—for example, in the concept of thermal age (5). Unsurprisingly, permafrost environments have yielded the oldest credible records of DNA survival, including short stretches o