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

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.

Fig. 1.

Fragment size recovery in DNA extraction. A constructed DNA ladder of sizes relevant to ancient DNA was run through a previously published extraction method (n = 2), as well as the current, optimized method (n = 4). Recovered DNA was then quantified against a control ladder. Error bars represent one standard deviation.

Fig. 2.

Length distributions and damage patterns. (A) Fragment length distributions of shotgun sequences (blue) and captured mitochondrial sequences (orange) as the fraction of sequences in each size bin. (B) Substitution patterns at the 5′ and 3′ ends of the aligned sequences. (C) DNA fragmentation patterns inferred from the reference base composition around alignment start and end points.

Fig. 3.

Phylogenetic position of U. deningeri within Late Pleistocene cave bears. ML reconstruction of cave bear relationships is shown. Numbers on branches represent bootstrap support. The tree is rooted with five brown bear sequences as outgroup (branch not depicted). Age of the samples is provided in brackets where known (r and s denote radiocarbon and stratigraphic dates, respectively).