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With Tweaks to Approach, Researchers Reconstruct Cave Bear Mitochondrial Genome from Small Fragments

NEW YORK (GenomeWeb News) – An international team of researchers has pieced together the mitochondrial genome of a Middle Pleistocene cave bear from very small DNA fragments.

As it reported in the Proceedings of the National Academy of Sciences today, the team led by Matthias Meyer from Max Planck Institute for Evolutionary Anthropology optimized a current ancient DNA extraction approach and paired it with a library-based amplification technique, which they then applied to a more than 300,000-year-old cave bear bone sample. By analyzing the resulting mitochondrial genome and comparing it to other cave bear sequences as well as brown bear sequences, the team found that this cave bear, Ursus deningeri, came from a sister lineage that diverged from the other bear lines.

"Here we present improvements to a widely used silica-based DNA extraction technique that, in combination with single-stranded library preparation, allows ancient DNA molecules as short as 30 [basepairs] to be efficiently recovered and sequenced," Meyer and his colleagues wrote. "We describe the results from applying these methods to a bone sample of a Middle Pleistocene cave bear (Ursus deningeri) from Sima de los Huesos [in Spain]." That cave has also been the source of other fossils, including from hominins, they noted.

While an ancient horse dug up in Canada's Yukon Territory dating back some 700,000 years was recently sequenced, samples from regions other than the tundra that have been sequenced are typically younger, no more than 100,000 to 120,000 years old. For example, in 2008, a team of French and Dutch researchers sequenced the mitochondrial genome of U. spelaeus, another cave bear species, from 32,000-year-old bone fragments found in France. For this study, Meyer and his team reached further back in time.

The researchers suspected that older, non-permafrost DNA samples could be strung together from small bits of DNA, including ones shorter than 40 basepairs.

To analyze such short stretches of DNA, Meyer and his colleagues turned to a combination of a better extraction approach and library preparation approaches to amplify the fragments.

Using a test assay of synthetic DNA fragments, the researchers determined that the common ancient DNA extraction technique is length-dependent, decreasing from 72 percent at 150 bp to 22 percent at 35 bp. By tweaking some of the conditions — using a binding buffer with a different chemical makeup, altering the proportion of that buffer to the extraction buffer, and using a silica spin column rather than a silica suspension — the researchers were able to recover fragments of all lengths at nearly the same rates.

After using that fine-tuned extraction approach to obtain cave bear DNA, the researchers converted those fragments into Illumina sequencing libraries and sequenced them using the MiSeq platform.

The consensus sequence covered 16,305 bp of the cave bear mitochondrial genome, the researchers reported. Most of the 480 or so missing basepairs are from the D loop, which consists of an approximately 320 bp stretch of repetitive sequence.

The researchers also noted that the samples contained an excess of cytosine deamination, in which terminal cytosines become uracils. While cytosine deamination is expected in ancient samples, they noted that about 62 percent and 66 percent of terminal ends, 5' and 3' respectively, were deaminated, which is higher than other reports. They hypothesized that difference could be due to ancient DNA samples having longer 3' overhangs, an asymmetry they noted was present to a lesser degree in Denisovan and Neandertal samples.

Using a maximum-likelihood approach, Meyer and his colleagues constructed a phylogeny for cave bears, drawing on sequence data from the Late Pleistocene U. spelaeus, U. ingressus, U. d. kudarensis, and their new Middle Pleistocene U. deningeri mitochondrial genome as well as brown bears as an outgroup.

From this, the researchers noted that U. deningeri branches off from the other cave bears basal to the common ancestor of U. spelaeus and U. ingressus as a sister lineage to Western European Late Pleistocene cave bears. "It is noteworthy, however, that the Sima de los Huesos sequence is located on a branch of substantial length," they added. "Sequences from additional specimens will therefore be needed to determine how closely the Sima de los Huesos population is related to the U. deningeri population that gave rise to Late Pleistocene cave bears."

Overall, the researchers said that their technique could be applied to other fossil remains from the Middle Pleistocene. In addition, they noted that the lower limit of fragment sizes that can be sequenced has yet to be systematically explored.

"It is therefore possible that even shorter molecules can be made available for sequencing in the future, by using library-based techniques as described here or directly via single-molecule sequencing," Meyer and his colleagues said. "However, in addition to further optimizations of the DNA extraction method, such attempts will have to include improvements to hybridization enrichment of very short molecules and the development of new sequence analysis strategies that allow for confidently aligning very short sequences to a reference genome while discriminating endogenous sequences from contaminating environmental DNA."

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