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New Sample Prep Technique Enables Analysis of DNA from Contaminated Neanderthal Samples

NEW YORK – Using a new sample prep method, researchers have analyzed DNA from two Neanderthals whose remains were found in Gibraltar in the mid-1800s and early 1900s.

While these Neanderthals were among the earliest specimens uncovered, most ancient hominins whose genomes have been analyzed have hailed from regions with climates where DNA is better preserved.

Using an improved library construction approach, researchers from the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, and the Natural History Museum in London pieced together parts of the Gibraltar Neanderthals' nuclear and mitochondrial genomes. As they reported today in the Proceedings of the National Academy of Sciences, the researchers found that one of these Neanderthals — dubbed the Forbes' Quarry individual — is genetically more similar to Neanderthals that lived about 120,000 years ago in what is now Germany and Belgium than to those that lived more recently, between 50,000 and 70,000 years ago, in Russia and Spain.

"This suggests that the Forbes' Quarry fossil predates the latter Neanderthals," the researchers wrote in their paper.

The Forbes' Quarry fossil was found in 1848 and is thought to be from a female who lived to an advanced age. The other sample, called the Devil's Tower individual, was found in 1926 and is thought to have come from a male child who died at approximately three to five years of age. The ages of the samples, though, were unclear, with estimates ranging from 30,000 years to 130,000 years old.

Both samples were also highly contaminated with modern human DNA. To focus on ancient DNA, the researchers used a library preparation method that targeted molecules containing uracil, as that is indicative of damage due to cytosine deamination that is frequently found in ancient DNA.

Using this approach, the researchers sequenced the specimens and aligned the resulting reads to the human reference genome and an archaic version of the human genome that includes alleles from high-coverage Neanderthal and Denisovan genomes. In all, they generated 70 Mb of sequence for the Forbes' Quarry individual and 0.4 Mb of sequence for the Devil's Tower individual. Based on coverage of the X chromosome and autosomes, they determined the Forbes' Quarry individual to be female and the Devil's Tower individual to be male, as was expected.

The researchers, meanwhile, also generated 11-fold coverage of the Forbes' Quarry individual's mitochondrial genome. A phylogenetic analysis of that data, along with data from 23 other Neanderthals, 25 modern humans, four Denisovans, and the Sima de los Huesos hominin, placed the Forbes' Quarry individual squarely among Neanderthals, the researchers reported.

A comparison of the Gibraltar Neanderthals' nuclear genomes with the Altai Neanderthal that lived about 130,000 years ago in Russia, as well as a Denisovan individual, a modern-day individual from Africa, and four great apes likewise found that the Forbes' Quarry and Devil's Tower individuals shared more alleles with the Altai Neanderthal than the others. The degree of sharing, though, was lower for the Devil's Tower individual than the Forbes' Quarry individual.

When the researchers focused on the Forbes' Quarry individual's relationship with other Neanderthals, they found that it shared more alleles with the Vindija 33.19 and Chagyrskaya 8 individuals — which lived about 44,000 years and 88,000 years ago, respectively — than with the Altai Neanderthal, similar to other Neanderthal samples that have indicated a replacement of Altai-like Neanderthals with Vindija 33.19-like Neanderthals about 90,000 years ago. They noted, though, that the Forbes' Quarry individual did not share more alleles with Vindija 33.19 than with the Chagyrskaya 8, unlike other Neanderthals, suggesting that the Forbes' Quarry individual was equally related to these two Neanderthals.

Additionally, the researchers said the Forbes' Quarry individual likely predates the 49,000-year-old El Sidrón 1253 individual and possibly the 60,000-year to 70,000-year-old Mezmaiskaya individual.

Their analysis shows it is possible to analyze archaic human DNA from a warm coastal climate like that of Gibraltar, they noted, and suggests that samples from other less-than-ideal climatic regions could also be analyzed in the future.