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Researchers Develop Method for Distinguishing Ancient Human DNA from Modern Day Contaminants

NEW YORK (GenomeWeb News) – Researchers from the Max Planck Institute for Evolutionary Anthropology and the Russian Academy of Sciences have come up with a way to overcome modern human DNA contamination — a major obstacle in past ancient human DNA sequencing efforts.

In a recent online, ahead of print paper in Current Biology, the team, led by Max Planck researcher Svante Pääbo, reported that they have successfully sequenced the mitochondrial genome of a 30,000-year-old modern human found in Russia after using Neandertal sequences to find fragmentation and DNA break patterns that could be used to differentiate between ancient DNA and present day contaminants.

Along with the insights the current study offers into relationships between the ancient European individual and existing populations, the method "really opens up a whole new field of looking at early modern humans," lead author Johannes Krause, a researcher at the Max Planck Institute, told GenomeWeb Daily News.

Previous attempts to sequence and characterize ancient human DNA have been complicated by contamination with modern day human genetic material. For instance, the researchers noted, attempts to amplify mtDNA sequences from 6,500-year-old European skeletons using PCR identified only a few rare or previously undetected mitochondrial sequence variants that are believed to be authentic.

Such PCR-based approaches have inherent limitations for working with very old sequences, Krause noted, because they preferentially amplify longer sequences and miss many of the shorter DNA fragments found in very old samples.

Instead, Krause, Pääbo and colleagues came up with a method for distinguishing ancient DNA from modern DNA based on the fragmentation patterns, fragment size, and DNA damage profiles in ancient DNA sequence capture libraries representing Neandertals from Germany, Russia, and Uzbekistan. The libraries were contaminated with modern human and other DNA, providing an opportunity to compare the characteristics of each.

For instance, the ancient DNA tended to have far more purine nucleotide bases before DNA breaks than modern DNA — a pattern that may reflect how DNA breaks down, Krause explained. Ancient DNA also tends to undergo cytosine deamination leading to cytosine to thymine substitutions at the 5' ends of genes.

They then used ancient DNA sequence capture and deep sequencing with the Illumina Genome Analyzer II to sequence the mitochondrial genome of the so-called Markina Gora skeleton, found in Kostenki, Russia in the 1950s.

When they compared this mitochondrial genome sequence with present day mitochondrial sequences, the researchers found that the early modern human mitochondrial sequence resembles a haplogroup called U2, which still exists in Europe, Asia, and Northern Africa today. That suggests there has been at least some continuity between populations living in Europe tens of thousands of years ago and those alive today, Krause said.

But while the ancient Russian sequence was similar to the existing haplogroup, it was shorter — and contained fewer mutations — than mitochondrial sequences today, he noted, consistent with the age of the sample.

Based on the DNA patterns present in mitochondria, the researchers concluded that the Russian remains are roughly 30,000 years old — in the same range as previous estimates that put the skeleton's age at between 30,000 and 33,000 years old.

The team is currently trying to collect more samples from early modern human populations. And the new method may have applications for studies of historical population patterns throughout Europe and elsewhere, Krause explained, such as the effects of an ice age occurring in Europe around 20,000 years ago.

"You can look at historical events that are much further away than other records available," he said.

The approach may eventually lead to nuclear genome sequencing of early modern human samples as well, though researchers are still weighing the costs, benefits, and feasibility of undertaking such an effort, which is expected to be even more challenging than the Neandertal genome sequencing project.

"We can now do what I thought was impossible just a year ago — determine reliable DNA sequences from modern humans — but this is still possible only from very well preserved specimens," Pääbo said in a statement.