Ancient DNA

Problems and errors

Degradation processes

Due to degradation processes (including cross-linking, deamination and fragmentation), ancient DNA is of lower quality than modern genetic material. The damage characteristics and ability of aDNA to survive through time restricts possible analyses and places an upper limit on the age of successful samples. There is a theoretical correlation between time and DNA degradation, although differences in environmental conditions complicate matters. Samples subjected to different conditions are unlikely to predictably align to a uniform age-degradation relationship. The environmental effects may even matter after excavation, as DNA decay-rates may increase, particularly under fluctuating storage conditions. Even under the best preservation conditions, there is an upper boundary of 0.4 to 1.5 million years for a sample to contain sufficient DNA for contemporary sequencing technologies.

Research into the decay of mitochondrial and nuclear DNA in moa bones has modelled mitochondrial DNA degradation to an average length of 1 base pair after 6,830,000 years at −5 °C. The decay kinetics have been measured by accelerated aging experiments, further displaying the strong influence of storage temperature and humidity on DNA decay. Nuclear DNA degrades at least twice as fast as mtDNA. Early studies that reported recovery of much older DNA, for example from Cretaceous dinosaur remains, may have stemmed from contamination of the sample.

Age limit

A critical review of ancient DNA literature through the development of the field highlights that few studies have succeeded in amplifying DNA from remains older than several hundred thousand years. A greater appreciation for the risks of environmental contamination and studies on the chemical stability of DNA have raised concerns over previously reported results. The alleged dinosaur DNA was later revealed to be human Y-chromosome. The DNA reported from encapsulated halobacteria has been criticized based on its similarity to modern bacteria, which hints at contamination, or they may be the product of long-term, low-level metabolic activity.

aDNA may contain a large number of postmortem mutations, increasing with time. Some regions of polynucleotide are more susceptible to this degradation, allowing erroneous sequence data to bypass statistical filters used to check the validity of data. Due to sequencing errors, great caution should be applied to interpretation of population size. Substitutions resulting from deamination of cytosine residues are vastly over-represented in the ancient DNA sequences. Miscoding of C to T and G to A accounts for the majority of errors.

Contamination

Another problem with ancient DNA samples is contamination by modern human DNA and by microbial DNA (most of which is also ancient). New methods have emerged in recent years to prevent possible contamination of aDNA samples, including conducting extractions under extreme sterile conditions, using special adapters to identify endogenous molecules of the sample (distinguished from those introduced during analysis), and applying bioinformatics to resulting sequences based on known reads in order to approximate rates of contamination.

Authentication of aDNA

Development in the aDNA field in the 2000s increased the importance of authenticating recovered DNA to confirm that it is indeed ancient and not the result of recent contamination. As DNA degrades over time, the nucleotides that make up the DNA may change, especially at the ends of the DNA molecules. The deamination of cytosine to uracil at the ends of DNA molecules has become a way of authentication. During DNA sequencing, the DNA polymerases will incorporate an adenine (A) across from the uracil (U), leading to cytosine (C) to thymine (T) substitutions in the aDNA data. These substitutions increase in frequency as the sample gets older. Frequency measurement of the C-T level, ancient DNA damage, can be made using various software such as mapDamage2.0 or PMDtools and interactively on metaDMG. Due to hydrolytic depurination, DNA fragments into smaller pieces, leading to single-stranded breaks. Combined with the damage pattern, this short fragment length can also help differentiate between modern and ancient DNA.

Non-human aDNA

Despite the problems associated with aDNA, a wide and ever-increasing range of aDNA sequences have now been published from a range of animal and plant taxa. Tissues examined include artificially or naturally mummified animal remains, bone, shells, paleofaeces, alcohol preserved specimens, rodent middens, dried plant remains, and recently, extractions of animal and plant DNA directly from soil samples.

In June 2013, a group of researchers including Eske Willerslev, Marcus Thomas Pius Gilbert and Orlando Ludovic of the Centre for Geogenetics, Natural History Museum of Denmark at the University of Copenhagen, announced that they had sequenced the DNA of a 560–780 thousand year old horse, using material extracted from a leg bone found buried in permafrost in Canada's Yukon territory. A German team also reported in 2013 the reconstructed mitochondrial genome of a bear, Ursus deningeri, more than 300,000 years old, proving that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. The DNA sequence of even older nuclear DNA was reported in 2021 from the permafrost-preserved teeth of two Siberian mammoths, both over a million years old.

Researchers in 2016 measured chloroplast DNA in marine sediment cores, and found diatom DNA dating back to 1.4 million years. This DNA had a half-life significantly longer than previous research, of up to 15,000 years. Kirkpatrick's team also found that DNA only decayed along a half-life rate until about 100 thousand years, at which point it followed a slower, power-law decay rate.

Human aDNA

Due to the considerable anthropological, archaeological, and public interest directed toward human remains, they have received considerable attention from the DNA community. There are also more profound contamination issues, since the specimens belong to the same species as the researchers collecting and evaluating the samples.

Results

Taking preventative measures in their procedure against such contamination though, a 2012 study analyzed bone samples of a Neanderthal group in the El Sidrón cave, finding new insights on potential kinship and genetic diversity from the aDNA. In November 2015, scientists reported finding a 110,000-year-old tooth containing DNA from the Denisovan hominin, an extinct species of human in the genus Homo.

The research has added new complexity to the peopling of Eurasia. A study from 2018 showed that a Bronze Age mass migration had greatly impacted the genetic makeup of the British Isles, bringing with it the Bell Beaker culture from mainland Europe.

It has also revealed new information about links between the ancestors of Central Asians and the indigenous peoples of the Americas. In Africa, older DNA degrades quickly due to the warmer tropical climate, although, in September 2017, ancient DNA samples, as old as 8,100 years old, have been reported.

Moreover, ancient DNA has helped researchers to estimate modern human divergence. By sequencing African genomes from three Stone Age hunter gatherers (2000 years old) and four Iron Age farmers (300 to 500 years old), Schlebusch and colleagues were able to push back the date of the earliest divergence between human populations to 350,000 to 260,000 years ago.

As of 2021, the oldest completely reconstructed human genomes are ~45,000 years old. Such genetic data provides insights into the migration and genetic history – e.g. of Europe – including about interbreeding between archaic and modern humans like a common admixture between initial European modern humans and Neanderthals.

Researchers specializing in ancient DNA

• Erika Hagelberg
• Alan Cooper
• Kirsten Bos
• Joachim Burger
• M. Thomas P. Gilbert
• Johannes Krause
• Svante Pääbo
• Hendrik Poinar
• David Reich
• Beth Shapiro
• Mark G. Thomas
• Eske Willerslev
• Carles Lalueza-Fox
• Turi King