Introduction to Ancient DNA
Ancient DNA (aDNA) refers to the genetic material extracted from biological remains such as bones, hair bulbs, skin fragments, teeth, sediments, etc., dating to the last tens of thousands of years or older. Due to its great age and environmental exposure, aDNA is subject to degradation, particularly fragmenting into short 50-100 base pairs, undergoing post-mortem mutations (e.g., cytosine deamination), and environmental contamination 3 . These considerations complicate the research and result in additional methodical approaches to avoid misleading conclusions 3 . Initial aDNA research began in 1984 with the extraction and sequencing of short mitochondrial fragments from the quagga ( Equus quagga quagga ), an extinct subspecies of zebra 9 , followed by Svante Pääbo’s paper where the first sequence of ancient human DNA from an Egyptian mummy was published 11 . Pääbo has been a pioneer in aDNA studies and has contributed greatly to the expansion and recognition of the field of paleogenomics.
The fragile nature of aDNA makes it difficult to analyse the samples by introducing various methodological assumption liberties, difficulties in dating samples (for example, by carbon dating), and ethical considerations relating to ownership.
Decoding aDNA: Methods, Limits & Applications
While vague in definition, generally the term aDNA encompasses DNA recovered from any postmortem material which has not been specifically preserved for later use. As described above, these samples are subject to degradation, meaning that it is often challenging to establish a precise timeframe of the sample based on the genetic material alone 5 . Apart from the biological material itself, the environment where the remains have been found can be associated with a particular time period, providing an indirect age for the sample. The combination of multidisciplinary DNA genotyping, radiocarbon dating, and dendrochronology assessments are often used and compared to other historical evidence to provide a narrow date range for the age of the sample 6 .
When dealing with trace quantities of preserved DNA, standard methods of genetic sequencing such as PCR introduce a major risk of accidentally sequencing contaminants as opposed to the aDNA itself 5 . Nowadays, the technology used includes utilisation of sterile facilities, next-generation high throughput sequencing which has been shown to work most efficiently on short DNA sequences, as well as novel statistical tools that can reliably estimate the sequence damage in aDNA 5 . While there have been attempts to standardise criteria in the design of aDNA research, these have often proved impractical and a ‘common-sense approach’ acknowledging the contamination issue appears to be significantly more feasible for a larger number of studies 5 . Another crucial consideration involves the awareness of taphonomic bias, which relates to burial, decay, and preservation processes, and analytical procedures bias relating to the extraction, amplification, and identification of the aDNA samples 7 . The main origin of bias in the study of ancient samples revolve around the source of the material, the methodology and efficiency of the DNA transfer, the preservation conditions of the sample, and the assumption that these have remained constant throughout time 7 .
On a more technical scale, a controversial aspect of aDNA research is the assumption of molecular rates of change. These include genetic diversity, the mutation rates of natural alleles, substitution rates, etc., which often assume an equilibrium between mutation and genetic drift over time 5 . Most of the aDNA work utilises mtDNA (mitochondrial DNA), as it is relatively easy to amplify. Prior to aDNA research,, studies focusing on the rates of change of mitochondrial loci sometimes resulted in conflicting values; for example, estimates for the mutation rate of the hypervariable region 1 of the mitochondrial genome have ranged between 2% to 10% per million years; however, recent estimates over shorter timeframes for intraspecific comparisons that use aDNA methodology differ greatly from those obtained via phylogenetic methods using DNA from living individuals 5 . Promisingly, findings like these have the capacity to be rectified as novel bioinformatics tools enable the re-evaluation of published data. The BMC Genomic Data in particular encourages the submission of ancient DNA Data Notes 3 .
Alongside recent advancements in next-generation sequencing technologies and bioinformatic research, the analyses of aDNA have achieved significant scholarly attention as they have the capacity to revolutionise our perception of human evolution, plant and animal domestication, the origins and the evolution of pathogens and microbiomes, migration patterns, and many more (see Figure 1) 3 . At present, genome-scale data has been obtained from thousands of palaeobiological samples, and the number of ancient biological tissues that are suitable for genome sequencing is continuously increasing, hence promising realistic near-future advancements 3 .
Figure 1: Applications of ancient DNA analysis . This chart outlines possible areas whereby ancient DNA implementation would shape the fields of anthropology and palaeobiology 3 .
Recent Discoveries in aDNA Research
Research team led by the Francis Crick Institute have identified a prehistoric person with mosaic Turner syndrome who is assumed to have lived around 2500 years ago, an Early Medieval Period individual with Jacob’s syndrome, three people with Klinefelter’s syndrome, and an Iron Age infant with Down’s syndrome 2 . These individuals have been analysed as a part of the Thousand Ancient British Genomes project and have been found at sites in Oxford, Somerset, Yorkshire, and Lincoln. All have been buried within the guidelines of society’s customs at the time, but without any possessions that could inform us about their lives. The findings surrounding atypical autosomal and sex chromosome karyotypes aided in the understanding of past perceptions of biological sex, as well as diverse biological traits 2 .
Since polygenic risk scores are already utilised as routine assessments by the NHS, ancient genomic data bring about the potential to reconstruct the history of human health over time, particularly the sought-after link between genomic diversity and disease. The oldest nuclear genome from the Homo genus was retrieved from a 430,000-year-old-Neanderthal 10 . While aDNA has been obtained from various parts of the world, there is a general northern hemisphere bias in the data which needs to be addressed. The potential of studying Neanderthal DNA arises from the introgression of Neanderthal DNA which forms a small proportion of modern human DNA, where research has revealed genes of major physiological relevance 10 . Recent paleogenomic studies have determined that 3% of people of European ancestry have a TYK2 P1104A tuberculosis risk variant which has evolved under strong selection pressure over two millennia. This finding reflects the pressure imposed by M. tuberculosis epidemics and would not have been brought to attention with sole modern DNA studies 10 . Interestingly, it has been established that a Neanderthal haplotype encompassing the antiviral OAS1 gene is linked to protection against severe COVID-19 12 . These findings have the potential to redefine our understanding of immunity and shape our strategies for tacking infectious diseases.
Cultural Sensitivity: Ownership of aDNA Data
Arguably the biggest concern surrounding aDNA is lack of means to obtained informed consent from test subjects and ownership of genomic data. The general principle is to ensure that paleogenomic researchers consider the ethical and cultural issues before, during, and after the study has been undertaken, and they must consult with the communities the remains have been found around 4 .
In the past, destruction of relics, defiling of holy places, and misrepresenting the remains have discouraged ethnic communities from consenting to such studies 4 . For example, in 1996, archaeologists assembled a nearly complete skeleton consisting of more than 300 bones found along the edge of the Columbia River, an area local to at least four Indigenous groups. The remains have been referred to as the Kennewick Man, or the Ancient One. One archaeologist called James C. Chatters, has suggested that Kennewick Man may have been European due to certain “Caucasoid” features like his skull morphology. When the remains have been found to be older than Chatters expected, the realised that this is not the person he suspected Kennewick Man to be. By suggesting that Kennewick Man was European, Chatters undermined the claim of Indigenous groups which stated that the skeletal remains belonged to them under the Native American Graves Protection and Repatriation Act (NAGPRA) 8 .
This act clearly declares that the remains must be returned to a tribe if they were found to be culturally related. Court rulings denied the tribes’ claims and granted scientists access to the bones, while Chatters was not part of the lawsuit, he was able to study the remains and published his book in 2002. Jerome Rose, an osteologist, was called to analyse the remains, concluded that the skeleton belongs to a Native American from the Great Plains, which is consistent with the time and location where the remains were found 8 . Paulette Steeves, an Indigenous (Cree-Métis) archaeologist at Algoma University in Canada (and one of Rose's former students) said this when referring to Rose's work on Kennewick Man: “[the archaeologists] tried to disenfranchise Kennewick Man from the local Indigenous communities” 8 .
In 2015, thanks to modern genome sequencing technology using one of the individual’s hand bones, Kennewick Man has been definitively linked with, and returned to, the Indigenous Columbia Basin tribes. At his burial site, more than 200 tribal members have gathered, displaying the impact of this situation on their culture. Paulette Steeves, Rose’s former student, said “Genetics . . . obviously showed later on that this person definitely was linked to those tribal communities and he was buried, but for how many years did [they] suffer? That’s the kind of damage that researchers and archaeologists and geneticists can do.” 8
Chatters argues that he did not deny the tribe ownership, instead he did not agree with the refusal to not let Kennewick Man to tell his story. The burial itself provided psychological relief to the tribal group; however, due to the unpleasant circumstances surrounding it, the stigma prohibited scientists from reviewing the burial site and answering important historical questions 8 . Furthermore, literature reports that genetic findings may result in repression of such communities through communal or personal convictions, or perpetrating falsifying narratives through the misuse of genetic data for determining group belonging 1 .
Some experts believe that it is not appropriate for these data to be used as an “arbiter of identity” and to avoid exercising extreme and misguided determination of claim to ancient heritage 1 . In addition, when conducting culturally sensitive studies, researchers should inform the communities that the genetic findings may be inconsistent with the communities’ beliefs. While the scientific analyses are reported as scholarly outputs, a study highlights that “they do not discredit, diminish, or decrease the importance of traditional expertise and deeply held beliefs”, and other lines of evidence should be reported alongside genetic analyses to reflect the complexity of factors that played a role in shaping those traditions 1 .
To address these ethical concerns, protective guidelines like NAGPRA ensure an alliance between scientists and Indigenous communities, where ethical and cultural implications are considered to develop strategies regarding communication and handling of the sensitive data, Indigenous community engagement, making sure they are fully aware of the process to maximise informed consent, and also tackling long-term responsibilities that arise from the genetic findings 4 . In these studies, the main priority lies with community engagement that involves Indigenous scholars and stakeholders 4 . More specifically, the widespread argument is that “(1) researchers must ensure that all regulations were followed in the places where they work and from which the human remains derived; (2) researchers must prepare a detailed plan prior to beginning any study; (3) researchers must minimize damage to human remains; (4) researchers must ensure that data are made available following publication to allow critical re-examination of scientific findings; and (5) researchers must engage with other stakeholders from the beginning of a study and ensure respect and sensitivity to stakeholder perspectives” 1 . Every aDNA researcher must commit to adhering to these and is expected to promote a high ethical standard in aDNA research on human remains 1 .
In conclusion, the field of ancient DNA has changed enormously since its beginnings, both in aspects of technological advancements and in ethical considerations. Ever since the field gained historic recognition through Svante Pääbo’s paleogenetic research on Neanderthals, it has consistently reached new milestones in terms of increased technical accuracy and sparking curiosity for insights into human migration patterns, cultural heritage, and even the impact of genetic diversity on infectious diseases. The field continues to expand, and with that, scientists learn to develop initiatives to establish and strengthen the dialogues between researchers, Indigenous peoples, and interdisciplinary specialists, ultimately elevating the domain of knowledge while ensuring a respectful and considerate approa
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