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aDNA analysis

Ancient DNA refers to DNA which is recovered from archaic and old biological material from the archaeological record. When it comes to the study of hominins and humans, it is often retrieved from hair, dental remains, coprolites, and bones.

Furthermore, DNA is preserved best in environments with cooler mean annual temperatures, neutral to slightly alkaline pH, and dry conditions, but it can also be found in anoxic wet ones. The oldest DNA in the archaeological record was found in permafrost and studied by Hoss et al in 1994 and Schwarz et al in 2009.

The first-ever study of aDNA was conducted by Russ Higuchi et al at the University of California, Berkeley. Ancient DNA was extracted from an extinct species of zebra-like animal known as the quagga (Equus quagga). The group of researchers were given a sample of dried muscle from a museum specimen, and extracted DNA from this tissue, including mtDNA which was then compared to the corresponding sequences of mtDNA from a mountain zebra. In 1987, the PCR methods was discovered by Mullis and Faloona, which made the study of aDNA much more efficient and yielding more positive results. Due to the instability of DNA, it often becomes fragmented due to damage over time. Thanks to advancements in the field of genetics, DNA fragments could now be amplified by PCR and studied.

Ancient DNA has been fundamental for a variety of scientific discoveries, including the discovery of new hominins: the Denisovans and the Homo luzonensis. In addition to this, it is useful for the reconstruction of both ancient lifeways and environments, providing a snapshot into the past from which we can act in the present and change the future.

However, the main uses of ancient DNA are for the study of infections and disease through time in the field of epidemiology, and for anthropological reasons which allow for the understanding of historical patterns and the evolution of human behaviour (such as the link between genetics and language).

Ancient DNA is vital in the field of palaeomicrobiology, as developments in molecular biology techniques in the 1990s have allowed for the analysis of aDNA from human bones and mummies, providing a greater depth of understanding of pathogens to researchers, doctors, etc. Important research that has developed useful findings include the study of Mycobacterium tuberculosis (commonly known as tuberculosis) by Helen D Donoghue et al in 2004, Mycobacterium leprae (leprosy) by Rafi et al in 1994, and the human papillomavirus by Fornaciari et al in 2003.

There are a variety of ethical issues that arise from the use of aDNA analysis. It is an inherently destructive process as it requires the use of precious ancient remains, often human ones, that are damaged in the name of scientific research. The ethical aspect is a growing priority, highlighting the importance of communication and consultation with descendant communities as a vital step in the process. Furthermore, when a study is being conducted with rare samples, extra consideration must be taken in terms of purpose of the testing and methodology used.

Method

DNA is extracted, captured, sequenced, and modern quality control is done on the samples to ensure there is no contamination from other DNA. First studies on aDNA aimed at successfully extracting and sequencing DNA, often fragments of mtDNA (Hagelberg and Clegg, 1991).

The basic technique is comprised of sample preparation and DNA extraction which involve decontamination of the outer layer of the material used, and extraction using, for example, phenol-chloroform and silica-based filtration protocols. Then, PCR/Sanger sequencing is used, or older methods such as DNA library preparation. Finally, NGS or target capture are used to analyse the sample. This must all be done in designated ancient DNA laboratories which are not near any modern ones.

Sample preparation occurs in a designated area in ancient DNA with a filtered, enclosed airflow system with UV lighting. This must be done with gloves, masks, gowns, and sterile tools (scalpels, saws, drills, containers). All work surfaces and tools must be sterilised regularly with bleach, ethanol, molecular grade H2O, as well being UV irradiated both before and after each use. Samples are stored at a constant temperature in a cool and dark place with low humidity due to DNA degrading over time in different conditions. Removal of surface contaminants must be done in a designated enclosed area to prevent any bone dust and debris from contaminating the extraction area, which would affect the results of DNA extraction. Surface contaminants are removed by grinding/cutting material (that are usually exposed layers of bone or tooth root), or by sterilising the surface with UV light/hydrochloric acid/bleach solution. The sample is then broken into dust or small pieces using a scalpel/hammer/bone mill/slow-speed drill to increase the surface area of the ancient material. This is to maximise the amount of DNA extracted during the next step of the process.

During DNA extraction, the sample is treated with a variety of solutions to make sure the DNA is accessible, and any unwanted material/contaminants have been thoroughly removed (this includes biological molecules such as amino acids). Most extraction techniques use ethylenediaminetetraacetic acid (EDTA) and the enzyme proteinase K in order to inactivate DNases and digest proteins which may hinder extraction. DNA is then separated from the proteins as it is hydrophilic (most proteins are hydrophobic), and this is done by using a binding agent that attaches to the DNA so the other elements can be easily washed away; this is known as phenol-chloroform extraction. On the other hand, the silica-based extraction methods use chaotropic salts (guanidium isothiocyanate or guanidium hydrochloride) in order to catalyse the adsorption of DNA to silica and help with the digestion of proteins (involving the use of silica column or silica-binding buffer). Many researchers prefer using this method as it maximises recovery of endogenous DNA. Any method of DNA extraction recovers all DNA from a sample, which can include both host and pathogen DNA, as well as handler DNA or DNA from the environment. The latter usually occurs when a sample is contaminated or not prepared properly.

The penultimate step is the analysis of the ancient DNA itself. Next-Generation Sequencing (NGS) is a sequencing technology used to determine DNA/RNA sequences for both genomes and regions of interest. This was developed in response to the human genome project to increase the amount of DNA sequence analyses. The shotgun technique sequences all adapter-ligated DNA in the sample, while target capture uses a modern genome as ‘bait’ to extract taxonomically similar aDNA. Capture methods most often use in-solution hybridization using single-stranded DNA or RNA to find complementary sequences in the sample, and it results in a reduction in costs as one doesn’t end up with unwanted sequences. Both require the construction of a DNA library which is amplified. Sequencing is done in a laboratory either at the same site of the extraction or the sample can be shipped to a different sequencing centre. Sequencing can take hours to months depending on the sample and method used.

Lastly, data is filtered to remove low-quality sequences, duplicates, and any contamination from modern DNA sources. It is then mapped to reference sequences - or newly assembled when references are not available - to create a consensus sequence which has identifiable variants needed for following phylogenetic and population genetic analyses that use special computer programmes and scripts (alongside high-performance computers).

Examples

Denisovan DNA

• In 2017, researchers Viviane Slon et al collected samples from the Denisova Cave in Russia, from which DNA was extracted and recorded as single-strand DNA libraries, which were shotgun sequenced and analysed with a taxonomic-binning approach. While the majority of the DNA belonged to a variety of ancient faunal and floral species of the area, hominin DNA was also targeted specifically.

• Using computer programmes, they were able to filter 8822 DNA fragments which were cleared through as exclusively human. Previously used DNA libraries were then enriched for human mtDNA, and sequences were merged from each sediment sample to obtain enough hominin DNA fragments to use for analysis. The main process used to identify hominin DNA is known as hybridisation capture.

• The researchers identified ‘diagnostic’ positions on the mtDNA genome which have changed in different branches of the phylogenetic tree of species related to modern humans, Neanderthals, and Denisovans. 84% of the samples from the Denisova Cave were found to carry Denisovan-specific variants of mtDNA, indicating the DNA fragments were of Denisovan origin.

Tuberculosis

• Characterised by lesions which tend to contain residual microbial DNA. This type of DNA has an increased amount of guanine and cytosine, which makes it more stable and therefore more likely to survive. Furthermore, mycobacterium cell walls are high in lipid content, which protects the cell from lytic enzymes. Furthermore, with tests using PCR, false positives are much less likely than older methods due to its use of species-specific primers, and with M. tuberculosis it does not give false positives for other species of mycobacteria.

• In 1993, Spigelman and Lemma studied 4 (out of a total of 11) human specimens from the archaeological record of Turkey, Europe, and Borneo, which were diagnosed with tuberculosis. The mycobacterium DNA was found through the use of PCR primers which are specific to M. tuberculosis, M. bovis, and M. africanum. The presence of M. tuberculosis was verified with sequencing. The bones from the bodies were dated to be a range of 300-1400 years old.

• Method used: a few milligrams of the central cortex of the infected bones were taken by using a lacrimal probe to collect the bone dust, which ultimately means that the shape is not damaged or changed. To know whether the bone area from which the sample is taken is infected, X-rays were taken and assessed carefully first to determine the best place to take a biopsy from. The preparation of the bones was carried out following Boom et al’s method. PCR estimation of the samples’ supernatant was then done, which confirmed that some of the bones exhibited Pott’s disease and had tuberculosis DNA.

References