Ancient DNA sample

Ancient DNA refers to DNA that is extracted from ancient specimens, such as skeletal materials, mummified tissues, or other specimens that have not been preserved specifically for further DNA analysis.

Ancient DNA is exciting to study because it can provide access to an evolutionary point in the past! DNA can be extracted from almost any cell. However, ancient DNA will have undergone a certain amount of degradation due to temperature exposure, water exposure, and other factors. The older the DNA sample is, the more damage it will likely have incurred but, due to recent advances in DNA analysis technology, even ancient DNA can be analyzed using Next Generation Sequencing. Because of this damage, the number of cycles in a PCR is usually limited. With every cycle, there is a possibility to introduce errors.

Several characteristics of ancient DNA are as follows: (a) single strand breaks, (b) cross-linking, and (c) oxidative and hydrolytic modification of bases.

Figure 1. Several characteristics of ancient DNA are as follows: (a) single strand breaks, (b) cross-linking, and (c) oxidative and hydrolytic modification of bases. (Willerslev, E. and Cooper, A., 2005, Proc.R.Soc.B, 272,3–16)

Ancient DNA characteristics

Ancient DNA samples share some characteristics that are caused by the damage they have undergone. By identifying these characteristics, we can recognize an ancient DNA sequence from a modern, contaminating DNA sequence (for example, the DNA sample of an individual who did not handle the sample properly before analysis in the laboratory). Below are some of the ancient DNA characteristics that are normally used in a Next Generation Sequencing analysis.

• Hydrolytic damages

Hydrolytic damage is responsible for damage in the sugar backbone; it could result in single-strand breaks or loss of DNA bases. When purines (adenine or guanine) are lost, the event is referred to as depurination. Hydrolysis can result in a conversion of cytosine to uracil by releasing ammonia in the process. This event is referred to as deamination. Deamination may also occur in other bases (cytosine, adenine, or guanine).

• Base substitution pattern

There are common base substitution patterns that are observed in ancient DNA samples. Base substitutions of C > T and G > A are observed higher in ancient DNA sample compared to modern DNA samples. However, these substitutions are not equally distributed along the DNA molecules. The C > T substitutions are found most commonly at the 5' end of the DNA molecule, whereas the G > A substitutions are found most commonly at the 3' end of the DNA molecule.

• Oxidative damages

Oxidative damages are caused by the direct interaction of ionizing radiation with DNA or the interaction of free radicals (created from water molecules by ionizing radiation) with DNA, resulting in modified bases. Some oxidative damage results in lesions that block the polymerase enzyme and promote chimeric sequences through "jumping PCR".

• Cross-linking

Cross-linking can occur between bases on the same DNA strand, also called as interstrand cross-links, or between DNA and other molecules, also called intermolecular cross-links. In intermolecular cross-links, DNA can be cross-linked to another DNA strands or a protein. Cross-links may prevent the amplification of endogenous template molecules, but it could also stabilize the DNA molecule over time, hence reducing the fragmentation.

• Fragmentation

As described above, ancient DNA accumulates many types of damage, including strand breaks and depurination, which results in fragmentation to short DNA molecules. Because the DNA strands are already fragmented, we do not need to expose the DNA sample to further fragmentation in the Next Generation Sequencing sample preparation step. According to recent studies, the average length of ancient DNA fragments varies from 50-150 bp.