Paleogenomics is a field of science based on the reconstruction and analysis of genomic information in extinct species. Improved methods for the extraction of ancient DNA (aDNA) from museum artifacts, ice cores, archeological or paleontological sites, and next-generation sequencing technologies have spurred this field. It is now possible to detect genetic drift, ancient population migration and interrelationships, the evolutionary history of extinct plant, animal and Homo species, and identification of phenotypic features across geographic regions. Scientists can also use paleogenomics to compare ancient ancestors against modern-day humans. The rising importance of paleogenomics is evident from the fact that the 2022 Nobel Prize in physiology or medicine was awarded to a Swedish geneticist Svante Pääbo [1955-], who worked on paleogenomics.

Initially, aDNA sequencing involved cloning small fragments into bacteria, which proceeded with low efficiency due to the oxidative damage the aDNA suffered over millennia. aDNA is difficult to analyze due to facile degradation by nucleases; specific environments and postmortem conditions improved isolation and analysis. Extraction and contamination protocols were necessary for reliable analyses. With the development of the Polymerase Chain Reaction (PCR) in 1983, scientists could study DNA samples up to approximately 100,000 years old, a limitation of the relatively short isolated fragments. Through advances in isolation, amplification, sequencing, and data reconstruction, older and older samples have become analyzable. Over the past 30 years, high copy number mitochondrial DNA was able to answer many questions; the advent of NGS techniques prompted far more. Moreover, this technological revolution allowed the transition from paleogenetics to paleogenomics.

PCR, NGS second generation, and various library methods are available for sequencing aDNA, besides many bioinformatics tools. When dealing with each of these methods it is important to consider that aDNA can be altered post-mortem. Specific alterations arise from:

Specific patterns and onset of these alterations help scientists to estimate the sample's age.

Formerly, scientists diagnosed post-mortem damages using enzymatic reactions or gas chromatography associated with mass spectroscopy; in more recent years scientists began to detect them by exploiting mutational sequence data. This strategy allows to identify excess of C->T mutations following treatment with uracil DNA glycosylase. Nowadays, one uses high-throughput sequencing (HTS) to identify depurination (a process that drives post-mortem DNA fragmentation, younger samples present more adenine than guanine), single strand breaks in double helix of DNA and abasic site (created by C->T mutation).
A single fragment of aDNA can be sequenced in its full length with HTS. With these data we can create a distribution representing a size decay curve that enables a direct quantitative comparison of fragmentation across specimens through space and environmental conditions. Throughout the decay curve it is possible to obtain the median length of the given fragment of aDNA. This length reflects the fragmentation levels after death, which generally increases with depositional temperature.

Two different libraries can be performed for aDNA sequencing using PCR for genome amplification:

The first one is created using the blunt-end approach. This technique uses two different adaptors: these adaptors bind randomly the fragment and it can then be amplified. The fragment that does not contain both adaptors cannot be amplified causing an error source. To reduce this error, Illumina T/A ligation was introduced: this method consists in inserting the A tailing in DNA sample to facilitate the ligation of T tailed adaptors. In this methods we optimize the amplification of the aDNA.

To obtain ssDNA libraries, DNA is first denatured with heat. The obtained ssDNA is then ligated to two adaptors in order to generate the complementary strand and finally PCR is applied.