Article series: Applications of next-generation sequencing

Reconstructing ancient genomes and epigenomes

Journal name:
Nature Reviews Genetics
Year published:
Published online


Research involving ancient DNA (aDNA) has experienced a true technological revolution in recent years through advances in the recovery of aDNA and, particularly, through applications of high-throughput sequencing. Formerly restricted to the analysis of only limited amounts of genetic information, aDNA studies have now progressed to whole-genome sequencing for an increasing number of ancient individuals and extinct species, as well as to epigenomic characterization. Such advances have enabled the sequencing of specimens of up to 1 million years old, which, owing to their extensive DNA damage and contamination, were previously not amenable to genetic analyses. In this Review, we discuss these varied technical challenges and solutions for sequencing ancient genomes and epigenomes.

At a glance


  1. Major advances in ancient genomics.
    Figure 1: Major advances in ancient genomics.

    The major methodological advances described in this Review are presented with respect to milestones in paleogenomics, including whole-genome sequencing and the characterization of transcriptomes, epigenomes and proteomes. Average genome fold-coverage (×) and sequencing platforms are indicated where applicable. aDNA, ancient DNA; ssDNA, single-stranded DNA.

  2. Typical ancient DNA molecules.
    Figure 2: Typical ancient DNA molecules.

    A diverse range of degradation reactions affect DNA post-mortem and result in extensive fragmentation (preferentially at purine nucleotides) and base modifications. The most common base modification identified in high-throughput sequencing data sets is deamination of cytosines into uracils (red), or thymines (blue) when cytosines were methylated (mC). Such deaminations occur much faster at overhanging ends. Other modifications include abasic sites (green) and single-strand breaks (vertical lines). The chemical structures of three damage by-products (uracils, thymines and abasic sites) are shown. R, purine; Y, pyrimidine.

  3. Constructing ancient DNA libraries.
    Figure 3: Constructing ancient DNA libraries.

    The three most common types of ancient DNA (aDNA) libraries are shown. 5′-phosphate groups are indicated with black circles, single-strand DNA breaks are shown as vertical lines, biotinylated adaptor groups are shown in red, and streptavidin-coated beads are shown in grey. a | To construct a double-stranded DNA (dsDNA) library, aDNA is first end-repaired. It is then ligated to double-stranded adaptors (blue), and the resultant nicks are filled in to construct library templates devoid of single-strand breaks. b | To construct an A-tailed DNA library, aDNA is end-repaired and then A-tailed (that is, an adenine is added to the 3′ ends of the strands) to facilitate subsequent ligation to T-tailed adaptors while disfavouring ligation between adaptor pairs. The adaptors are typically Y-shaped (that is, they are complementary at the T-tailed end but have non-complementary arms at the other end). The use of such adaptors results in aDNA strands being flanked by distinct non-complementary adaptor sequences at each end to enable subsequent unidirectional sequencing through the aDNA fragment. Nicks resulting from ligation are filled-in through PCR post-ligation. c | To construct a single-stranded DNA (ssDNA) library, aDNA is first denatured into single strands using heat and then ligated to biotinylated single-stranded adaptors. The original DNA strand is then copied using DNA polymerase extension, and a second adaptor is ligated to enable further PCR amplification and sequencing. Purification steps are performed using streptavidin-coated paramagnetic beads. Part c adapted with permission from Ref. 16, American Association for the Advancement of Science.

  4. Enriching DNA libraries for ancient inserts.
    Figure 4: Enriching DNA libraries for ancient inserts.

    a | Selective uracil enrichment is shown. 5′-phosphate groups are indicated with black circles, single-strand DNA breaks are shown as vertical lines, biotinylated adaptor groups are shown in red, and streptavidin-coated beads are shown in grey. A single-stranded DNA (ssDNA) library is built until the polymerase extension step. DNA is then phosphorylated to enable the ligation of the second adaptor. This contrasts with the ssDNA library procedure, in which the ligation occurs between the 5′ end of the second adaptor and the 3′ end of the newly synthesized strand (Fig. 3c). DNA is then treated with uracil DNA glycosylase and endonuclease VIII (USER mix) to generate and then cleave out abasic sites at cytosines that were deaminated into uracils post-mortem. The 3′-phosphate groups at these new termini are then removed (not shown). The resulting 3′-OH ends now serve to prime an extension with a DNA polymerase, which copies throughout the whole length of the strand complementary to where the damage was. As a result, the supernatant now contains double-stranded DNA (dsDNA) library templates corresponding to the original deaminated strands. Other library templates remain unaffected and can be separated, as they remain bound to streptavidin-coated paramagnetic beads. b | In whole-genome in-solution capture (WISC), ssDNA templates from an ancient DNA (aDNA) library are prepared. The target, endogenous aDNA is shown as thin black lines, whereas the exogenous contaminating DNA is shown as thin green lines; adaptors are shown as thick blue lines. In parallel, a probe DNA library is prepared from fresh modern DNA extracts (thin red lines) and used to generate biotinylated RNA probes through in vitro transcription. T7 adaptors to enable in vitro transcription are shown in thick purple lines. The aDNA library is annealed to the RNA probes, low-complexity DNA and adaptor blockers (the latter two are not shown for simplicity). The library fraction of interest is then recovered following elution from streptavidin-coated paramagnetic beads. Part a adapted with permission from Ref. 68, Cold Spring Harbor Laboratory Press. Part b adapted with permission from Ref. 78, The American Society of Human Genetics.

  5. Tracking ancient nucleosome and methylation maps.
    Figure 5: Tracking ancient nucleosome and methylation maps.

    DNA wrapped around nucleosomes can be protected post-mortem and over-represented in high-throughput sequencing (HTS) data. Therefore, depth-of-coverage patterns along the genome can be exploited to position the location of nucleosomes on ancient genomes. Similarly, post-mortem deamination at CpG sites transforms methylated CpG (mCpG) sites into TpG sites but transforms unmethylated CpG sites into UpG sites. With molecular tools disabling the sequencing of the UpGs, CpGright arrowTpG mutations in HTS data provides an opportunity to detect ancient mCpGs, with hypomethylated regions showing low CpGright arrowTpG conversion rates and hypermethylated regions showing high CpGright arrowTpG conversion rates. Adapted with permission from Ref. 38, American Association for the Advancement of Science.


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  1. Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5–7, Copenhagen 1350C, Denmark.

    • Ludovic Orlando,
    • M. Thomas P. Gilbert &
    • Eske Willerslev
  2. Université de Toulouse, University Paul Sabatier (UPS), Laboratoire AMIS, CNRS UMR 5288, 37 allées Jules Guesde, 31000 Toulouse, France.

    • Ludovic Orlando
  3. Trace and Environmental DNA Laboratory, Department of Environment and Agriculture, Curtin University, Perth, Western Australia 6102, Australia.

    • M. Thomas P. Gilbert

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  • Ludovic Orlando

    Ludovic Orlando received his Ph.D. in molecular genetics from the University of Lyon, France, 20 years after the first ancient DNA molecule was sequenced. He opened his laboratory in 2010 at the Centre of Excellence in GeoGenetics at Copenhagen University, Denmark, focusing on the development of innovative molecular and computational methods tailored to the analysis of ancient genomes. His recent work includes the characterization of the oldest mammalian genome and the construction of the first ancient human epigenomic maps. He is now reconstructing the genomes of ancient horses, spanning different stages of the domestication process to better understand the origins of the modern horse. Ludovic Orlando's homepage.

  • M. Thomas P. Gilbert

    M. Thomas P. Gilbert holds a D.Phil. from the University of Oxford, UK, where his research focused on overcoming the challenges in ancient human DNA analyses in the pre-high-throughput sequencing era. His interests in ancient DNA range from pathogens (for example, HIV-1 and Phytophthora infestans) to plants (for example, maize and grapes) and animals (including humans), and much of his research focuses on the optimization of methods to maximize the information potential that such samples hold for archaeological and evolutionary biological studies.

  • Eske Willerslev

    Eske Willerslev is an evolutionary geneticist. He is currently a Lundbeck Foundation professor at the University of Copenhagen, Denmark, and the Prince Philip Professor of Ecology and Evolutionary Biology elect at the University of Cambridge, UK. He is Director of the Centre of Excellence in GeoGenetics, Copenhagen. He focuses his research on understanding the processes that resulted in the genetic and cultural diversity among humans today.

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