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Ancient DNA analysis

Abstract

Although the first ancient DNA molecules were extracted more than three decades ago, the first ancient nuclear genomes could only be characterized after high-throughput sequencing was invented. Genome-scale data have now been gathered from thousands of ancient archaeological specimens, and the number of ancient biological tissues amenable to genome sequencing is growing steadily. Ancient DNA fragments are typically ultrashort molecules and carry extensive amounts of chemical damage accumulated after death. Their extraction, manipulation and authentication require specific experimental wet-laboratory and dry-laboratory procedures before patterns of genetic variation from past individuals, populations and species can be interpreted. Ancient DNA data help to address an entire array of questions in anthropology, evolutionary biology and the environmental and archaeological sciences. The data have revealed a considerably more dynamic past than previously appreciated and have revolutionized our understanding of many major prehistoric and historic events. This Primer provides an overview of concepts and state-of-the-art methods underlying ancient DNA analysis and illustrates the diversity of resulting applications. The article also addresses some of the ethical challenges associated with the destructive analysis of irreplaceable material, emphasizes the need to fully involve archaeologists and stakeholders as part of the research design and analytical process, and discusses future perspectives.

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Fig. 1: Analytical milestones in aDNA research.
Fig. 2: Experimental workflow.
Fig. 3: Post-mortem DNA damage and data authentication.
Fig. 4: Examples of standard aDNA statistical analyses applied to human and microbiome data.
Fig. 5: Geographical and temporal distribution of ancient specimens analysed at the genome scale.

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Acknowledgements

The authors thank A. Hübner for assistance with figure 4c. L.O., P.S., P.W.S. and C.W. received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreements ERC-2015-CoG 681605-PEGASUS, ERC-2018-StG 852558-AGRICON, ERC-2015-StG 678901-FoodTransforms and ERC-2017-StG 804844-DAIRYCULTURES, respectively). L.O. was also supported by ANR (LifeChange) and the Simone et Cino Del Duca Foundation (HealthTimeTravel). P.S. was also supported by the Francis Crick Institute core funding (FC001595) from Cancer Research UK, the UK Medical Research Council and the Wellcome Trust, a Wellcome Trust Investigator award (217223/Z/19/Z) and the Vallee Foundation. C.W. also received funding from the Max Planck Society, the Deutsche Forschungsgemeinschaft (EXC 2051 #390713860) and the Siemens Foundation (Paleobiochemistry).

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Authors and Affiliations

Authors

Contributions

Introduction (L.O., A.C.S. and C.W.); Experimentation (L.O., R.A., C.D.S., P.W.S., A.C.S. and C.W.); Results (L.O., P.S., R.A., P.W.S., M.C.A.-A. and C.W.); Applications (L.O., R.A., P.W.S., C.D.S., M.C.A.-A., Q.F., J.K., E.W., A.C.S. and C.W.); Reproducibility and data deposition (L.O. and M.C.A.-A.); Limitations and optimizations (L.O.); Outlook (L.O., P.W.S., A.C.S. and C.W.); Overview of the Primer (L.O. and C.W.).

Corresponding author

Correspondence to Ludovic Orlando.

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Nature Reviews Methods Primers thanks T. Günther, L. Matisoo-Smith, R. Pinhasi, N. Rawlence, A. Zink and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Ancient Human DNA uMap: https://umap.openstreetmap.fr/en/map/ancient-human-dna_41837#6/51.000/2.000

Bitbucket: https://bitbucket.org/product

European Nucleotide Archive (ENA): https://www.ebi.ac.uk/ena

GitHub: https://github.com

International Symposium for Biomolecular Archaeology (ISBA): https://isba9.sciencesconf.org

Max Planck Harvard Research Center for the Archaeoscience of the Ancient Mediterranean: https://www.archaeoscience.org/

Protocols.io: https://www.protocols.io

Reich laboratory: https://reich.hms.harvard.edu/datasets

Sequence Read Archive (SRA): https://www.ncbi.nlm.nih.gov/sra

Standards and Precautions and Advances in Ancient Metagenomics (SPAAM):https://github.com/SPAAM-workshop

Glossary

Ancient DNA

(aDNA). Ultrashort and degraded DNA fragments that are preserved in subfossil material, including hard tissues, such as bones, teeth and shells, and soft tissues, such as mummified skin and hair, as well as sediments.

Holobiomes

The total sum of the DNA fragments making up the genome of a host organism and all of its microbiota.

DNA library

A molecular construction in which DNA fragments are ligated to DNA adapters of known sequences in order to be amplified and optionally captured prior to sequencing; different sequencing platforms require different library constructs.

DNA barcoding

The taxonomic assignment of metagenomic DNA content on the basis of DNA fragments that show limited intra-specific sequence diversity but large inter-specific sequence diversity.

Shotgun sequencing

Non-targeted sequencing of DNA library content.

DNA ligases

A class of enzymes that are capable of stitching together different DNA fragments.

Ascertainment bias

Statistical bias resulting from the collection of genetic data at a subset of loci that do not reflect the overall genetic diversity present at the whole-genome scale.

Demultiplexing

A process by which pools of sequences originating from different DNA libraries are assigned back to their original samples on the basis of short synthetic sequences added during library indexing.

Outgroup

An individual, a population or a group of populations and/or species that are genetically close but different from those under study.

Identity by descent

DNA segments between two or more individuals are identical by descent when they are inherited from a common ancestor in the absence of recombination.

Procrustes analysis

Also known as Procrustes superimposition. A statistical method allowing the translation, rotation and scaling of multidimensional objects within a single analytical space where they can be compared.

16S meta-barcodes

Selected variable regions of the 16S ribosomal RNA gene whose sequence provides taxonomic resolution amongst bacteria and archaea.

DNA methylation

A biological process by which the activity of a DNA segment is modified without changing the underlying sequence but by adding methyl groups to the DNA molecule.

Bisulfite conversion

A chemical reaction using sodium bisulfite that converts unmethylated CpG dinucleotides into UpGs but leaves methylated CpGs intact, thereby allowing the detection of DNA methylation by sequencing.

Immunoprecipitation

A molecular laboratory technique by which specific molecules are purified on the basis of their chemical affinities for particular protein groups, such as antibodies.

Population replacement

A population process by which the gene pool of one local population is at least partially replaced by that coming from another, genetically distinct, population.

Environmental DNA

(eDNA). Fragments of DNA that are preserved within sediments and water that can be used for a fast, cost-effective monitoring of the ecology of a given region.

Stratigraphic leaching

The migration of DNA across strata in sediments caused by water movement, microorganism growth or bioturbation and compromising the reliability of the stratigraphy, that is, the order, position and age of the geological layers formed by the different piles of sediments.

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Orlando, L., Allaby, R., Skoglund, P. et al. Ancient DNA analysis. Nat Rev Methods Primers 1, 14 (2021). https://doi.org/10.1038/s43586-020-00011-0

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