Ancient DNA analysis | Nature Reviews Methods Primers

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

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS THE ALLEN ANCIENT DNA RESOURCE (AADR) A CURATED COMPENDIUM OF ANCIENT HUMAN GENOMES Article Open access 10 February 2024 MORE

THAN A DECADE OF GENETIC RESEARCH ON THE DENISOVANS Article 18 September 2023 ACCURATE DETECTION OF IDENTITY-BY-DESCENT SEGMENTS IN HUMAN ANCIENT DNA Article Open access 20 December 2023

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Preprint at _bioRxiv_ https://doi.org/10.1101/2020.09.02.279570 (2020). Article  Google Scholar  Download references 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). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Centre d’Anthropobiologie et de Génomique de

Toulouse, Université Paul Sabatier Toulouse III, Faculté de Médecine Purpan, Toulouse, France Ludovic Orlando & Clio Der Sarkissian * School of Life Sciences, University of Warwick,

Coventry, UK Robin Allaby * Ancient Genomics Laboratory, Francis Crick Institute, London, UK Pontus Skoglund * Institute for Pre- and Protohistoric Archaeology and Archaeology of the Roman

Provinces, Ludwig Maximilian University, Munich, Germany Philipp W. Stockhammer * Department of Archaeogenetics, Max Planck Institute for the Science of Human History, Jena, Germany Philipp

W. Stockhammer, Johannes Krause & Christina Warinner * International Laboratory for Human Genome Research, National Autonomous University of Mexico, Queretaro, Mexico María C.

Ávila-Arcos * Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Center for Excellence in Life and Paleoenvironment,

Chinese Academy of Sciences, Beijing, China Qiaomei Fu * Lundbeck Foundation GeoGenetics Center, GLOBE Institute, University of Copenhagen, Copenhagen, Denmark Eske Willerslev * Welcome

Trust, Sanger Institute, Hinxton, UK Eske Willerslev * The Danish Institute for Advanced Study, University of Southern Denmark, Odense, Denmark Eske Willerslev * Department of Zoology,

University of Cambridge, Cambridge, UK Eske Willerslev * School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, USA Anne C. Stone * Department of Anthropology,

Harvard University, Cambridge, MA, USA Christina Warinner Authors * Ludovic Orlando View author publications You can also search for this author inPubMed Google Scholar * Robin Allaby View

author publications You can also search for this author inPubMed Google Scholar * Pontus Skoglund View author publications You can also search for this author inPubMed Google Scholar * Clio

Der Sarkissian View author publications You can also search for this author inPubMed Google Scholar * Philipp W. Stockhammer View author publications You can also search for this author

inPubMed Google Scholar * María C. Ávila-Arcos View author publications You can also search for this author inPubMed Google Scholar * Qiaomei Fu View author publications You can also search

for this author inPubMed Google Scholar * Johannes Krause View author publications You can also search for this author inPubMed Google Scholar * Eske Willerslev View author publications You

can also search for this author inPubMed Google Scholar * Anne C. Stone View author publications You can also search for this author inPubMed Google Scholar * Christina Warinner View author

publications You can also search for this author inPubMed Google Scholar 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.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _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. PUBLISHER’S NOTE Springer Nature remains neutral

with regard to jurisdictional claims in published maps and institutional affiliations. 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. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

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 Download citation * Accepted: 16

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