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Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing

Abstract

DNA preserved in ancient bones, teeth and sediments is typically highly fragmented and present only in minute amounts. Here, we provide a highly versatile silica-based DNA extraction protocol that enables the retrieval of short (≥35 bp) or even ultrashort (≥25 bp) DNA fragments from such material with minimal carryover of substances that inhibit library preparation for high-throughput sequencing. DNA extraction can be performed with either silica spin columns, which offer the most convenient choice for manual DNA extraction, or silica-coated magnetic particles. The latter allow a substantial cost reduction as well as automation on liquid-handling systems. This protocol update replaces a now-outdated version that was published 11 years ago, before high-throughput sequencing technologies became widely available. It has been thoroughly optimized to provide the highest DNA yields from highly degraded samples, as well as fast and easy handling, requiring not more than ~15 min of hands-on time per sample.

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Fig. 1
Fig. 2: DNA fragment length distribution in libraries prepared from extracts generated with the protocol options described here.
Fig. 3: Boxplots showing the fraction of fragments ≥35 bp in libraries prepared from extracts using the protocol options described here (n = 12).
Fig. 4: Influence of the extraction method on the informative sequence content of the libraries.
Fig. 5: Boxplots showing the influence of the extraction method on library preparation efficiency, as inferred from the conversion rate of the control oligonucleotide that was spiked into each library preparation reaction (n = 6).

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Acknowledgements

We thank S. Pääbo and D. Reich for their support; A. Weihmann and B. Schellbach for performing the sequencing runs; J. Kelso and J. Visagie for help with raw data processing; N. Broomand, M. Ferry, M. Michel, J. Oppenheimer and K. Stewardson for support in the lab; and S. Mallick for bioinformatics processing of initial experiments. We also thank P. Rudan, C. Verna, T. Kutznetsova, K. Post, G. Rabeder, M. Shunkov, R. Roberts, A. Derevianko, R. Miller, J. Stewart and M. Soressi for providing the samples. This work was funded by the Strategic Innovation Fund of the Max Planck Society and ERC grant agreement no. 694707 to S. Pääbo.

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Authors

Contributions

N.R., I.G., A.A.-P. and M.M. designed experiments. N.R., I.G. and A.A.-P. performed experiments. N.R., I.G. and M.M. analyzed the data and wrote the paper.

Corresponding authors

Correspondence to Nadin Rohland or Isabelle Glocke.

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The authors declare no competing interests.

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Related links

Key references using this protocol

1. Slon, V. et al. Science 356, 605–608 (2017): https://doi.org/10.1126/science.aam9695

2. Meyer, M. et al. Nature 505, 403–406 (2014): https://doi.org/10.1038/nature12788

3. Olalde, I. et al. Nature 555, 190–196 (2018): https://doi.org/10.1038/nature25738

This protocol is an update to: Nat. Protoc. 2, 1756–1762 (2007): https://doi.org/10.1038/nprot.2007.247

Supplementary information

Supplementary Table 1

Summary of sequencing results

Reporting Summary

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Rohland, N., Glocke, I., Aximu-Petri, A. et al. Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing. Nat Protoc 13, 2447–2461 (2018). https://doi.org/10.1038/s41596-018-0050-5

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