Isolating the human cochlea to generate bone powder for ancient DNA analysis

Article metrics


The cortical bone that forms the structure of the cochlea, part of the osseous labyrinth of the inner ear, is now one of the most frequently used skeletal elements in analyses of human ancient DNA. However, there is currently no published, standardized method for its sampling. This protocol describes the preparation of bone powder from the cochlea of fragmented skulls in which the petrous pyramid of the temporal bone is accessible. Using a systematic process of bone removal based on distinct anatomical landmarks and the identification of relevant morphological features, a petrous pyramid is cleaned with a sandblaster, and the cochlea is located, isolated, and reduced to a homogeneous bone powder. All steps are carried out in dedicated ancient DNA facilities, thus reducing the introduction of contamination. This protocol requires an understanding of ancient DNA clean-room procedures and basic knowledge of petrous pyramid anatomy. In 50–65 min, it results in bone powder with endogenous DNA yields that can exceed those from teeth and other bones by up to two orders of magnitude. Compared with drilling methods, this method facilitates a more precise targeting of the cochlea, allows the user to visually inspect the cochlea and remove any residual sediment before the generation of bone powder, and confines the damage to the inner ear region and surface of the petrous portion of fragmentary crania.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Step-by-step walkthrough of the isolation and cleaning of the cochlea for a DNA analysis.
Fig. 2: Comparative yields of endogenous ancient DNA obtained from the cochlea alone and from the cochlea plus the surrounding dense bone.

Data availability

The raw sequencing data that support the findings of this study have been deposited in the Sequence Read Archive under accession code SRP058345.


  1. 1.

    Rasmussen, M. et al. Ancient human genome sequence of an extinct Palaeo-Eskimo. Nature 463, 757–762 (2010).

  2. 2.

    Slatkin, M. & Racimo, F. Ancient DNA and human history. Proc. Natl. Acad. Sci. USA 113, 6380–6387 (2016).

  3. 3.

    Pickrell, J. & Reich, D. Towards a new history and geography of human genes informed by ancient DNA. Trends Genet. 30, 377–389 (2014).

  4. 4.

    Huerta-Sánchez, E. et al. Altitude adaptation in Tibetans caused by introgression of Denisovan-like DNA. Nature 512, 194–197 (2014).

  5. 5.

    Lazaridis, I. et al. Genomic insights into the origin of farming in the ancient Near East. Nature 536, 419–424 (2016).

  6. 6.

    Mathieson, I. et al. Genome-wide patterns of selection in 230 ancient Eurasians. Nature 528, 499–503 (2015).

  7. 7.

    Pinhasi, R. et al. Optimal ancient DNA yields from the inner ear part of the human petrous bone. PLoS One 10, e0129102 (2015).

  8. 8.

    Skoglund, P. et al. Genomic insights into the peopling of the Southwest Pacific. Nature 538, 510–513 (2016).

  9. 9.

    Jónsson, H., Ginolhac, A., Schubert, M., Johnson, P. L. F. & Orlando, L. mapDamage2.0: fast approximate Bayesian estimates of ancient DNA damage parameters. Bioinformatics 29, 1682–1684 (2013).

  10. 10.

    Ginolhac, A., Rasmussen, M., Gilbert, M. T. P., Willerslev, E. & Orlando, L. mapDamage: testing for damage patterns in ancient DNA sequences. Bioinformatics 27, 2153–2155 (2011).

  11. 11.

    Briggs, A. W. et al. Patterns of damage in genomic DNA sequences from a Neandertal. Proc. Natl. Acad. Sci. USA 104, 14616–14621 (2007).

  12. 12.

    Bollongino, R., Tresset, A. & Vigne, J.-D. Environment and excavation: pre-lab impacts on ancient DNA analyses. Comptes Rendus Palevol 7, 91–98 (2008).

  13. 13.

    Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl. Acad. Sci. USA 110, 15758–15763 (2013).

  14. 14.

    Gansauge, M.T. et al. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. Nucleic Acids Res 45, e79 (2017).

  15. 15.

    Damgaard, P. B. et al. Improving Access to Endogenous DNA in Ancient Bones and Teeth (Springer, New York, 2015).

  16. 16.

    Korlević, P. et al. Reducing microbial and human contamination in DNA extractions from ancient bones and teeth. Biotechniques 59, 87–93 (2015).

  17. 17.

    Boessenkool, S. et al. Combining bleach and mild predigestion improves ancient DNA recovery from bones. Mol. Ecol. Resour. 17, 742–751 (2016).

  18. 18.

    Nieves-Colón Maria, A. et al. Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments. Am. J. Phys. Anthropol. 166, 824–836 (2018).

  19. 19.

    Hajdinjak, M. et al. Reconstructing the genetic history of late Neanderthals. Nature 555, 652–656 (2018).

  20. 20.

    Götherström, A., Collins, M. J., Angerbjörn, A. & Lidén, K. Bone preservation and DNA amplification. Archaeometry 44, 395–404 (2002).

  21. 21.

    Sosa, C. et al. Association between ancient bone preservation and DNA yield: a multidisciplinary approach. Am. J. Phys. Anthropol. 151, 102–109 (2013).

  22. 22.

    Ottoni, C. et al. Preservation of ancient DNA in thermally damaged archaeological bone. Naturwissenschaften 96, 267–278 (2009).

  23. 23.

    Lipson, M. et al. Ancient genomes document multiple waves of migration in Southeast Asian prehistory. Science 361, 92–95 (2018).

  24. 24.

    Lipson, M. et al. Population turnover in Remote Oceania shortly after initial settlement. Curr. Biol. 28, 1157–1165 (2018).

  25. 25.

    Skoglund, P. et al. Reconstructing prehistoric African population structure. Cell 171, 59–71 (2017).

  26. 26.

    Standring, S. Gray’s Anatomy E-Book: The Anatomical Basis of Clinical Practice (Elsevier Health Sciences, Philadelphia, 2015).

  27. 27.

    Gamba, C. et al. Genome flux and stasis in a five millennium transect of European prehistory. Nat. Commun. 5, 1–9 (2014).

  28. 28.

    Hansen, H. B. et al. Comparing ancient DNA preservation in petrous bone and tooth cementum. PLoS One 12, e0170940 (2017).

  29. 29.

    Hernandez, C. J., Majeska, R. J. & Schaffler, M. B. Osteocyte density in woven bone. Bone 35, 1095–1099 (2004).

  30. 30.

    Rask‐Andersen, H. et al. Human cochlea: anatomical characteristics and their relevance for cochlear implantation. Anat. Rec. 295, 1791–1811 (2012).

  31. 31.

    Douka, K. et al. Direct radiocarbon dating and DNA analysis of the Darra-i-Kur (Afghanistan) human temporal bone. J. Hum. Evol. 107, 86–93 (2017).

  32. 32.

    Lazaridis, I. et al. Genetic origins of the Minoans and Mycenaeans. Nature 548, 214–218 (2017).

  33. 33.

    Mathieson, I. et al. The genomic history of southeastern Europe. Nature 555, 197–203 (2018).

  34. 34.

    Prendergast, M. E. & Sawchuk, E. Boots on the ground in Africa’s ancient DNA ‘revolution’: archaeological perspectives on ethics and best practices. Antiquity 92, 803–815 (2018).

  35. 35.

    Sirak, K. A. et al. A minimally-invasive method for sampling human petrous bones from the cranial base for ancient DNA analysis. Biotechniques 62, 283–289 (2017).

  36. 36.

    Ponce de León, M. S. et al. Human bony labyrinth is an indicator of population history and dispersal from Africa. Proc. Natl. Acad. Sci. USA 115, 4128–4133 (2018).

  37. 37.

    Jeffery, N. & Spoor, F. Prenatal growth and development of the modern human labyrinth. J. Anat. 204, 71–92 (2004).

  38. 38.

    Richard, C. et al. New insight into the bony labyrinth: a microcomputed tomography study. Auris Nasus Larynx 37, 155–161 (2010).

  39. 39.

    Benjamin, O. et al. Sexual dimorphism of the bony labyrinth: a new age-independent method. Am. J. Phys. Anthropol. 151, 290–301 (2013).

  40. 40.

    Spoor, F. The semicircular canal system and locomotor behaviour, with special reference to hominin evolution. Courier Forschungsinstitut Senckenberg 243, 93–104 (2003).

  41. 41.

    Spoor, F., Wood, B. & Zonneveld, F. Implications of early hominid labyrinthine morphology for evolution of human bipedal locomotion. Nature 369, 645–648 (1994).

  42. 42.

    Philipp, G., Marissa, R., Melanie, K., Jean-Jacques, H. & Fred, S. The mammalian bony labyrinth reconsidered, introducing a comprehensive geometric morphometric approach. J. Anat. 220, 529–543 (2012).

  43. 43.

    Hublin, J. J., Spoor, F., Braun, M., Zonneveld, F. & Condemi, S. A late Neanderthal associated with Upper Palaeolithic artefacts. Nature 381, 224–226 (1996).

  44. 44.

    Jackler, R. K. & Dillon, W. P. Computed tomography and magnetic resonance imaging of the inner ear. Otolaryngol. Head Neck Surg. 99, 494–504 (1988).

  45. 45.

    Rohland, N., Glocke, I., Aximu-Petri, A. & Meyer, M. Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing. Nat. Protoc. 13, 2447–2461 (2018).

  46. 46.

    Meyer, M. & Kircher, M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb. Protoc. 2010, pdb.prot5448 (2010).

  47. 47.

    Cooper, A. & Poinar, H. N. Ancient DNA: do it right or not at all. Science 289, 1139 (2000).

  48. 48.

    Knapp, M., Clarke, A. C., Horsburgh, K. A. & Matisoo-Smith, E. A. Setting the stage—building and working in an ancient DNA laboratory. Ann. Anat. 194, 3-6 (2012).

  49. 49.

    Pääbo, S. et al. Genetic analyses of ancient DNA. Ann. Rev. Genet. 38, 645–679 (2004).

  50. 50.

    Poinar, H. N. The top 10 list: criteria of authenticity for DNA from ancient and forensic samples. Int. Cong. Ser. 1239, 575–579 (2003).

  51. 51.

    Fulton, T. L. Setting up an ancient DNA laboratory. in Ancient DNA (eds Shapiro, B. & Hofreiter, M.) 1–11 (Humana Press, New York, 2012).

  52. 52.

    Champlot, S. et al. An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications. PLoS One 5, e13042 (2010).

  53. 53.

    Peltzer, A. et al. EAGER: efficient ancient genome reconstruction. Genome Biol. 17, 60 (2016).

Download references


We thank E. Harney, P. Skoglund, and D. Reich for their comments on the manuscript. Support for this project was provided by ERC starting grant ADNABIOARC (263441) to R.P.

Author information

R.P. conceived and developed the concept, and provided supervision. D.F., O.C., and K.S. designed the protocol and contributed expertise. R.P., D.F., O.C., and K.S. contributed equally to the writing and editing of the manuscript.

Correspondence to Ron Pinhasi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Pinhasi, R. et al. PLoS One 10, e0129102 (2015):

Skoglund, P. et al. Nature 538, 510–513 (2016):

Lazaridis, J. et al. Nature 536, 419–424 (2016):

Mathieson, I. et al. Nature 528, 499–503 (2015):

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pinhasi, R., Fernandes, D.M., Sirak, K. et al. Isolating the human cochlea to generate bone powder for ancient DNA analysis. Nat Protoc 14, 1194–1205 (2019) doi:10.1038/s41596-019-0137-7

Download citation


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.