Ancient DNA pp 163-194 | Cite as

Authentication and Assessment of Contamination in Ancient DNA

  • Gabriel Renaud
  • Mikkel Schubert
  • Susanna Sawyer
  • Ludovic OrlandoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1963)


Contamination from both present-day humans and postmortem microbial sources is a common challenge in ancient DNA studies. Here we present a suite of tools to assist in the assessment of contamination in ancient DNA data sets. These tools perform standard tests of authenticity of ancient DNA data including detecting the presence of postmortem damage signatures in sequence alignments and quantifying the amount of present-day human contamination.

Key words

Contamination Ancient DNA Postmortem damage Schmutzi DICE mapDamage2.0 



We would like to thank Fernando Racimo for comments and suggestions and José Victor Moreno Mayar and Thorfinn Sand Korneliussen for their insights into the contamination method using the X chromosome. This work was supported by the Danish Council for Independent Research, Natural Sciences (Grant 4002-00152B); the Danish National Research Foundation (Grant DNRF94); Initiative d’Excellence Chaires d’attractivité, Université de Toulouse (OURASI); the Villum Fonden miGENEPI research project; and the European Research Council (ERC-CoG-2015-681605).


  1. 1.
    Ermini L, Der Sarkissian C, Willerslev E, Orlando L (2015) Major transitions in human evolution revisited: a tribute to ancient DNA. J Hum Evol 79:4–20. Scholar
  2. 2.
    Llamas B, Fehren-Schmitz L, Valverde G et al (2016) Ancient mitochondrial DNA provides high-resolution time scale of the peopling of the. Am Sci Adv.
  3. 3.
    Librado P, Der Sarkissian C, Ermini L et al (2015) Tracking the origins of Yakutian horses and the genetic basis for their fast adaptation to subarctic environments. Proc Natl Acad Sci U S A 112:E6889–E6897. Scholar
  4. 4.
    Frantz LAF, Mullin VE, Pionnier-Capitan M et al (2016) Genomic and archaeological evidence suggest a dual origin of domestic dogs. Science 352:1228–1231. Scholar
  5. 5.
    MacHugh DE, Larson G, Orlando L (2016) Taming the past: ancient DNA and the study of animal domestication. Annu Rev Anim Biosci.
  6. 6.
    Der Sarkissian C, Ermini L, Schubert M et al (2015) Evolutionary genomics and conservation of the endangered Przewalski’s horse. Curr Biol 25:2577–2583. Scholar
  7. 7.
    Da Fonseca RR, Smith BD, Wales N, et al (2015) The origin and evolution of maize in the Southwestern United States. Nat Plants. doi:
  8. 8.
    Bos KI, Schuenemann VJ, Golding GB et al (2011) A draft genome of Yersinia pestis from victims of the Black Death. Nature.
  9. 9.
    Wagner MR, Lundberg DS, Coleman-Derr D et al (2015) Corrigendum to Wagneret al.: natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative. Ecol Lett.
  10. 10.
    Ramos-Madrigal J, Smith BD, Moreno-Mayar JV et al (2016) Genome sequence of a 5,310-year-old maize cob provides insights into the early stages of maize domestication. Curr Biol 26:3195–3201. Scholar
  11. 11.
    Rasmussen S, Allentoft ME, Nielsen K et al (2015) Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. Cell 163:571–582. Scholar
  12. 12.
    Orlando L, Ginolhac A, Zhang G et al (2013) Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499:74–78. Scholar
  13. 13.
    Dabney J, Knapp M, Glocke I et al (2013) Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc Natl Acad Sci U S A 110:15758–15763. Scholar
  14. 14.
    Meyer M, Arsuaga J-L, de Filippo C et al (2016) Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins. Nature 531:504–507. Scholar
  15. 15.
    Meyer M, Fu Q, Aximu-Petri A et al (2014) A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature 505:403–406. Scholar
  16. 16.
    Hofreiter M, Serre D, Poinar HN et al (2001) Ancient DNA. Nat Rev Genet 2:353–359. Scholar
  17. 17.
    Briggs AW, Stenzel U, Johnson PL et al (2007) Patterns of damage in genomic DNA sequences from a Neandertal. Proc Natl Acad Sci U S A 104:14616–14621. Scholar
  18. 18.
    Green RE, Malaspinas A-S, Krause J et al (2008) A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing. Cell 134:416–426. Scholar
  19. 19.
    Gilbert MTP, Wilson AS, Bunce M, Hansen AJ, Willerslev E, Shapiro B, Higham TFG, Richards MP, O’Connell TC, Tobin DJ, Janaway RC, Cooper A (2004) Ancient mitochondrial DNA from hair. Curr Biol 14:R463–R464CrossRefGoogle Scholar
  20. 20.
    Pilli E, Modi A, Serpico C et al (2013) Monitoring DNA contamination in handled vs. directly excavated ancient human skeletal remains. PLoS One.
  21. 21.
    Korlević P, Gerber T, Gansauge M-T et al (2015) Reducing microbial and human contamination in DNA extractions from ancient bones and teeth. BioTechniques 59:87–93. Scholar
  22. 22.
    Guschanski K, Krause J, Sawyer S et al (2013) Next-generation museomics disentangles one of the largest primate radiations. Syst Biol 62:539–554. Scholar
  23. 23.
    Pruvost M, Schwarz R, Correia VB et al (2007) Freshly excavated fossil bones are best for amplification of ancient DNA. Proc Natl Acad Sci U S A 104:739–744. Scholar
  24. 24.
    Champlot S, Berthelot C, Pruvost M et al (2010) An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications. PLoS One.
  25. 25.
    Serre D, Langaney A, Chech M, et al (2004) No evidence of Neandertal mtDNA contribution to early modern humans. PLoS Biol.
  26. 26.
    Brown S, Higham T, Slon V et al (2016) Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis. Sci Rep.
  27. 27.
    Briggs AW, Good JM, Green RE et al (2009) Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325:318–321. Scholar
  28. 28.
    Sawyer S, Renaud G, Viola B et al (2015) Nuclear and mitochondrial DNA sequences from two Denisovan individuals. Proc Natl Acad Sci U S A 112:15696–15700. Scholar
  29. 29.
    Lazaridis I, Patterson N, Mittnik A et al (2014) Ancient human genomes suggest three ancestral populations for present-day Europeans. Nature 513:409–413. Scholar
  30. 30.
    Fu Q, Li H, Moorjani P et al (2014) Genome sequence of a 45,000-year-old modern human from western Siberia. Nature 514:445–449. Scholar
  31. 31.
    Allentoft ME, Sikora M, Sjögren K-G et al (2015) Population genomics of Bronze Age Eurasia. Nature 522:167–172. Scholar
  32. 32.
    Haak W, Lazaridis I, Patterson N et al (2015) Massive migration from the steppe was a source for Indo-European languages in Europe. Nature 522:207–211. Scholar
  33. 33.
    Krause J, Briggs AW, Kircher M et al (2010) A complete mtDNA genome of an early modern human from Kostenki, Russia. Curr Biol 20:231–236. Scholar
  34. 34.
    Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–715. Scholar
  35. 35.
    Seguin-Orlando A, Schubert M, Clary J et al (2013) Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes. PLoS One.
  36. 36.
    Meyer M, Kircher M, Gansauge M-T et al (2012) A high-coverage genome sequence from an archaic Denisovan individual. Science 338:222–226. Scholar
  37. 37.
    Wales N, Ramos Madrigal J, Cappellini E et al (2016) The limits and potential of paleogenomic techniques for reconstructing grapevine domestication. J Archaeol Sci.
  38. 38.
    Seguin-Orlando A, Hoover CA, Vasiliev SK et al (2015) Amplification of TruSeq ancient DNA libraries with AccuPrime Pfx: consequences on nucleotide misincorporation and methylation patterns. Sci Technol Archaeol Res.
  39. 39.
    Ginolhac A, Rasmussen M, Gilbert MTP et al (2011) mapDamage: testing for damage patterns in ancient DNA sequences. Bioinformatics 27:2153–2155. Scholar
  40. 40.
    Jónsson H, Ginolhac A, Schubert M et al (2013) mapDamage2.0: fast approximate Bayesian estimates of ancient DNA damage parameters. Bioinformatics 29:1682–1684. Scholar
  41. 41.
    Wall JD, Kim SK (2007) Inconsistencies in Neanderthal genomic DNA sequences. PLoS Genet 3:1862–1866. Scholar
  42. 42.
    Prüfer K, Meyer M (2015) Anthropology. Comment on “Late Pleistocene human skeleton and mtDNA link Paleoamericans and modern Native Americans”. Science.
  43. 43.
    Weiß CL, Dannemann M, Prüfer K, Burbano HA (2015) Contesting the presence of wheat in the British Isles 8,000 years ago by assessing ancient DNA authenticity from low-coverage data. eLife.
  44. 44.
    Schubert M, Ermini L, Der Sarkissian C et al (2014) Characterization of ancient and modern genomes by SNP detection and phylogenomic and metagenomic analysis using PALEOMIX. Nat Protoc 9:1056–1082. Scholar
  45. 45.
    Kircher M (2012) Analysis of high-throughput ancient DNA sequencing data. Methods Mol Biol 840:197–228. Scholar
  46. 46.
    Schubert M, Lindgreen S, Orlando L (2016) AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res Notes.
  47. 47.
    Renaud G, Stenzel U, Kelso J (2014) leeHom: adaptor trimming and merging for Illumina sequencing reads. Nucleic Acids Res.
  48. 48.
    Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. Scholar
  49. 49.
    O’Connell J, Schulz-Trieglaff O, Carlson E et al (2015) NxTrim: optimized trimming of Illumina mate pair reads. Bioinformatics 31:2035–2037. Scholar
  50. 50.
    Sturm M, Schroeder C, Bauer P (2016) SeqPurge: highly-sensitive adapter trimming for paired-end NGS data. BMC Bioinformatics.
  51. 51.
    Zhang J, Kobert K, Flouri T, Stamatakis A (2014) PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30:614–620. Scholar
  52. 52.
    Magoč T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963. Scholar
  53. 53.
    Mielczarek M, Szyda J (2016) Review of alignment and SNP calling algorithms for next-generation sequencing data. J Appl Genet 57:71–79. Scholar
  54. 54.
    Li HH, Durbin RR (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754–1760. Scholar
  55. 55.
    Kerpedjiev P, Frellsen J, Lindgreen S, Krogh A (2014) Adaptable probabilistic mapping of short reads using position specific scoring matrices. BMC Bioinformatics.
  56. 56.
    Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. Scholar
  57. 57.
    Nomiyama H, Fukuda M, Wakasugi S et al (1985) Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA. Nucleic Acids Res 13:1649–1658. Scholar
  58. 58.
    Lopez JV, Yuhki N, Masuda R et al (1994) Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat. J Mol Evol 39:174–190PubMedGoogle Scholar
  59. 59.
    Li H, Handsaker B, Wysoker A et al (2008) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079CrossRefGoogle Scholar
  60. 60.
    Dozmorov MG, Adrianto I, Giles CB et al (2015) Detrimental effects of duplicate reads and low complexity regions on RNA- and ChIP-seq data. BMC Bioinformatics.
  61. 61.
    McKenna A, Hanna M, Banks E et al (2010) The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303. Scholar
  62. 62.
    Briggs AW, Stenzel U, Meyer M et al (2010) Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA. Nucleic Acids Res.
  63. 63.
    Meyer M, Kircher M (2010) Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb Protoc.
  64. 64.
    Krause J, Unger T, Noçon A et al (2008) Mitochondrial genomes reveal an explosive radiation of extinct and extant bears near the Miocene-Pliocene boundary. BMC Evol Biol.
  65. 65.
    Rohland N, Harney E, Mallick S et al (2015) Partial uracil-DNA-glycosylase treatment for screening of ancient DNA. Philos Trans R Soc Lond B Biol Sci.
  66. 66.
    Pedersen JS, Valen E, Velazquez AMV et al (2014) Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome. Genome Res 24:454–466. Scholar
  67. 67.
    Gokhman D, Lavi E, Prüfer K et al (2014) Reconstructing the DNA methylation maps of the Neandertal and the Denisovan. Science 344:523–527. Scholar
  68. 68.
    Hanghøj K, Seguin-Orlando A, Schubert M et al (2016) Fast, accurate and automatic ancient nucleosome and methylation maps with epiPALEOMIX. Mol Biol Evol 33:3284–3298. Scholar
  69. 69.
    Renaud G, Slon V, Duggan AT, Kelso J (2015) Schmutzi: estimation of contamination and endogenous mitochondrial consensus calling for ancient DNA. Genome Biol.
  70. 70.
    Schuenemann VJ, Singh P, Mendum TA et al (2013) Genome-wide comparison of medieval and modern Mycobacterium leprae. Science 341:179–183. Scholar
  71. 71.
    Sawyer S, Krause J, Guschanski K et al (2012) Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA. PLoS One.
  72. 72.
    Parks M, Lambert D (2015) Impacts of low coverage depths and post-mortem DNA damage on variant calling: a simulation study. BMC Genomics.
  73. 73.
    Skoglund P, Northoff BH, Shunkov MV et al (2014) Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proc Natl Acad Sci U S A 111:2229–2234. Scholar
  74. 74.
    Green RE, Briggs AW, Krause J et al (2009) The Neandertal genome and ancient DNA authenticity. EMBO J 28:2494–2502. Scholar
  75. 75.
    Zhang H, Paijmans JLA, Chang F et al (2013) Morphological and genetic evidence for early Holocene cattle management in northeastern China. Nat Commun.
  76. 76.
    Weissensteiner H, Pacher D, Kloss-Brandstätter A et al (2016) HaploGrep 2: mitochondrial haplogroup classification in the era of high-throughput sequencing. Nucleic Acids Res 44:W58–W63. Scholar
  77. 77.
    Rasmussen M, Sikora M, Albrechtsen A et al (2015) The ancestry and affiliations of Kennewick Man. Nature 523:455–458. Scholar
  78. 78.
    Korneliussen TS, Albrechtsen A, Nielsen R (2014) ANGSD: analysis of next generation sequencing data. BMC Bioinformatics.
  79. 79.
    Racimo F, Renaud G, Slatkin M (2016) Joint estimation of contamination, error and demography for nuclear DNA from ancient humans. PLoS Genet.
  80. 80.
    Skoglund P, Storå J, Götherström A, Jakobsson M (2013) Accurate sex identification of ancient human remains using DNA shotgun sequencing. J Archaeol Sci.
  81. 81.
    Abecasis GR, Auton A, Brooks LD, et al with 1000 Genomes Project Consortium (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491:56–65. Scholar
  82. 82.
    Louvel G, Der Sarkissian C, Hanghøj K, Orlando L (2016) metaBIT, an integrative and automated metagenomic pipeline for analysing microbial profiles from high-throughput sequencing shotgun data. Mol Ecol Resour 16:1415–1427. Scholar
  83. 83.
    Renaud G, Hanghøj K, Willeslev E, Orlando L (2016) gargammel: a sequence simulator for ancient DNA. Bioinformatics 33(4):577–579. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Gabriel Renaud
    • 1
  • Mikkel Schubert
    • 1
  • Susanna Sawyer
    • 1
  • Ludovic Orlando
    • 1
    • 2
    Email author
  1. 1.Centre for GeoGenetics, Natural History Museum of DenmarkUniversity of CopenhagenCopenhagen KDenmark
  2. 2.Laboratoire d’Anthropobiologie Moléculaire et d’Imagerie de SynthèseCNRS UMR 5288, Université de Toulouse, University Paul SabatierToulouseFrance

Personalised recommendations