Conservation Genetics Resources

, Volume 11, Issue 3, pp 345–353 | Cite as

Experimental approaches for ancient DNA extraction and sample preparation for next generation sequencing in ultra-clean conditions

  • A. D. Matsvay
  • I. E. Alborova
  • E. V. Pimkina
  • M. L. Markelov
  • K. KhafizovEmail author
  • K. K. Mustafin
Methods and Resources Article


In this study, we aimed to develop an approach for genetic material extraction and sample preparation of ancient DNA for next generation sequencing that minimizes intra-laboratory contamination. A special module system consisting of four sterile gloveboxes connected by antechambers, which maintain a high purity atmosphere and allow conducting all stages of sample preparation in an isolated clean environment, starting with the processing of archaeological material, was developed. Furthermore, a fast and cost-effective double-stranded library preparation protocol for massive parallel sequencing was developed using non-standard modified sequencing adaptors, which enabled obtaining a library of fragments from extremely low amounts of starting degraded material. Bioinformatics data processing showed that the distribution of substitution frequencies in a subset of reads, mapped to the human reference genome, completely coincides with the pattern of postmortem modifications expected when the double-stranded library preparation method is employed. This is a strong evidence for the endogenous origin of the sequenced fragments of the human genome. Thus, the results of ancient sample and negative control sample sequencing, indicated the absence of visible contamination during sample preparation procedure.


Ancient DNA Bioinformatics Human genetics Next generation sequencing DNA extraction 



This work was supported by the Endowment Fund.


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • A. D. Matsvay
    • 1
    • 2
  • I. E. Alborova
    • 1
  • E. V. Pimkina
    • 2
  • M. L. Markelov
    • 2
  • K. Khafizov
    • 1
    • 2
    Email author
  • K. K. Mustafin
    • 1
  1. 1.Moscow Institute of Physics and TechnologyDolgoprudnyRussian Federation
  2. 2.Central Research Institute of EpidemiologyMoscowRussian Federation

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