Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA

It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3′ ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5′ adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30−35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired.

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Data availability

All raw data points underlying Fig. 3 are listed in Supplementary Table 1.

Code availability

Electronic protocol files for automated library preparation and auxiliary files are available at the Zenodo website (https://zenodo.org/record/3631147).

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Acknowledgements

We thank L. Bokelmann, E. Essel, L. Lippik, J. Richter, B. Schellbach and A. Weihmann for help in the lab, D. Massilani, S. Pääbo, B. Vernot and E. Zavala for helpful discussions, I. Bünger for help with installing software, J. Kelso and J. Visagie for help with data processing and L. Jauregui for comments on the manuscript. This work was funded by the Max Planck Society.

Author information

Authors and Affiliations

  1. Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany Marie-Theres Gansauge, Ayinuer Aximu-Petri, Sarah Nagel & Matthias Meyer
  1. Marie-Theres Gansauge