Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol Update
  • Published:

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

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic overview of the workflow described in this protocol.
Fig. 2: Size selection of amplified libraries.
Fig. 3: Quantitative PCR results obtained after the automated preparation of 96 single-stranded DNA libraries.

Similar content being viewed by others

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).

References

  1. Meyer, M. et al. A high-coverage genome sequence from an archaic Denisovan individual. Science 338, 222–226 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Gansauge, M. T. & Meyer, M. Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA. Nat. Protoc. 8, 737–748 (2013).

    PubMed  Google Scholar 

  3. Prüfer, K. et al. The complete genome sequence of a Neanderthal from the Altai Mountains. Nature 505, 43–49 (2014).

    PubMed  Google Scholar 

  4. Prüfer, K. et al. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science 358, 655–658 (2017).

    PubMed  PubMed Central  Google Scholar 

  5. Mafessoni, F. et al. A high-coverage Neandertal genome from Chagyrskaya Cave. Preprint at https://www.biorxiv.org/content/10.1101/2020.03.12.988956v1 (2020).

  6. Fu, Q. et al. Genome sequence of a 45,000-year-old modern human from western Siberia. Nature 514, 445–449 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Meyer, M. et al. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature 505, 403–406 (2014).

    CAS  PubMed  Google Scholar 

  9. Meyer, M. et al. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins. Nature 531, 504–507 (2016).

    CAS  PubMed  Google Scholar 

  10. Slon, V. et al. Neandertal and Denisovan DNA from Pleistocene sediments. Science 356, 605–608 (2017).

    CAS  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Gansauge, M. T. & Meyer, M. A method for single-stranded ancient DNA library preparation. Methods Mol. Biol. 1963, 75–83 (2019).

  14. Glocke, I. & Meyer, M. Extending the spectrum of DNA sequences retrieved from ancient bones and teeth. Genome Res. 27, 1230–1237 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. de Filippo, C., Meyer, M. & Prüfer, K. Quantifying and reducing spurious alignments for the analysis of ultra-short ancient DNA sequences. BMC Biol. 16, 121 (2018).

    PubMed  PubMed Central  Google Scholar 

  16. Bennett, E. A. et al. Library construction for ancient genomics: single strand or double strand? Biotechniques 56, 289–290 (2014).

    CAS  PubMed  Google Scholar 

  17. Wales, N. et al. New insights on single-stranded versus double-stranded DNA library preparation for ancient DNA. Biotechniques 59, 368–371 (2015).

    CAS  PubMed  Google Scholar 

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

  19. Caroe, C. et al. Single-tube library preparation for degraded DNA. Methods Ecol. Evol. 9, 410–419 (2018).

    Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Skoglund, P. et al. Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proc. Natl Acad. Sci. USA 111, 2229–2234 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Gansauge, M. T. & Meyer, M. Selective enrichment of damaged DNA molecules for ancient genome sequencing. Genome Res. 24, 1543–1549 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Bokelmann, L. et al. A genetic analysis of the Gibraltar Neanderthals. Proc. Natl Acad. Sci. USA 116, 15610–15615 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Stiller, M. et al. Single-strand DNA library preparation improves sequencing of formalin-fixed and paraffin-embedded (FFPE) cancer DNA. Oncotarget 7, 59115–59128 (2016).

    PubMed  PubMed Central  Google Scholar 

  25. Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M. & Shendure, J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164, 57–68 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Burnham, P. et al. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma. Sci. Rep. 6, 27859 (2016).

  27. Turchinovich, A. et al. Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA. RNA Biol. 11, 817–828 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Karlsson, K. et al. Amplification-free sequencing of cell-free DNA for prenatal non-invasive diagnosis of chromosomal aberrations. Genomics 105, 150–158 (2015).

    CAS  PubMed  Google Scholar 

  29. Raine, A., Manlig, E., Wahlberg, P., Syvanen, A. C. & Nordlund, J. SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing. Nucleic Acids Res. 45, e36 (2017).

    PubMed  Google Scholar 

  30. Wu, J., Dai, W., Wu, L. & Wang, J. SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states. BMC Genomics 19, 143 (2018).

    PubMed  PubMed Central  Google Scholar 

  31. Ding, J., Taylor, M. S., Jackson, A. P. & Reijns, M. A. Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq. Nat. Protoc. 10, 1433–1444 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Petryk, N. et al. Replication landscape of the human genome. Nat. Commun. 7, 10208 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Tin, M. M., Economo, E. P. & Mikheyev, A. S. Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics. PLoS ONE 9, e96793 (2014).

    PubMed  PubMed Central  Google Scholar 

  34. Troll, C. J. et al. A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos. BMC Genomics 20, 1023 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 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).

    CAS  PubMed  Google Scholar 

  36. Kampmann, M. L., Borsting, C. & Morling, N. Decrease DNA contamination in the laboratories. Forensic Sci. Int. Genet. 6, E577–E578 (2017).

    Google Scholar 

  37. 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).

    CAS  PubMed  Google Scholar 

  38. Prüfer, K. snpAD: an ancient DNA genotype caller. Bioinformatics 34, 4165–4171 (2018).

    PubMed  PubMed Central  Google Scholar 

  39. Varshney, U. & van de Sande, J. H. Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase. Biochemistry 30, 4055–4061 (1991).

    CAS  PubMed  Google Scholar 

  40. Burbano, H. A. et al. Targeted investigation of the Neandertal genome by array-based sequence capture. Science 328, 723–725 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Fu, Q. et al. DNA analysis of an early modern human from Tianyuan Cave, China. Proc. Natl Acad. Sci. USA 110, 2223–2227 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Haak, W. et al. Massive migration from the steppe was a source for Indo-European languages in Europe. Nature 522, 207–211 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Kircher, M., Sawyer, S. & Meyer, M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res 40, e3 (2012).

    CAS  PubMed  Google Scholar 

  44. Dabney, J. & Meyer, M. Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. Biotechniques 52, 87–94 (2012).

    CAS  PubMed  Google Scholar 

  45. Thompson, J. R., Marcelino, L. A. & Polz, M. F. Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Res. 30, 2083–2088 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Renaud, G., Stenzel, U. & Kelso, J. leeHom: adaptor trimming and merging for Illumina sequencing reads. Nucleic Acids Res. 42, e141 (2014).

    PubMed  PubMed Central  Google Scholar 

  47. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors

Contributions

M.-T.G. and M.M. developed the manual protocol. A.A.-P. and M.M. developed the automated protocol version, which was tested and optimized by S.N. M.M. and M.-T.G. wrote the paper with help from A.A-P. and S.N.

Corresponding authors

Correspondence to Marie-Theres Gansauge or Matthias Meyer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Eva-Maria Geigl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Related links

Key references using this protocol

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

Hajdinjak, M. et al. Nature 555, 652−656 (2018): https://doi.org/10.1038/nature26151

Slon, V. et al. Nature 561, 113–116 (2018): https://doi.org/10.1038/s41586-018-0455-x

Protocol update to:

Gansauge, M. & Meyer, M. Nat. Protoc. 8, 737–748 (2013): https://doi.org/10.1038/nprot.2013.038

This protocol is an update to Nat. Protoc. 8, 737–748 (2013): https://doi.org/10.1038/nprot.2013.038

Extended data

Supplementary information

Supplementary Manual

Automated protocol of single-stranded library preparation, including Supplementary Table 1 (Characterization of single-stranded DNA libraries by quantitative PCR and shallow shotgun sequencing).

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gansauge, MT., Aximu-Petri, A., Nagel, S. et al. 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. Nat Protoc 15, 2279–2300 (2020). https://doi.org/10.1038/s41596-020-0338-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-0338-0

This article is cited by

Comments

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.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research