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
  • Published:

Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA

This article has been updated

Abstract

This protocol describes a method for converting short single-stranded and double-stranded DNA into libraries compatible with high-throughput sequencing using Illumina technology. This method has primarily been developed to improve sequence retrieval from ancient DNA, but it is also applicable to the sequencing of short or degraded DNA from other sources, and it can also be used for sequencing oligonucleotides. Single-stranded library preparation is performed by ligating a biotinylated adapter oligonucleotide to the 3′ ends of heat-denatured DNA. The resulting strands are then immobilized on streptavidin-coated beads and copied with a polymerase. A second adapter is attached by blunt-end ligation, and library preparation is completed by PCR amplification. We estimate that intact DNA strands are recovered in the library with 50% efficiency. Libraries can be generated from up to 12 DNA or oligonucleotide samples in parallel within 2 d.

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

Access options

Buy this article

Purchase on Springer Link

Instant access to full article PDF

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

Figure 1: Schematic overview of single-stranded library preparation, exemplified by one strand of a double-stranded DNA molecule containing a uracil and a single-strand break.
Figure 2

Similar content being viewed by others

Change history

  • 15 May 2013

     In the version of this article initially published, the sequence of oligonucleotide CL78 reported in Table 1 as “(AGATCGGAAG[C3Spacer]10[TEG-biotin] (TEG = triethylene glycol spacer))” is incorrect. The correct sequence is “[Phosphate]AGATCGGAAG[C3Spacer]10[TEG-biotin] (TEG = triethylene glycol spacer)”. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Miller, W. et al. Sequencing the nuclear genome of the extinct woolly mammoth. Nature 456, 387–390 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Green, R.E. et al. A draft sequence of the Neandertal genome. Science 328, 710–722 (2010).

    Article  CAS  Google Scholar 

  4. Reich, D. et al. Genetic history of an archaic hominin group from Denisova Cave in Siberia. Nature 468, 1053–1060 (2010).

    Article  CAS  Google Scholar 

  5. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

    Article  CAS  Google Scholar 

  6. Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

    Article  CAS  Google Scholar 

  7. McKernan, K.J. et al. Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding. Genome Res. 19, 1527–1541 (2009).

    Article  CAS  Google Scholar 

  8. Rothberg, J.M. et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature 475, 348–352 (2011).

    Article  CAS  Google Scholar 

  9. Briggs, A.W. et al. Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325, 318–321 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Maricic, T., Whitten, M. & Pääbo, S. Multiplexed DNA sequence capture of mitochondrial genomes using PCR products. PLoS ONE 5 e14004 (2010).

  12. Avila-Arcos, M.C. et al. Application and comparison of large-scale solution-based DNA capture-enrichment methods on ancient DNA. Sci. Rep. 1, 74 (2011).

    Article  Google Scholar 

  13. DeAngelis, M.M., Wang, D.G. & Hawkins, T.L. Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 23, 4742–4743 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. Gilbert, M.T. et al. The isolation of nucleic acids from fixed, paraffin-embedded tissues—which methods are useful when? PLoS ONE 2, e537 (2007).

    Article  Google Scholar 

  17. Shi, S.R. et al. DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH. J. Histochem. Cytochem. 50, 1005–1011 (2002).

    Article  CAS  Google Scholar 

  18. Hou, Y. et al. Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell 148, 873–885 (2012).

    Article  CAS  Google Scholar 

  19. Adey, A. et al. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genom. Biol. 11, R119 (2010).

    Article  CAS  Google Scholar 

  20. Li, T.W. & Weeks, K.M. Structure-independent and quantitative ligation of single-stranded DNA. Anal. Biochem. 349, 242–246 (2006).

    Article  CAS  Google Scholar 

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

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

    Article  CAS  Google Scholar 

  23. Brotherton, P. et al. Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of postmortem miscoding lesions. Nucleic Acids Res. 35, 5717–5728 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Briggs, A.W. et al. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA. Nucleic Acids Res. 38, e87 (2010).

    Article  Google Scholar 

  26. Krause, J. et al. A complete mtDNA genome of an early modern human from Kostenki, Russia. Curr. Biol. 20, 231–236 (2010).

    Article  CAS  Google Scholar 

  27. Sawyer, S., Krause, J., Guschanski, K., Savolainen, V. & Pääbo, S. Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA. PLoS ONE 7, e34131 (2012).

    Article  CAS  Google Scholar 

  28. Rohland, N. & Hofreiter, M. Comparison and optimization of ancient DNA extraction. Biotechniques 42, 343–352 (2007).

    Article  CAS  Google Scholar 

  29. Willerslev, E. & Cooper, A. Ancient DNA. Proc. Biol. Sci. 272, 3–16 (2005).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  31. Kircher, M., Heyn, P. & Kelso, J. Addressing challenges in the production and analysis of Illumina sequencing data. BMC Genom. 12, 382 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank I. Glocke for help in the lab; B. Höber, M. Kircher, S. Pääbo and U. Stenzel for helpful discussions; J. Dabney and S. Sawyer for comments on the manuscript; and S. Tüpke for help with the figures. This work was funded by the Max Planck Society.

Author information

Authors and Affiliations

Authors

Contributions

M.-T.G. and M.M. developed the protocol and wrote the paper.

Corresponding authors

Correspondence to Marie-Theres Gansauge or Matthias Meyer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Sequences of adapters, amplification and sequencing primers. Adapter sequences of a regular single-indexed Illumina multiplex library (as obtained using Illumina's TruSeq DNA sample preparation kit, cat. no. FC-121-2001/2002, or following the protocol provided in ref. 21 of the main text) are shown on top. Libraries prepared from single-stranded DNA differ by a deletion of 5 bp in the P5 adapter. Both library types are compatible with double-indexed sequencing (see ref. 22 of the main text for details). Amplification and sequencing primers are indicated by arrows. Primers with prefix 'IS' are further described in ref. 21 of the main text. (PDF 364 kb)

Supplementary Figure 2

Characterization of two DNA libraries prepared with and without uracil removal from a Neanderthal DNA extract (panels on the right and left, respectively). A) Fragment length distributions of the amplified libraries obtained from chip electrophoresis using the Bioanalyzer 2100. B) Fragment size distributions obtained from sequencing (the fraction of mapped sequences is indicated by a dotted line). C) Frequency of C to T substitutions around 5′and 3′ends of Neanderthal sequences. D) Average GC-content of Neanderthal sequences as a function of fragment size. (PDF 397 kb)

Supplementary Figure 3

Quality control of single-stranded adapter oligonucleotide CL78. From two independently synthesized batches of the oligonucleotide, 10 pmol were loaded onto a 10% denaturing polyacrylamide gel, next to a size marker (lane 1; 20/100 ladder). The gel was run for 35 min at 200V and stained with SybrGold dye. While no impurities were detected in the first batch (lane 2), the second batch (lane 3) is dominated by a double-length artifact, representing an extreme example of poor oligonucleotide synthesis quality. (PDF 197 kb)

Supplementary Figure 4

Determining the optimal cycle number for indexing PCR using the qPCR amplification plots. Shown are the amplification plots obtained from quantifying the libraries prepared in the experiment described in 'Anticipated results'. The saturation phase of PCR starts after cycle 18 (sample libraries, red and blue) and cycle 23 (blank library, green), respectively. Assuming full amplification efficiency (i.e. a doubling of library molecules in each cycle), the optimal cycle number for indexing PCR can be determined as follows by correcting for differences in reaction volumes and the amount of template DNA: (i) qPCR was performed in 25 μl reactions, whereas indexing PCR is performed in 100 μl volume. Thus, 2 cycles should be added to allow for 4 times more end product. (ii) One microliter of a 1:20 library dilution was used for measurement, whereas 24 μl of the library are used for indexing PCR (480 times as much). This corresponds to 8.9 (rounded 9) cycles that should be deducted. Thus, 11 and 16 were estimated to be the optimal cycle numbers for indexing PCR. (PDF 278 kb)

Supplementary Table 1

Program settings of Cooling-ThermoMixer MKR13 recommended for single-stranded library preparation. The device may also be used to replace the vortexer in bead resuspension steps (use 'short mix' button). (PDF 255 kb)

Supplementary Table 2

P5 and P7 indexing primers. An identical set of P7 indexing primers was published before in ref. 21 of the main text. (PDF 331 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gansauge, MT., Meyer, M. Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA. Nat Protoc 8, 737–748 (2013). https://doi.org/10.1038/nprot.2013.038

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.038

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

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing