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.
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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.
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Authors and Affiliations
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.
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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
Extended Data Fig. 1 Overview of the adapter sequences and library amplification primers used in this protocol.
All sequences are shown in 5′–3′ orientation.
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).
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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
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DOI: https://doi.org/10.1038/s41596-020-0338-0
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