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Preparation of Next-Generation Sequencing Libraries from Damaged DNA

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Book cover Ancient DNA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 840))

Abstract

Next-generation sequencing (NGS) has revolutionized ancient DNA research, especially when combined with high-throughput target enrichment methods. However, attaining high sequencing depth and accuracy from samples often remains problematic due to the damaged state of ancient DNA, in particular the extremely low copy number of ancient DNA and the abundance of uracil residues derived from cytosine deamination that lead to miscoding errors. It is therefore critical to use a highly efficient procedure for conversion of a raw DNA extract into an adaptor-ligated sequencing library, and equally important to reduce errors from uracil residues. We present a protocol for NGS library preparation that allows highly efficient conversion of DNA fragments into an adaptor-ligated form. The protocol incorporates an option to remove the vast majority of uracil miscoding lesions as part of the library preparation process. The procedure requires only two spin column purification steps and no gel purification or bead handling. Starting from an aliquot of DNA extract, a finished, highly amplified library can be generated in 5 h, or under 3 h if uracil removal is not required.

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, MA, USA

    Adrian W. Briggs

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany

    Patricia Heyn

Authors
  1. Adrian W. Briggs
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  2. Patricia Heyn
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Correspondence to Adrian W. Briggs .

Editor information

Editors and Affiliations

  1. Dept. of Ecology and Evolutionary Biolog, University of California Santa Cruz, A414 Earth & Marine Sciences, Santa Cruz, 16802, California, USA

    Beth Shapiro

  2. , Department of Biology (Area 2), University of York, Heslington, York, YO 105DD, United Kingdom

    Michael Hofreiter

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Briggs, A.W., Heyn, P. (2012). Preparation of Next-Generation Sequencing Libraries from Damaged DNA. In: Shapiro, B., Hofreiter, M. (eds) Ancient DNA. Methods in Molecular Biology, vol 840. Humana Press. https://doi.org/10.1007/978-1-61779-516-9_18

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  • DOI: https://doi.org/10.1007/978-1-61779-516-9_18

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-515-2

  • Online ISBN: 978-1-61779-516-9

  • eBook Packages: Springer Protocols

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