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M Ethods in M Olecular B Iology M ETHODS IN M OLECULAR B IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Ancient DNA Methods and Protocols Second Edition Edited by Beth Shapiro Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA, USA Axel Barlow Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Peter D. Heintzman Tromsø University Museum, UiT—The Arctic University of Norway, Tromsø, Norway Michael Hofreiter, Johanna L. A. Paijmans Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany André E. R. Soares Laboratório Nacional de Computação Científica, Petrópolis, RJ, Brazil Editors Beth Shapiro Axel Barlow Department of Ecology and Evolutionary Institute for Biochemistry and Biology Biology University of Potsdam University of California Santa Cruz Potsdam, Germany Santa Cruz, CA, USA Michael Hofreiter Peter D. Heintzman Institute for Biochemistry and Biology Tromsø University Museum University of Potsdam UiT—The Arctic University of Norway Potsdam, Germany Tromsø, Norway Andre´ E. R. Soares Johanna L. A. Paijmans Laborato´rio Nacional de Computac¸a˜o Cientı´fica Institute for Biochemistry and Biology Petro´polis, RJ, Brazil University of Potsdam Potsdam, Germany ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9175-4 ISBN 978-1-4939-9176-1 (eBook) https://doi.org/10.1007/978-1-4939-9176-1 Library of Congress Control Number: 2019933331 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Preface Research in ancient DNA began during the early 1980s with the publication of short mitochondrial DNA sequence fragments from a quagga, an extinct subspecies of the plains zebra. Following this, several institutions invested resources to develop both the laboratory infrastructure and research expertise to recover trace amounts of degraded DNA from ancient samples. For many years, the field remained small, as researchers grappled with the challenges of inhibition, degradation, and contamination of ancient DNA extracts. Today’s broad application of ancient DNA as a research tool is owed to two technical innovations within the last 25 years: the invention of the polymerase chain reaction, or PCR, and the development of economical and high-throughput sequencing technologies. PCR allowed the targeted retrieval of small fragments of DNA that, in fortunate circumstances, are preserved in fossil and museum specimens. High-throughput sequencing approaches made the recovery of many millions of preserved molecules economically viable and practi- cal, which has pushed the field into the realm of genomics. These key innovations have allowed ancient DNA to become an increasingly widespread research tool, with the capacity to expand both the temporal and taxonomic breadth of questions that can be asked in ecological and evolutionary research. As we noted in the first edition of this book that was published in 2012, progress in ancient DNA research has been inherently technology driven. In our first collection of protocols, we attempted to summarize the most common approaches toward the retrieval and analysis of ancient DNA sequences. We began with guidelines for establishing an ancient DNA laboratory and described extraction protocols that had been optimized for a wide range of different substrates. We included instructions for ancient DNA-specific approaches to DNA extraction, preparing and performing PCR and genomic library preparation, and suggested analytical approaches for population genetic analyses of ancient DNA and the initial quality control of data recovered from high-throughput sequencing. Many of the protocols included in the first edition remain relevant today. However, new protocols for both ancient DNA recovery and analysis have been introduced during the time since publication, and these have expanded significantly the range of samples from which ancient DNA can be recovered. These new protocols are the focus of this second edition. We include in this second edition protocols that address the most challenging aspects of experimental work in ancient DNA: preparing ancient samples for DNA extraction, the DNA extraction itself, and transforming extracted ancient DNA molecules so that they can be sequenced on the different available sequencing platforms, which is also known as sequencing library preparation. We also include several chapters that discuss the analysis of high-throughput sequencing data recovered from ancient specimens, which, because of the degraded nature of ancient DNA and common co-extraction of contaminant DNA, has challenges that are unique compared to data recovered from modern specimens. We begin with a chapter that discusses procedures for setting up an ancient DNA laboratory and authenticating recovered ancient DNA. Next, we include two chapters that describe methods to pretreat samples prior to ancient DNA extraction, both of which lead to improvements in the relative abundance of endogenous versus contaminant DNA. We then present four chapters that describe the latest innovations in retrieving ancient DNA from organic remains and environmental samples. We include updated protocols presenting v vi Preface double-stranded and single-stranded approaches to prepare genomic libraries, and several approaches enrich ancient DNA extracts for molecular targets of interest. Finally, we include a chapter on data authenticity assessment, which is critical for all ancient DNA studies, and a chapter that includes a practical approach and troubleshooting advice for those aiming to assemble ancient genomes from next-generation sequencing data. As in the first edition, the goal of this second edition is to present these protocols in a manner that makes them easily accessible for everyday use in the lab. We hope this book will be another useful source of information for both beginning and experienced researchers hoping to add ancient DNA to their professional toolkit. We express our sincere thanks to all the authors for their willingness to share their time and their trade secrets, and to Prof. John Walker at Humana Press for giving us the opportunity to assemble this collection of protocols. Santa Cruz, CA, USA Beth Shapiro Potsdam, Germany Axel Barlow Tromsø, Norway Peter D. Heintzman Potsdam, Germany Michael Hofreiter Potsdam, Germany Johanna L. A. Paijmans Petropolis, RJ, Brazil Andre´ E. R. Soares Contents Preface . ................................................................... v Contributors................................................................. ix 1 Setting Up an Ancient DNA Laboratory ................................... 1 Tara L. Fulton and Beth Shapiro 2 Pretreatment: Removing DNA Contamination from Ancient Bones and Teeth Using Sodium Hypochlorite and Phosphate . ............... 15 Petra Korlevic´ and Matthias Meyer 3 Pretreatment: Improving Endogenous Ancient DNA Yields Using a Simple Enzymatic Predigestion Step ..................................... 21 Hannes Schroeder, Peter de Barros Damgaard, and Morten E. Allentoft 4 Extraction of Highly Degraded DNA from Ancient Bones and Teeth . ........ 25 Jesse Dabney and Matthias Meyer 5 Sampling and Extraction of Ancient DNA from Sediments................... 31 Laura S. Epp, Heike H. Zimmermann, and Kathleen R. Stoof-Leichsenring 6 Extraction of Ancient DNA from Plant Remains ............................ 45 Nathan Wales and Logan Kistler 7 DNA Extraction from Keratin and Chitin .................................. 57 Paula F. Campos and M. Thomas P. Gilbert 8 Double-Stranded Library Preparation for Ancient and Other Degraded Samples . .................................................... 65 Kirstin Henneberger, Axel Barlow, and Johanna L. A. Paijmans 9 A Method for Single-Stranded Ancient DNA Library Preparation............. 75 Marie-Theres Gansauge and Matthias Meyer 10 Sequencing Library Preparation from Degraded Samples for Non-illumina Sequencing Platforms ..................................
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