This fully updated second edition explores protocols that address the most challenging aspects of experimental work in ancient DNA, such as preparing ancient samples for DNA extraction, the DNA extraction itself, and transforming extracted ancient DNA molecules for sequencing library preparation. The volume also examines 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.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.