Analysis of Ancient DNA in Microbial Ecology
The development of next-generation sequencing has led to a breakthrough in the analysis of ancient genomes, and the subsequent genomic analyses of the skeletal remains of ancient humans have revolutionized the knowledge of the evolution of our species, including the discovery of a new hominin, and demonstrated admixtures with more distantly related archaic populations such as Neandertals and Denisovans. Moreover, it has also yielded novel insights into the evolution of ancient pathogens. The analysis of ancient microbial genomes allows the study of their recent evolution, presently over the last several millennia. These spectacular results have been attained despite the degradation of DNA after the death of the host, which results in very short DNA molecules that become increasingly damaged, only low quantities of which remain. The low quantity of ancient DNA molecules renders their analysis difficult and prone to contamination with modern DNA molecules, in particular via contamination from the reagents used in DNA purification and downstream analysis steps. Finally, the rare ancient molecules are diluted in environmental DNA originating from the soil microorganisms that colonize bones and teeth. Thus, ancient skeletal remains can share DNA profiles with environmental samples and identifying ancient microbial genomes among the more recent, presently poorly characterized, environmental microbiome is particularly challenging. Here, we describe the methods developed and/or in use in our laboratory to produce reliable and reproducible paleogenomic results from ancient skeletal remains that can be used to identify the presence of ancient microbiota.
Key wordsAncient DNA NGS Double-stranded library Single-stranded library IonTorrent Illumina Contamination
- 1.Pruvost M, Schwarz R, Correia VB, Champlot S, Braguier S, Morel N et al (2007) Freshly excavated fossil bones are best for amplification of ancient DNA. Proc Natl Acad Sci U S A 104:739–744Google Scholar
- 2.Fortea J, de la Rasilla M, Garcia-Tabernero A, Gigli E, Rosas A, Lalueza-Fox C (2008) Excavation protocol of bone remains for Neandertal DNA analysis in El Sidron Cave (Asturias, Spain). J Hum Evol 55:353–357Google Scholar
- 8.Geigl EM (2005) Why ancient DNA research needs taphonomy. In: O’Connor T (ed) Biosphere to lithosphere, new studies in vertebrate taphonomy. Oxbow Books, Oxford, pp 79–86Google Scholar
- 30.Watanabe I, Kodama Y, Harayama S (2001) Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting. Journal of Microbiological Methods 44:253–262Google Scholar
- 31.Pruvost M, Grange T, Geigl EM (2005) Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies. Biotechniques 38 (4):569–575Google Scholar