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Methods

Identification of ancient remains through genomic sequencing

¹ U.S. Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA; ² Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA; ³ Ancient DNA Laboratory, Department of Archaeology, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada; ⁴ Division of Paleolithic Studies, Department of Archaeology, Institute of Archaeology and Ethnography, Novosibirsk 630090, Russia

Studies of ancient DNA have been hindered by the preciousness of remains, the small quantities of undamaged DNA accessible, and the limitations associated with conventional PCR amplification. In these studies, we developed and applied a genomewide adapter-mediated emulsion PCR amplification protocol for ancient mammalian samples estimated to be between 45,000 and 69,000 yr old. Using 454 Life Sciences (Roche) and Illumina sequencing (formerly Solexa sequencing) technologies, we examined over 100 megabases of DNA from amplified extracts, revealing unbiased sequence coverage with substantial amounts of nonredundant nuclear sequences from the sample sources and negligible levels of human contamination. We consistently recorded over 500-fold increases, such that nanogram quantities of starting material could be amplified to microgram quantities. Application of our protocol to a 50,000-yr-old uncharacterized bone sample that was unsuccessful in mitochondrial PCR provided sufficient nuclear sequences for comparison with extant mammals and subsequent phylogenetic classification of the remains. The combined use of emulsion PCR amplification and high-throughput sequencing allows for the generation of large quantities of DNA sequence data from ancient remains. Using such techniques, even small amounts of ancient remains with low levels of endogenous DNA preservation may yield substantial quantities of nuclear DNA, enabling novel applications of ancient DNA genomics to the investigation of extinct phyla.

⁶ Corresponding author.

E-mail emrubin{at}lbl.gov; fax (510) 486-4229.

[Supplemental material is available online at www.genome.org.]

Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.076091.108.

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