Density separation is a process routinely used to segregate minerals, organic matter, and even microplastics, from soils and sediments. Here we apply density separation to archaeological bone powders before DNA extraction to increase endogenous DNA recovery relative to a standard control extraction of the same powders. Using nontoxic heavy liquid solutions, we separated powders from the petrous bones of 10 individuals of similar archaeological preservation into eight density intervals (2.15 to 2.45 g/cm3, in 0.05 increments). We found that the 2.30 to 2.35 g/cm3 and 2.35 to 2.40 g/cm3 intervals yielded up to 5.28-fold more endogenous unique DNA than the corresponding standard extraction (and up to 8.53-fold before duplicate read removal), while maintaining signals of ancient DNA authenticity and not reducing library complexity. Although small 0.05 g/cm3 intervals may maximally optimize yields, a single separation to remove materials with a density above 2.40 g/cm3 yielded up to 2.57-fold more endogenous DNA on average, which enables the simultaneous separation of samples that vary in preservation or in the type of material analyzed. While requiring no new ancient DNA laboratory equipment and fewer than 30 min of extra laboratory work, the implementation of density separation before DNA extraction can substantially boost endogenous DNA yields without decreasing library complexity. Although subsequent studies are required, we present theoretical and practical foundations that may prove useful when applied to other ancient DNA substrates such as teeth, other bones, and sediments.

Centre for Applied Bioanthropology Institute for Anthropological Research 10000 Zagreb Croatia

Centre for Forensic Research School of Criminology Simon Fraser University Burnaby British Columbia V5A 1S6 Canada

CIAS Department of Life Sciences University of Coimbra 3000 456 Coimbra Portugal

Department of Anthropology National Museum Prague 115 79 Czech Republic

Department of Archaeology University of Cambridge Cambridge CB2 3DZ United Kingdom

Department of Evolutionary Anthropology University of Vienna 1030 Vienna Austria

Department of Evolutionary Anthropology University of Vienna 1030 Vienna Austria;

Department of Genetics Harvard Medical School Boston Massachusetts 02115 USA

Department of History and Art History Utrecht University 3512 BS Utrecht The Netherlands

Department of Human Evolutionary Biology Harvard University Cambridge Massachusetts 02138 USA

Dipartimento di Biologia Ambientale Sapienza Università di Roma Rome 00185 Italy

Dipartimento di Scienze dell'Antichità Sapienza Università di Roma Rome 00185 Italy

Human Evolution and Archaeological Sciences Forschungsverbund University of Vienna 1030 Vienna Austria

Institute of Archaeology of the Czech Academy of Sciences Prague 118 00 Czech Republic

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