In the past decades ancient DNA research has brought numerous insights to archaeological research where traditional approaches were limited. The determination of sex in human skeletal remains is often challenging for physical anthropologists when dealing with incomplete, juvenile or pathological specimens. Molecular approaches allow sexing on the basis of sex-specific markers or by calculating the ratio of DNA derived from different chromosomes. Here we propose a novel approach that relies on the ratio of X chromosome-derived shotgun sequencing data to the autosomal coverage, thus establishing the probability of an XX or XY karyotype. Applying this approach to the individuals of the Upper Paleolithic triple burial of Dolní Věstonice reveals that all three skeletons, including the individual DV 15, whose sex has long been debated due to a pathological condition, were male.

Department of Anthropology Faculty of Science Masaryk University Kotlářská 61137 Brno Czech Republic

Department of Genetics Harvard Medical School Boston Massachusetts 02115 United States of America

Institute for Archeological Sciences University of Tübingen D 72070 Tübingen Germany

Institute of Archaeology Academy of Science of the Czech Republic 69129 Dolní Věstonice Czech Republic

Max Planck Institute for the Science of Human History D 07745 Jena Germany

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