aDNA Analysis of Paleolithic Metatarsal

Only the small 3rd metatarsal 3150 from layer 4c of Grotta Paglicci (direct ¹⁴C date: 14,372–13,759 cal. yr bp) was selected for aDNA analysis. The oldest specimens are too small to extract enough quantity of bone powder without significantly damaging them. DNA analysis was carried out in the Molecular Anthropology Laboratory of the University of Florence, exclusively dedicated to ancient DNA analysis. Blanks as negative controls were used in all of the experimental steps to monitor the absence of contaminants in reagents and environment. To remove potential contamination, the outer layer of the bone was mechanically taken out using a dentist drill with disposable tip. After brushing, sample was irradiated by ultraviolet light for 45 min in a Biolink DNA Crosslinker (Biometra). The DNA was extracted from approximately 50 mg of bone powder following a published silica-based protocol^{50 (link),80 (link)} and eluted in 100 µl of TET buffer (10 nM Tris, 1 mM EDTA and 0.05% Tween-20). 20 μl of DNA extract were transformed into genetic library following a double-stranded DNA protocol^{81 (link)} using a unique combination of two indexes. Sample and negative controls were checked with Agilent 2100 Bioanalyzer DNA 1000 chip. Libraries were then enriched for mitochondrial DNA following a capture protocol^{81 (link),82 (link)} and sequenced on an Illumina MiSeq run for 2 × 75 + 8 + 8 cycles.