All pre-PCR procedures were carried out in a dedicated Ancient DNA facility in the Australian Research Centre for Human Evolution, Griffith University. The facility is sealed, geographically isolated from any modern molecular laboratory, and has one-way airflow under positive pressure, and the air is high-efficiency particulate air (HEPA)–filtered. The skeletal remains and hair samples were processed within an ultraviolet-sterilized ultralow particulate air (ULPA)–filtered vertical laminar flow cabinet (used for this purpose only). Each sample was initially treated with 10% bleach to remove any surface contaminants and then washed with UltraPure DNase/RNase-Free Distilled Water (Invitrogen) to remove any remaining bleach.

Skeletal material was processed using a Dremel rotary tool with a high-speed diamond cutter head or manually with a sterilized scalpel blade, where the outer surface was discarded. DNA was extracted from ~50 mg of bone or tooth powder, as previously described (13). Extraction blanks were included throughout all procedures. Hair samples were processed in 2 to 4 ml of digestion buffer, as previously described (14). This solution was incubated in a rotating incubator oven for 24 hours at 45°C. After complete digestion, the samples were centrifuged at 9000g for 3 min. The supernatant was combined with 10× volume of a modified binding buffer [500 ml of buffer PB (phosphate buffer; Qiagen), 1:250 pH indicator I, 15 ml of 3 M NaOAC (pH 5.2), and 1.25 ml of NaCl]. Extractions were purified using the MinElute Reaction Cleanup Kit (Qiagen) following the manufacturer’s protocol and eluted using 100 μl of buffer EB (elution buffer; Qiagen) after incubation for 10 min at 37°C.

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