DNA extraction and sequencing

Six specimens consisting of intact multi-rooted teeth (labeled as Cap-C58-1, Cap-C58-2, Cap-C58-3, Cap-C58-5, Cap-C58-6, and Cap-C58-9) were sampled and stored for genetic analysis during the archaeological excavation. All procedures leading to Next Generation Sequencing (NGS) were performed in dedicated aDNA facilities of the Molecular Anthropology Laboratory, University of Florence. Standard criteria for the analysis of aDNA³⁹ were rigorously followed and strict precautionary measures were undertaken to avoid contamination with exogenous DNA. All experimental steps were implemented following the protocol described in detail by Modi et al.⁴⁰ and Tassi et al.⁴¹. In brief, each tooth sample was cleaned by mechanical abrasion of the surface using a dental micro-drill, followed by UV irradiation (254 nm) for 45 minutes, on each side. The powdered tissue used for the isolation of genetic material was obtained from the tooth roots using the same dental device at a very low rotation speed. For each sample, DNA was extracted from 50 mg of biological material following a silica-based protocol, specially designed to target ultra-short molecules⁴². Illumina libraries were constructed using a previously developed protocol⁴³. No enzymatic treatment for damage repair was applied in order to preserve and consequently analyze the degradation patterns of DNA fragments. The libraries were amplified to reach plateau and enriched for human mitochondrial DNA using a selective bead-capture approach by hybridization of aDNA with probes consisting of long-range PCR products¹⁴. A negative control was processed during all analytical phases to monitor for possible contamination. The enriched libraries were pooled in equimolar ratios with libraries from other samples and paired-end sequenced (2 × 75 + 8 + 8 cycles) using the Illumina MiSeq platform.