Heritage Cases

Methodology (short): Genome sequence data for hafting mastics will be generated. DNA will be extracted from the samples and DNA libraries will be prepared. Well established and published ancient DNA laboratory procedures such as Günther et al (2018) will be followed, albeit with minor changes: there will be a reliance on extraction methods for ‘dirty’ samples and the use of dual indexing. Also, a different sequencing instrument using the same technology as in the previous studies (Next Generation Sequencing / NovaSeq) will be used for DNA sequencing. Residual adapters will be trimmed, and the reads will be mapped to the reference human genome. Authenticity of the sequences will be evaluated using well-established methods. The aim will be to bring 20% (10) of the samples to >1X coverage, which will allow for genotype imputation to discover diploid genotypes. Two samples will be brought to >10X, and for these high covered samples, GATK’s Best Practices will be followed to discover diploid genotypes with some modifications to deal with the nature of ancient DNA. The project will benefit from the toolbox assembled and developed within the ATLAS project, and 1000 Ancient Genomes, such as the systems used to authenticate ancient DNA with a general assessment based on, in-sample variation of mtDNA and sex-chromosomal DNA (to assess a general contamination level), and a specific assessment based on damage patterns as analysed by the PMD-tools. When these systems are in agreement for an ancient single-source of origin, they present a strong case for authenticity. The toolbox also includes well-established downstream analyses for demographic purposes such as D-statistics, F-statistics, ADMIXTURE, and Principal Component Analysis (PCA). Confirmation/permit by museum: Curator letter Damage/destructive analysis? (if yes, explain in detail) Samling: The mastic pieces / residues will be irradiated in a crosslinker, at about 6 J/cm2 at 254 nm. The outer shell of the mastics / residues will be discarded to avoid surface contaminants. The use of a Dremel drill or a scalpel will allow the collection of material which will be collected into 2ml plastic tubes. The weight of the obtained “powder” should be between 50 and 200 mg depending on the sample. Extraction: For DNA extraction, we will perform several incubations on the samples. At each incubation the samples will be kept in rotation. First, the samples will be pre-digested at 45 °C for 15 min in 1000 μL of extraction buffer, consisting of Urea, EDTA (0.5 M) (VWR) and 10 μL of Proteinase K (10 mg/mL) (VWR). A negative control will be added during this step and taken through the work process. The supernatant from the predigestion step will be removed and a fresh extraction buffer (same as above) with proteinase is added to the samples and left for digestion overnight at 37 °C. The supernatant from this step will be stored, and more extraction buffer and proteinase will be added to the samples (same as above) and left rotating at 55 °C for 4 h. The final supernatant will be collected and combined with the previous one (around 2000 μL) and spun down to 100 μL using membrane filters (Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-30 from Millipore). The extract will be purified using MinElute spin columns and a buffer set (both Qiagen). We will modify the Qiagen protocol (reducing the PE buffer volume to 600 μL and performing two elutions using 55 μL of the EB buffer) and obtain about 110 μL of extract, which will be stored at −20 °C. After extraction the DNA is captured on a silica membrane of a spin column and purified, resulting in 100 μL of product which is then stored at −20 °C. Statement why this study cannot be done in South Africa: The expertise for extracting the unique genetic information from chewing gums and hafting mastics was pioneered at Stockholm University, which recently formed the Centre for Palaeogenetics (CPG) in collaboration with the Swedish Museum of Natural History. CPG researchers recently set the record for the oldest aDNA ever recovered, demonstrating their expertise as world leaders in the field of prehistoric genetic research. The CPG hosts a dedicated aDNA and ‘dirty’ samples laboratory, which is fully equipped and specialises in the extraction and analysis of aDNA from such samples. The application of these cutting-edge methods developed in Sweden, has the possibility to revolutionise our understanding of human evolution and development in Africa – the ‘Cradle of Humankind’.