Ancient DNA Extraction and Mitochondrial Enrichment

Bone samples were exposed to UV-light overnight to remove surface contamination. A sample of 50mg was removed from the inside of the longbone of each bone using a dentistry drill. DNA extraction was carried out using a guanidinium-silica based method24 (link). For each sample a DNA library was prepared according to published protocols25 (link). Sample-specific indexes were added to both library adapters to allow differentiation between individual samples after pooling and multiplex sequencing26 (link). Indexed libraries were amplified in 100 μl reactions followed by purification over Qiagen MinElute spin columns (Quiagen, Hilden, Germany) and quantification using Agilent 2100 Bioanalyzer DNA 1000 chip. Target enrichment of mitochondrial DNA was performed by capture of the pooled libraries using bait generated from modern roe deer (Capreolus capreolus) mitochondrial DNA27 (link). The bait was generated by use of three primer sets (table 1) designed with the Primer3Plus software package. All extractions and pre-amplification steps of the library preparation were performed in clean room facilities and one negative control was included for each reaction.