Fig. 3

Methodological principles for ancient DNA investigation. Documentation of tissue type (i.e., leaves, seeds, stems, wood), conservation (i.e., dessicated, waterlogged, charred), and age is followed by sample decontamination, DNA extraction (depending on DNA degradation, DNA size, quantity, and absence of inhibitors), and NGS library (single-stranded) preparation (depending on input quantity, aDNA fragment size, and the number of amplification cycles) performed in standardized clean conditions using laboratory procedures optimized for aDNA. Sequencing reads can be mapped against a reference genome (defining endogenous aDNA and unique mapped reads) and authenticated based on typical post-mortem damage patterns using bioinformatic pipelines tailored for aDNA. The exogenous DNA content can be evaluated using metagenomic analysis tools. Finally, the authenticated endogenous DNA can be compared to modern reference samples to unveil genomic footprints of species’ origin, migration, anthropogenic translocation, extinction, and hybridization events. Examples of bioinformatic tools used in investigating aDNA sequences are mentioned at the bottom of each panel (see main text for further details)