Advancements and optimization in ancient DNA protocols

The recovery of ancient DNA can only be achieved through the destructive sampling of archaeological remains. It is, therefore, of utmost importance to maximize the efficiency of both the laboratory protocols utilized and the data generated in this fashion. Here we present four manuscripts detailing a set of suggested best-practice sampling and analyses guidelines aimed at making the destructive sampling of ancient remains a more ethical and informed process. Manuscript 1 represents the first large-scale, systematic investigation of DNA preservation in Medieval skeletal remains spanning 23 possible sampling locations. We find DNA preservation to be best in cortical bone recovered from the cochlear region of the petrous pyramid, the vertebral body and superior vertebral arch of thoracic vertebrae, the exterior neck of the tali, the shaft and apical tuft of distal phalanges, as well as material recovered from the interior of the dental pulp chamber, dentin, and cementum of molars. In Manuscript 2 we provide the sampling protocols developed for the generation of bone powder from these sampling locations in both text and video formats. In Manuscript 3 we demonstrate the utility of extended screening of the datasets generated from the next generation sequencing of DNA recovered using these techniques by detailing two new high-coverage Yersinia pestis genomes recovered from these same samples. These genomes, despite not being recovered from a site exhibiting any epidemic context, provide strong evidence that Pestis secunda originated from a central European rodent refugia, continued to evolve, and later re-emerged to re-infect the human population and eventually spread back into eastern Eurasia. Manuscript 4 details a successful proof-of-concept manuscript detailing the feasibility of recovering very long DNA fragments ( > 8 kilobase pairs) from modern human dental calculus for use in the de novo assembly of high-quality oral microbiome reference genomes.