Reassessing DNA Preservation of Ancient Material from Poulton, UK and Testing and Optimizing of CATS: a Novel Single-Strand DNA Library Preparation Technique

Abstract

Sequencing of ancient DNA with low preservation is still a major challenge, despite the immense progress that the field of ancient genetic studies has made since its inception over thirty years ago. The main aim of this study was to reassess the DNA preservation of ancient medieval material from Poulton, Cheshire, UK, using standard dsDNA library preparation methods. We targeted cementum of teeth as the optimal substrate for DNA extraction, along with long bones for comparison. A second aim, using a newer single-stranded DNA (ssDNA) library method, namely Capture and Amplification by Tailing and Switching (CATS) was also tested for the potential of isolating/obtaining higher levels of endogenous DNA from Poulton material. Ten libraries built by the double-stranded DNA (dsDNA) method were sequenced successfully, displaying well-preserved DNA with good potential for high- coverage genome studies. Principal Component Analysis (PCA) and haplogroup data suggests that the Poulton samples group within modern European variation, though with some affinity to Southeastern Europe relative to modern British people. Sex determination of the Poulton samples presented four males and three females. The ssDNA CATS method holds potential for further development, but in our study did not perform better than the standard dsDNA library preparation method. We also compared the sequence quality of samples that were washed upon excavation with samples that were not washed. These results highlighted the sensitivity of ancient samples to washing and importance of following up-to-date guidelines for optimal protection of DNA in excavated ancient material.