Abstract
The Vác Mummy Collection (VMC) housed at the Hungarian Natural History Museum comprises 265 naturally mummified individuals from Hungary buried in a local crypt between 1731 and 1838 in pinewood coffins. Since their discovery in 1994, numerous studies have been conducted applying anthropological and molecular methods showing that the majority of the individuals suffered from latent or active Mycobacterium tuberculosis (MTB) infections, the causing agent of tuberculosis. Nevertheless, retrieving sufficient genomic pathogen sequencing data to perform in-depth analyses remains challenging due to the nature of ancient DNA and exogenous contamination by other microbial DNA. Therefore, we believe it is crucial to take a step back to investigate technical optimizations that could offer important insights to improve the study of the evolutionary history of the MTB complex. Here, we aimed to extend the collection of ancient genomic MTB data with particular emphasis on the detection of mixed MTB infections and optimization of the molecular workflow, specifically adapted for skeletal specimens, by applying modified sampling techniques and DNA extraction protocols. Out key findings show the following: (1) intra-bone variability of samples can affect pathogen DNA yield (2) DNA precipitation using linear polyacrylamide is highly purifying, and an effective alternative to silica-based DNA extractions, and (3) a mixed MTB infection in a 62-year-old midwife demonstrates the long-time prevalence of the European sublineages L4.1.2.1/Haarlem and L4.10/PGG3 within the local community. Finally, we highlight the vast potential of the VMC to study local epidemiological links and transmission events by combining genetic data and historical records.