Title: A leap in the dark – How intra-bone variability of pathogen load affects metagenomic detection of ancient Mycobacterium tuberculosis DNA in skeletal human remains

Authors: Heidi Y. Jäger¹, Frank Maixner¹, Albert Zink¹

Affiliation: ¹Institute for Mummy Studies, Eurac Research, Viale Druso 1, 39100 Bolzano, Italy

Abstract: While it is known that environmental conditions influence overall DNA preservation over time and the degree of exogenous contamination of a sample, little is known about how intra-bone variability could influence the yield and quality of extracted DNA. To shed a light on this matter, we selected four skeletal samples with PCR-based evidence for Mycobacterium tuberculosis (MTB) and applied a comparative metagenomic approach by sampling equal amounts of bone powder from multiple locations. All samples were subjected to the same molecular workflow including DNA extraction, library construction, and shotgun sequencing. General taxonomic profiles were generated, and MTB and human endogenous DNA content were evaluated. Additionally, PCR analysis targeting the multicopy insertion element IS6110 was performed. Even though microbial compositions within and across samples displayed low variances, we could confirm high intra-bone variability of up to 36.45- and 62.88-folds for authentic MTB and human reads. Furthermore, we highlight that MTB detection with PCR is highly sensitive and does not necessarily translate into metagenomic detection of MTB, yet sufficient coverage for genome level analyses. Hence, we believe that this multiple sampling approach could be effective as an initial screening method to gain a detailed picture of a bone sample. Once hotspots with high pathogen load are determined, additional optimization steps such as targeted enrichment or deep shotgun sequencing could be applied.