Abstract
Background: To date tuberculosis is one of the leading causes of deaths worldwide. Yet, despite being an ancient disease, the evolutionary history of its causative agent Mycobacterium tuberculosis remains enigmatic. Paleogenetic studies have been successful in proving tuberculous DNA in ancient human remains, but full genome wide reconstructions are still limited to well-preserved specimens with low environmental contamination and connected with extensive screening efforts. Hence, there is a high necessity to improve diagnostic tools to create well covered genome wide data.
Method: In this study, we designed a RNA:DNA hybridization capture assay using a unique RNA probe set (7.849 Mbs) targeting specifically M. tuberculosis complex (MTBC) and M. leprae DNA. We evaluated the assay efficiency on human remains with different endogenous contents. Thereby, we examined different parameters such as hybridization temperature and incubation time and created a comparative dataset with high throughput DNA sequencing and metagenomic analyses.
Results: In a preliminary test, the newly developed capture assay was examined for sensitivity, specificity, and coverage of MTBC DNA in tissue biopsies of five 18th century mummified human remains from Vac, Hungary. The test revealed a positive enrichment effect of up to 20-folds in four individuals. Importantly, the captured reads display an even distribution over the whole genome which excludes a capture bias on certain genome regions only.
Discussion: This study provides the framework for the development of a screening tool specifically for MTBC DNA in ancient human remains. If further optimized, it could be also interesting for clinical metagenomics to screen and characterize MTBC in patient samples on a full genome level.