Method development for screening archaeological samples for ancient pathogens

Recent advances in sequencing technology made it possible to retrieve DNA from archaeological samples that are hundreds or thousand years old Working with DNA retrieved from archaeological samples poses its own unique challenges. Firstly, DNA has a half-life and will decay after death, that means, only a very small part of a samples DNA content is endogenous DNA. Further the small amount of endogenous DNA that is left tends to be very short and fragmented. In this thesis, we address the main challenges faced by paleomicrobiology. With HOPS, we created a novel screening tool tailored to the characteristics of ancient DNA. HOPS produced reliable results both with in silico as well as real data for as few as 50 reads per species, which are an adequate representation of most screening Samples. While HOPS is designed to be still sensitive when working with low endogenous degraded DNA, it is dependent on the quality of the metagenomic reference database that is the foundation of the analysis. A novel tool DatabaseZen downloads and generates metagenomic databases from NCBI RefSeq, while using several filters to ensure database quality by removing contaminated sequences and avoid database bias by ensuring a balanced database composition. The low-endogenous DNA content in archaeological samples has negative consequences for paleomicrobiology, as most of the sequencing effort will go towards environmental contaminants instead of the remnants of the microbiome preserved in a sample. To that and we use a novel 16S/18S rRNA in-solution capture as an alternative to whole genome shotgun sequencing. The 16S capture was highly efficient in enriching for 16S/18S rRNA in all analyzed samples. The capture has the advantage of removing the length bias, which is present in 16S amplicon sequencing. Overall, we provided a screening tool for paleomicrobiology, an application to generate and clean reference databases and an alternative sequencing technology.