HuMAN-PaGe

The application of paleogenetics can provide novel insights into human and microbial evolutionary history. During the course of my PhD project, I aim to use ancient DNA to infer human kinship structures and population genetics, extending the analysis to the reconstruction of the human microbiome and human-associated pathogens. By using next generation sequencing, target-enrichment techniques and bioinformatical pipelines designed for ancient genetic data, I will analyse human and microbial DNA from ancient human remains.

This project is made up of three parts. Firstly, around 30 individuals from a 7000-year-old mass grave in Talheim, Germany, will be analysed to infer possible familiar relationships, as well as their genetic affinities and population genetics. Secondly, metagenomic analysis of ancient dental calculus from 17^th century Trentino/Alto Adige individuals will be performed. Together with our collaborators in Trento, the ancient microbial datasets will be compared to modern dental calculus in the same area, in order to discern human oral microbiome evolution. With the available anthropological data, it will also be possible to compare ancient healthy and non-healthy oral flora. Thirdly, specific pathogens such as Yersina pestis (the causative agent of the plague) and Mycobacterium tuberculosis from ancient human remains throughout northern Italy and Europe will be studied to reconstruct their evolutionary and epidemiological history. 

Description of the overall project

The recent years’ advances in genetic sequencing has opened up a new world of research when it comes to human history. We now have the tools to confirm and expand the archaeological and anthropological knowledge by extending human history with molecular and genetic analyses. In this PhD project, I aim to shed light on human and bacterial population genetics and evolution through the ages. The project consists of three parts: population structure and evolutionary history of 1) humans, 2) the human oral microbial flora, and 3) human pathogens. The methodology used in these studies will be established ancient DNA laboratory protocols, high-throughput sequencing, DNA target-enrichment techniques and bioinformatic analysis specialised in ancient DNA.

Genome-wide analysis of the early Neolithic mass-grave individuals from Talheim, Germany.

The Talheim mass grave, found in Baden-Württemberg, Germany, is one of the earliest evidence of violent massacres of Early Neolithic Farmers in Europe. An excavation in 1983 unearthed 34 individuals dated to the Linearbandkeramik Culture (LBK, approx. 5000 BC). Two-thirds of the individuals displayed severe head trauma, in positions on the back and right of the skull indicating that they were attacked from behind. Individuals of all age groups and both sexes were discovered suggesting that the mass grave contained an entire community, killed and buried at the same time. Anthropological and isotope analyses have suggested a possible kinship between some of the individuals. Altogether, the current data point to the massacre of a complete community from a late LBK village, and these individuals could give us a unique insight into the family structure of an LBK village.

Twenty-four petrous bones from the Talheim site have previously been sampled and screened for DNA using high-throughput sequencing. The samples yielded authentic ancient genetic data, which will be further analysed in this study. I will also analyse an additional five petrous bones, as well as perform resampling of some of the already sampled individuals to confirm the previous genetical results. In total, the study will include 29 of the 34 individuals. The in-depth genetical analysis will include shotgun sequencing and target-enrichment to capture human mitochondrial and autosomal DNA. Genetical analysis using bioinformatical tools designed for ancient DNA will be used to infer the individuals’ genetical sex, kinship structure to the familiar level, and population genetics to discern their genetic affinity to other ancient and modern populations. Anthropological data have been provided by Joachim Wahl of the State Office for Cultural Heritage Management Baden-Württemberg, Osteology, in Konstanz, Germany. A compilation of the existing literature, including the anthropological and stable isotope analyses, will also be performed.

Characterisation of the human oral flora in ancient dental calculus from Trento-Alto Adige, Italy

There are billions of bacteria living in and on the human body – the human microbiota. In the human oral cavity, the oral microbiome consists of a complex community of microorganisms, which plays an important part in dental health. However, the oral microbiota can also be the cause of dental disease; dental plaque – a biofilm of microorganisms – is one of the major causes of human oral disease like periodontitis and gingivitis, and the bacterium Streptococcus mutans is the major cause of dental caries. Dental calculus is formed by the calcification of dental plaque and contains an abundance of molecules from the microorganisms that formed it, including their DNA. These molecules have been shown to persist in the dental calculus several thousand years after the individual died. Therefore, by analysing ancient dental calculus, it is possible to study the human oral microbiota through history.

In this study, we aim to analyse the evolutionary history of the human oral microbiome. Ancient individuals recovered from different archaeological sites in Trentino-Alto Adige, spanning from the Neolithic to the Early Middle Age, have previously been sampled for dental calculus. The study of the human remains was previously authorised by the Archaeological Office of the Autonomous Province of Bolzano.

The dental calculus samples have been screened for ancient DNA, successfully generating oral microbial sequences from 20 individuals. Together with our collaborators at the University of Trento we will compare the oral flora of these ancient individuals with those of modern individuals from the same area, thus giving insight in how the microbiome have changed since the Neolithic, including strain level and phylogenetic analyses. By including anthropological data of the dental disease state of the ancient individuals, we also aim to compare healthy and non-healthy oral floras in both modern and ancient individuals. We also aim to perform analyses that compare the samples with other previously published ancient oral microbiomes, and search for antibiotic resistance genes that might have been present in these microorganisms.

Evolution of ancient pathogens

As opposed to the healthy human microbiome, there are other microorganisms that are negatively associated with human health – pathogens. Innumerable people have died due to bacterial infections through the course of human history. By studying ancient pathogens, it could be possible to infer their occurrence and epidemiology through human populations in the past.

Two diseases that have had a severe impact on the number of human deaths are the plague and tuberculosis. The plague raged through Eurasia in three big waves, killing up to 60 % of the population; the Justinian plague in the 6^th century; the Black Death in the 14^th century; and the bubonic plague in Asia in the middle of the 19^th century. Tuberculosis have been a known disease throughout known human history, reaching its most deadly during the 18^th and 19^th century, accounting for almost one in every four deaths, and is still killing 2 million people worldwide each year.

The causative agent for plague have been determined as the bacteria Yersinia pestis. This has been done using genetical analyses of human remains from suspected plague mass graves, where DNA sequences of Y. pestis have been found and reconstructed. Similar plague mass graves are present in Trentino and Alto Adige, but so far, the causative agent has not been confirmed. Using historical records to find the cause of deaths is difficult, since many diseases have similar symptoms to the plague, including typhus fever and smallpox. Tuberculosis is caused by the bacteria Mycobacterium tuberculosis, and its DNA have been found in human remains dating as far back as 7000 BCE.

The aim of this project is to reconstruct evolution and spread of human pathogens like Y. pestis and M. tuberculosis. We have access to the human remains from the 17^th century mass graves discovered in Bressanone Oratorio Don Bosco and in Naturno S. Procolo, South Tyrol, which we will analyse for Y. pestis.  Together with our collaborators at the Department of Biological Anthropology at the University of Szeged, Hungary, we will analyse 18^th and 19^th century mummies from Vác, Hungary, for M. tuberculosis. In order to retrieve as much endogenous pathogenic DNA as possible, sites not usually sampled for human DNA will be tested; inside the pulp chamber of teeth (Y. pestis) and ribs and lung tissue (M. tuberculosis). Using M. tuberculosis target enrichment, DNA sequences will be more easily detected from the vast flora of microorganisms surrounding the human body before and after death, thus increasing the amount of data to be analysed. The genetic sequences will be analysed against their modern counterparts, as well as other ancient strains, in order to infer their spread and infection routes. In the cases where the human individuals’ identity or relatedness to other individuals are known, it could also be possible to infer if relatives share the same strains of the pathogen, thus indicating a spread between family members. We also aim to perform genome reconstruction of the different pathogens to perform strain level comparisons and phylogenetic analyses.