The application of next generation sequencing to Archaeology

PhD Candidate:Niall O'Sullivan
Supervisor: Professor Ron Pinhasi
Funded By: The European Academy, Bolzano, Italy

Abstract

The aim of this PhD project is to apply NGS (next generation sequencing) techniques to archaeological samples to retrieve biological information about individuals that would be difficult or impossible using PCR based approaches (Meyer and Kircher 2010). I will sequence ancient DNA using techniques that continue to develop in the field of biological archaeology. For example, the IS6110 sequence is often PCR amplified in modern and ancient pathogen samples, the size of the fragment is key to identifying a strain of mycobacteria. However, in ancient DNA, the region between the primers is often too fragmented or degraded even where lesions on the bone or tissue are present. Using WGS (whole genome amplification) and NGS it is possible to sequence all the mycobacterial DNA present in an unbiased fashion and identify the presence of pathogen specific genotypes. In the last decade, sequencing and computing technologies have developed to an extent that ancient DNA can be affordably and rapidly studied at a masters or PhD level. Moreover, HTS (high throughput sequencing) gives increased statistical weight to data allowing confident inferences to be made about the degree of contamination, degradation and even the age of the DNA.

Presently, I am engaged in three research projects under the theme of bimolecular archaeology in collaboration with several research institutes. Firstly, analysis of the Iceman’s clothing for presence of animal DNA. The aim of this project is to identify the species of the leather and fur, and their relation to modern domestic breeds. This will be done by using phylogenetic analysis of the mitochondrial DNA. As part of this project a supposed blood was extracted from the Iceman’s coat to see if it contains DNA belonging to him. Some phylogenetic analysis has already been done (Olivieri et al 2012), but only used part of the mitochondria and was not assigned to particular clothing.

Secondly, I am engaged in the analysis of a medieval gravesite in southern Germany, of Alemannic origin. The samples I am analysing were discovered in 1962 and were in poor state of physical condition for osteological analysis. In addition, they were exposed to modern contamination for over 50 years that they were in storage. The arrangement of the bodies and the artefacts at the gravesite confounded archaeologists about the origin, sex and pathology of the individuals. However, by using targeted enrichment on extracted DNA (Maricic et al 2010) and then in silico statistical analysis on sequences it was possible to increase the proportion of endogenous DNA and remove modern environmental contaminants. The aims of this project are to show population genetics and pathology of the medieval individuals and to place them in a European context using a technique adapted from Raghavan et al (2010). We searched for traces of pathogens in the teeth by enriching extracted DNA for TB and Yersinia pestis. We continue to look at their relation to each other and modern European populations. Thirdly, the analysis of metagenomic DNA for the presence of pathogenic and commensal bacteria in ancient individuals. Analysis of calculus for presence of pathogens or other mycobacteria. Calculus will become an important material for the study of metagenomes from archaeological specimens. Calculus accumulates over an individual’s lifetime in the teeth and encases bacterial DNA and particles of food. Calculus is quite durable and has been demonstrated to contain high amounts of metagenomic DNA in relatively high quantity compared to other archaeological material. This project will analyse ancient calculus from individuals that are likely the victims of pathogens. Calculus has been largely untested so far but a growing body of publications demonstrates its usefulness in directly analysing the metagenome (Warriner et al 2014). Overall, the aim of this project will be to investigate the metagenome in archaeological samples and use shotgun libraries from soil as a control. Overall, the data will provide insight into the daily lives of ancient individuals by being a proxy for general health, diet and exposure to pathogens.

References

Maricic T., Whitten M. and Pääbo S. (2010) Multiplexed DNA sequence capture of mitochondrial genomes using PCR products. Plos One, 5: 11; 14004.

Meyer M. and Kircher M. (2010) Illumina Sequencing Library preparation for highly multiplexed target capture and sequencing. Cold spring harbour protocol.

Olivieri C, Ermini L, Rizzi E, Corti G, Luciani S, et al. (2012) Phylogenetic position of a copper age sheep (Ovis aries) mitochondrial DNA. Plos One 7: e33792.

Raghavan et al (2014) The genetic prehistory of the New World Arctic. Science 345: DOI: 10.1126/science.1255832.

Warinner C, Rodrigues JF, Vyas R, Trachsel C, Shved N, et al. (2014) Pathogens and host immunity in the ancient human oral cavity. Nat GenetDOI:10.1038/ng.2906.