Recovery of ancient DNA from Upper Nubian skeletal remains ABAGAIL M. BREIDENSTEIN 1,2 , ABIGAIL BOUWMAN 2 , GRETCHEN E. ZOELLER 3 , GEOFF EMBERLING 4 , FRANK RUEHLI 2 and ABIGAIL W. BIGHAM 1 1 Department of Anthropology, University of Michigan, 2 Institute of Evolutionary Medicine, University of Zurich, 3 Department of Anthropology, Indiana University, 4 Kelsey Museum of Archaeology, University of Michigan The use of ancient samples from the Nile River Valley for anthropological genetic studies has proven difficult due to adverse conditions for preservation of biomolecules like DNA. Recent advances in recovery methodologies of ancient biomolecules, however, have allowed for the exploration of populations in this region previously not included, namely Upper Nubians (modern Sudan). For this study, we attempted to recover ancient DNA (aDNA) from a sample of Medieval Nubians, excavated from the Christian settlement site of el-Kurru. Skeletons of twenty-eight individuals were exhumed from the adjacent cemetery using sterile techniques during recovery and post-field processing. aDNA was extracted and amplified using previously optimized protocols in a clean room setting. Initial results show human DNA is recoverable, despite poor preservation of the skeletal tissue and teeth due to thermal degradation in this climate and repeated inundations over many centuries a t el- Kurru. These results demonstrate the potential to explore further the genetic history of Nubia by including populations from theregion of Upper Nubia, thus expanding the use of aDNA throughout the ancient Nile Valley. With its rich and expansive history, the ability to examine the genetic makeup of the Nile Valley in a fuller capacity will undoubtedly provide valuable information allowing for an even deeper and more comprehensive understanding of this region for future research. Sample Prep • Photograph samples • Bleach, UV treatment, Decontamination • Tooth samples > Enamel removed, pulverized into powder, weighed • Petrous Bone samples > outer service removed, hardest tissue drilled and weighed Pre- Extract Digestion • Two extractions performed • 100mg powder digested with EDTA + Proteinase K solution with agitation • Second digestion used from DNA extraction (Pinhasi et al 2015) with modifications (Hansen et al 2017) DNA Extraction • Modified MinElute protocol using silica columns (QIAGEN) • Eluted in 60uL of buffer • Qubit Assay to quantify DNA concentration MtDNA Screening • Inhibition test with lambda DNA, serial dilutions if needed • Amplification of Hypervariable Region of MtDNA, using four optimized primer sets Sequencing • Purified samples via QIAGEN columns • Off-site, direct sequencing via GATC services (Konstanz, Germany) • Cloning next? • The Nubian site of el-Kurru (modern Sudan) lies along the Nile River about 140 km upstream of Old Dongola, the capital of the Medieval Christian kingdom of Makuria • First excavated by George Reisner (Harvard) in the 1910s, exploring the Royal pyramids and burials tombs of the Kushite Kings (25 th Dynasty, 8 th and 7 th centuries BCE) • International Kurru Archaeological Project excavated in 2013-17, centered on restoration and discovering the fortified Medieval settlement, dating to roughly 600-1000 CE SETTING • 28 Individuals excavated; sex and age estimations performed, pathologies noted in-field • 11 subadults, 16 adults (1 undetermined) of various ages • Undisturbed, fully articulated skeletons • Christian burials (cemetery organization, body treatment, grave fill) • Excavation of skeletons – exposed, photo-planned, lifted, sampled & catalogued MATERIALS • Ancient Nubia has been occupied since 7,000 BCE and spans from modern day Aswan in southern Egypt to Khartoum in Northern Sudan • Populations in this region of the Nile Valley were mobile with broad trade contacts since prehistoric times, through the empire of Kush, the Medieval Christian kingdoms, and the arrival of Islam in the 14th century CE • Nubian populations are well researched from a bioarchaeological stance and are the focus of several of the best studied from this region of the world INTRODUCTION We would like to thank the funding sources for all aspects of this project, including excavation and lab work: the University of Michigan Rackham International Research Fund, the African Studies Center, and Rackham Graduate Research Funds. Additional thanks to the National Corporation for the Antiquities and Museums in Khartoum, the including Dr. Abdelrahman Ali, the International Kurru Archaeological Project personnel, and the villagers of el-Kurru. A special thank you to those who helped gather data in el-kurru and at the IEM: namely, Annina Kruettli, Martin Uildriks, and members of the IKAP team. ACKNOWLEDGEMENTS 1. Skuldbøl T, Uildriks M, Rose K, Philips J, & Breidenstein A. “The Medieval Fortification, Settlement, and Cemetery” in El-Kurru 2015- 16: Preliminary Report. Sudan & Nubia, 20, 40-46. 2. Emberling, G., Dann, R. J., Abdelwahab Mohamed-Ali, M., Skuldbøl, T. B. B., Cheng, J., & Blinkhorn, E. (2013). New Excavations at El-Kurru: Beyond the Napatan Royal Cemetery. Sudan & Nubia, 17, 42-60. 3. Pinhasi, R., Fernandes, D., Sirak, K., Novak, M., Connell, S., Alpaslan-Roodenberg, S., ... & Anders, A. (2015). Optimal ancient DNA yields from the inner ear part of the human petrous bone. PloS one, 10(6), e0129102. 4. Hansen, H. B., Damgaard, P. B., Margaryan, A., Stenderup, J., Lynnerup, N., Willerslev, E., & Allentoft, M. E. (2017). Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum. PLoS One, 12(1), e0170940. LITERATIRE CITED • Intact teeth and petrous bones shipped to Universität Zürich, skeletons to U Michigan for further analyses • Ancient work was carried out in dedicated, isolated lab space at the Institute of Evolutionary Medicine, adhering to strict decontamination protocols and strict rules to prevent cross- contamination or introduction of exogenous material • Multiple negative controls were included with each step in clean and modern lab settings • Pipeline as follows: METHODS Sudan & Nubia, 19, 20 RESULTS • These experiments demonstrate the presence of MtDNA from excavated remains from el-Kurru • Generally, extractions had a low success rate for extraction of endogenous human DNA • No evidence of contamination during PCR amplification or molecular sexing (results not shown), additionally too little DNA • Qubit values between ~ 0.4 – 1.1 ug/mL • Inconclusive results at this point and warrant further investigation in methodology • Amplified Mt region spans from position 16225 to 16325, not including primers • Two individuals produced readable sequence data – Individual 209 (adolescent) and Individual 203 (middle adult male) • Almost complete (98%) match to human mitochondrial DNA via BLAST • Reactions seemed to be heavily inhibited - even with dilutions, amplifications were unsuccessful RESULTS • Despite a low success rate, some MtDNA was recovered from remains and petrous bone sequence data is much cleaner • Not enough sequence data for evaluation of full HVR • Very low yield - preservation of remains is sub-optimal, due to the wadi fan, proximity to modern agricultural land (which is irrigated by flooding), and likely the seasonal inundation of the archaeological site before the damming of the Nile • Continued work includes finishing sequencing pipeline with cloning and authentication analyses • Second, independent extractions for verification of previous work • Troubleshooting with other extraction techniques with powdered tissue samples • Further efforts to Radiocarbon date two individuals and further bioarchaeological analyses DISCUSSION & FUTURE WORK Ind. 203 C1F Tooth Ind. 209 C1R Tooth Ind. 206 C1F Petrous Ind. 206 C1R Petrous Ind. 202 Temporal Bone Ind. 204 Mandible with Teeth • Human DNA recovered from one individual – Ind. 206 (young adult female) • Complete match (100%) to human mitochondrial DNA via BLAST • 141 bp section of HVR amplified using optimized primer set • There are matches to many haplogroups, due to small size of amplicon