Validation of a Non-Destructive DNA Extraction Protocol for Ancient DNA Analyses

Validation of a Non-Destructive DNA Extraction Protocol for Ancient DNA Analyses Frankie L. Pack1,2, Kathryn E. Kulhavy2, and Graciela S. Cabana1,2 1Molecular Anthropology

I.

Laboratories, The University of Tennessee Knoxville; 2Department of Anthropology, The University of Tennessee Knoxville

IV. Results

Introduction

The destructive nature of traditional DNA extraction techniques presents one of the primary obstacles to accessing genetic information from museum and archaeological collections. Here we assess a recently published “non-destructive” DNA extraction protocol by Bolnick and colleagues in terms of the quality of DNA extracted from a set of samples of even greater antiquity than those tested in the original analysis as well as a more recent sample.

II. Materials • Eva Samples- The collection Eva Site (40BN12) collection, currently housed at the McClung Museum of Natural History and Culture (MMNHC) at The University of Tennessee, Knoxville, is a stratified Archaic shell midden from the western Tennessee River Valley.Teeth from 4 Eva individuals from Stratum IV, which was recently re-dated to ca. 8,450 – 8,200 cal BP (Bissett 2014), were collected from the MM. • Norris Farms #36 Samples - An additional 4 teeth from the Norris Farms #36 archaeological collection were used with permission from the Illinois State Museum. The Norris Farms #36 site is an Oneonta cemetery located in northern Illinois, dating to ca. 1300 AD (Stone & Stoneking, 1998).

Hypervariable regions I and II (HVR I and HVR II) from all 8 samples were successfully amplified and visualized on a 3% agarose gel. Paired t-tests demonstrate that there were no statistically significant differences in dental metrics nor weights as a result of the demineralization soak.

• Sequencing – All 4 Eva Collection HVR I and HVR II amplified samples were purified and taken to the Molecular Biology Research Facility at the University of Tennessee for Sanger sequencing. DNA sequences were analyzed using DNAstar SeqMan software. Of the four Eva samples sequenced, two (Samples 181 and 182) did not yield enough sequence data to determine haplogroup assignment. Samples 175 and 37, were assigned to Haplogroup B. All results are tentative, as analyses have not been confirmed by independent analysis. • Next-generation Sequencing – Eva Samples 175 and 37 were selected for high-throughput sequencing and taken to the Malhi Molecular Anthropology Laboratory at the University of Illinois at Urbana Champaign. Libraries were constructed using the Illumina TruSeq kit in the dedicated ancient laboratory. Using the MyBaits target enrichment kit by Mycroarray, whole mitochondrial genomes were captured for both samples. Samples were barcoded Sequencing was performed on the Illumina HiSeq platform. Sequence data were analyzed using DNAstar software and confirmed the Sanger sequencing results and assignment of both samples to Haplogroup B. Sample ID Eva 37 Eva 175

III. Methods • Sample Preparation – All teeth were decontaminated by soaking in 6% sodium hypochlorite for 15 minutes (after Kemp and Smith, 2005), rinsed with molecular grade water, and air dried. • Testing the Bolnick Protocol – All 8 teeth were soaked in a demineralization buffer (10ml 0.5M EDTA, 150ul proteinase K), following the Bolnick Protocol for 24 hours. Buffer was poured off and retained for extraction. Teeth were rinsed with molecular grade water and air dried for storage. The buffer solution was extracted following the silica-based protocol by Rohland and Hofreiter (2007) as recommended by Bolnick and colleagues (2012). • Amplification - All samples were amplified using a short mitochondrial DNA (mtDNA) primers (167 bp) from the HVI region, designed for degraded DNA (Kemp, 2006). • Metrics - Weight and standard dental metrics (crown height, mesiodistal length, and buccolingual width) were collected before and after soaking to evaluate the effect of the Bolnick Protocol on gross morphology of the teeth after full treatment. • Best Practices - Sample preparation/decontamination, extraction, and PCR setup all occurred in the Molecular Anthropology Laboratories’ dedicated Ancient DNA Laboratory (Unless otherwise noted. See below for contamination controls). PCR amplification and 3% agarose gel electrophoresis were performed in the MAL’s Modern DNA Laboratory at the University of Tennessee.

Contamination Controls at the Molecular Anthropology Laboratories at the University of Tennessee, Knoxville (MAL-UTK) All relevant precautions for preventing and detecting exogenous contamination were followed during this research (Kaestle & Horsburgh 2002; Knapp et al. 2012): DNA extractions and PCR set-up took place in a restricted access clean room that is dedicated to pre-PCR ancient DNA research and is equipped with dedicated equipment, overhead UV lights, positive air pressure, and HEPA-filtered ventilation. The post-PCR laboratory is located in a separate wing of the building and is on a separate air handling system. Personnel movement between facilities is unidirectional (from pre-PCR forensic genetic laboratory to post-PCR). A decontaminated face shield, mask, disposable coveralls with hood, shoe covers, and double gloves were worn at all times in the ancient DNA laboratory. Workspaces and equipment were regularly decontaminated with a 30% household bleach solution and/or DNA AWAYTM, and UV-irradiated between uses. All reagents and disposable tubes were certified DNA-free and/or molecular grade. Negative (blank) controls were included at all stages of extraction and amplification.

HVR I Polymorphisms 16187, 16189, 16217 16187, 16189

HVR II Polymorphisms 00073, 00263 N/A

Haplogroup Assignment B4 B

Above: Table depicting DNA polymorphisms from Sanger sequencing. Right: Tooth pictures, before and after soaking. Sample 37: Before (left),After (right)

Sample 175: Before (left),After (right)

V. Conclusions & Future Directions This research validates the utility of the Bolnick non-destructive DNA extraction protocol. Here, we corroborate that this method produces DNA extracts that can be successfully amplified and sequenced using traditional Sanger sequencing. Additionally, this research demonstrates that non-destructively extracted samples can be successfully used for high-throughput techniques, specifically, whole mitochondrial genome sequencing. We also show that the method is indeed “non-destructive.” Dental metrics and weights taken before and after extraction indicate no significant change in the teeth due to the extraction process. The use of the Bolnick protocol and other modified non-destructive extraction methods could not only expand the range of samples available for DNA analysis, as well as the downstream analyses possible.

References • • • • • • • •

Bissett TG. 2014. The Western Tennessee shell mound archaic: Prehistoric occupation in the lower Tennessee River Valley between 9000 and 2500 cal years BP. Doctoral dissertation, Bolnick DA, Bonine HM, Mata-Miguez J, Kemp BM, Snow MH, and LeBlanc SA. 2012. “Nondestructive sampling of human skeletal remains yields ancient nuclear and mitochondrial DNA.” American Journal of Physical Anthropology 147(2): 293-300. Kaestle FA, and Horsburgh KA. 2002. Ancient DNA in anthropology: Methods, applications, and ethics. Yearbook of Physical Anthropology 45: 92-130. Knapp M, Clarke AC, Horsburgh KA, and Matisoo-Smith. 2012. Setting the stage – building and working in an ancient DNA laboratory. Annals of Anatomy 194: 3-6. Kemp BM, and Smith DG. 2005. “Use of bleach to eliminate contaminating DNA from the surfaces of bones and teeth.” Forensic Science International 154: 53-61. Kemp BM. 2006. Mesoamerica and Southwest prehistory, and the entrance of humans into the Americas: mitochondrial DNA evidence. Ph.D. dissertation, University of California, Davis. Rohland N, and Hofreiter M. 2007. Ancient DNA extraction from bones and teeth. Nature Protocols 2: 1756-1762. Stone A, and Stoneking M. 1998. mtDNA analysis of a prehistoric Oneota population: Implications for the peopling of the New World. American Journal of Human Evolution 62:1153-1170.

Project Acknowledgements: We would like to thank the McClung Museum of Natural History and Culture for the opportunity to work with the Eva Collection samples. We also thank Dr. Deborah A. Bolnick for sharing her protocol and expertise for this project and Dr. Ripan Malhi for his support of the next generation sequencing portion of this research.