Heritage Cases

DNA analyses of sediment from South Africa: Klipdrift Cave, Klipdrift Shelter, and Blombos Cave

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Through the analyses of sediment from Klipdrift Cave (KDC), Klipdrift Shelter (KDS), and Blombos Cave (BBC), we propose to identify hominin groups and lineage assignments using sediment samples and, when possible, generate nuclear genomic data.

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1. Objectives of Study In the last few years, increasing amounts of ancient DNA sequences have become available from ancient humans, including full genomes from Neandertals, Denisovans and Early Modern Humans (e.g., Meyer et al., 2012; Fu et al., 2014: Prüfer et al., 2014; Prüfer et al., 2017). In 2017 our lab vastly expanded this pool of resources by showing that it is also possible to retrieve ancient DNA from sediment: not only ancient faunal DNA, but also ancient hominin (e.g. Neandertal) DNA, perhaps most notably from an archaeological site that had not previously yielded skeletal remains of Neandertals (Slon et al. 2017). Through the analyses of sediment from Klipdrift Cave (KDC), Klipdrift Shelter (KDS), and Blombos Cave (BBC), we propose to identify hominin groups and lineage assignments using sediment samples and, when possible, generate nuclear genomic data. 2. Materials & Methods A. Sampling techniques In the case of DNA extraction from sediment, ~ 50mg is used for a single extraction, but larger samples (0.5 to 1 gram) are requested as they enable repeat-experiments. Only 5-10 sediment samples are needed in the initial screening phase to establish whether ancient DNA is preserved at a site. These preliminary samples for screening DNA preservation from KDC, KDS, and BBC will be taken from areas scattered throughout the stratigraphy. While the exact origin of hominin DNA in sediment is still unclear, we will target the areas at the sites where one would identify a more intense occupation or use by hominins (artefacts, tools, hearths, etc.) in an attempt to increase the chances of retrieving their DNA. Sediment samples will be taken while wearing gloves and a face mask. The first 1-2cm of the surface material will be removed using a scalpel, and then with a sterile scalpel and new pair of gloves, the sediment sample will be collected into a sterile tube, then placed into a plastic bag, and stored in a cool location until transport. Transport of samples will be by DHL courier. B. Analytical procedures 1) First screening phase: preservation of ancient mammalian mtDNA DNA will be extracted in an ancient DNA cleanroom using a method developed for the isolation of highly degraded DNA from skeletal remains and sediments (Rohland et al. 2018). In the first step of DNA extraction, DNA that is bound to mineral particles is released. DNA is then isolated under conditions that minimize the co-extraction of inhibitory substances such as humic acids. This and all following steps are performed on Bravo NGS workstations (Agilent Technologies) using automated liquid handling. The purified DNA is converted into a DNA library using single-stranded DNA library preparation (Gansauge et al. 2017). The efficiency of library preparation is monitored using a synthetic oligonucleotide (Glocke and Meyer 2017). Libraries are then amplified and barcoded with two sample-specific indices (Kircher et al. 2012) and enriched for mitochondrial DNA (mtDNA) from more than 240 mammalian species, including hominins, by hybridization capture (Slon et al., 2016; Slon et al. 2017). Mammalian sequences will be identified through alignment to a reference database of mammalian mtDNA genomes. Sequences with similarity to mammalian DNA are then taxonomically classified on the family level through a comparison to a reference database of mammalian mtDNA genomes. Sequences assigned to each family will be evaluated separately for the presence of deamination-derived cytosine (C) to thymine (T) substitutions at their ends to determine whether they derive from genuine ancient DNA or recent contamination. If none of the samples from an archeological site yield mammalian mtDNA sequences with signals of deamination, the site will be excluded from further analyses. 2) Second screening phase: identification of ancient hominin mtDNA In a second screening phase, all libraries displaying evidence for ancient DNA preservation will be enriched specifically for hominin mtDNA (Fu et al., 2013; Slon et al., 2017). Hominin mtDNA sequences will be identified and evaluated for the presence of ancient DNA base damage as described above. Using sets of ‘diagnostic’ positions in the mtDNA genome that define each branch in the hominin mtDNA tree, we will then assign the sequences to the modern human, Neandertal, Denisovan or Sima de los Huesos lineages. If necessary, this analysis will be restricted to sequences showing evidence of deamination in order to disentangle genuine ancient sequences from modern human contamination (Meyer et al. 2014; Slon et al. 2017). This analysis also allows identification of hitherto unknown mtDNA lineages, which would contain mutations common to all hominins, but none of the mutations specific to any known hominin groups. 3) Capture and analysis of nuclear DNA As a single genetic locus that is exclusively maternally inherited, mtDNA functions well as a marker for larger hominin groups (e.g. Neandertals vs Denisovans), but is of limited value for reconstructing more detailed population histories. To do so requires the recovery of nuclear DNA. However, sequencing full nuclear genomes from sediment is not technically feasible, and identifying nuclear DNA sequences from hominins is more easily confounded by the presence of DNA from other mammals. We therefore will use hybridization capture to target approximately one million informative single nucleotide polymorphisms (SNPs) in the nuclear genome that are located in regions of high sequence divergence between primates and other mammals (Vernot et al., in prep). Targeting a large number of SNPs will allow the retrieval of meaningful amounts of information from libraries that contain much less than one-fold coverage of the human genome, and from libraries where analyses have to be restricted to deaminated DNA fragments due to contamination with present-day human DNA. C. Justification for destructive sampling & sample export Sediment from an archaeological site is oftentimes treated as a byproduct of the excavation process. This research collaboration aims to take advantage of this historically undervalued material that might otherwise be washed or sieved away. Our group in Leipzig has developed and is continually optimizing some of the most sensitive methods for ancient DNA recovery presently used worldwide and is currently the only group with expertise in the retrieval of hominin DNA from ancient sediments. Furthermore, as we have now automated all steps of sample preparation on liquid handling systems (Bravo NGS workstations, Agilent Technologies), our laboratory is uniquely equipped to handle large screenings for DNA preservation in sediment samples in a time and cost efficient manner (Fu et al. 2013; Gansauge et al. 2017; Slon et al. 2017; Rohland et al. 2018). 4. Duration of analysis, expected results and their importance The term of this research collaboration shall be for five (5) years from the date of the signed accompanying Research Collaboration Agreement. The Parties will collaborate to facilitate the analysis of DNA from sediment samples from the archaeological sites of KDC, KDS, and BBC to assess the preservation of ancient DNA from both ancient hominins and fauna, in order to address questions regarding their evolution and/or dispersal(s). If successful, this collaboration could produce valuable insights into African prehistory, all while preserving precious skeletal material or, in some cases, even generating data from archaeological sites that have yet to yield hominin fossils. We will regularly report about the progress of the project, including positive as well as negative results, to the responsible curators and researchers. Obviously, we will publish the results together with all curators and researchers involved in the study. The materials provided and extracts made from them will be used for this study only, and no material will be released to any third party without explicit written permission.

ApplicationDate: 

Monday, October 14, 2019 - 09:10

CaseID: 

14449

OtherReferences: 

ReferenceList: 

Citation Type Date Retrieved
Fu Q, Li H, Moorjani P, Jay F, Slepchenko SM, Bondarev AA, Johnson PL, Aximu-Petri A, Prüfer K, de Filippo C et al. 2014. Genome sequence of a 45,000-year-old modern human from western Siberia. Nature 514: 445-449. Journal
Fu Q, Meyer M, Gao X, Stenzel U, Burbano HA, Kelso J, Pääbo S. 2013. DNA analysis of an early modern human from Tianyuan Cave, China. Proceedings of the National Academy of Sciences of the United States of America 110: 2223-2227. Journal
Gansauge MT, Gerber T, Glocke I, Korlevic P, Lippik L, Nagel S, Riehl LM, Schmidt A, Meyer M. 2017. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. Nucleic acids research 45: e79. Journal
Glocke I, Meyer M. 2017. Extending the spectrum of DNA sequences retrieved from ancient bones and teeth. Genome Res 27: 1230-1237. Journal
Kircher M, Sawyer S, Meyer M. 2012. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic acids research 40: e3. Journal
Meyer M, Fu Q, Aximu-Petri A, Glocke I, Nickel B, Arsuaga JL, Martinez I, Gracia A, de Castro JM, Carbonell E et al. 2014. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature 505: 403-406. Journal
Meyer M, Kircher M, Gansauge MT, Li H, Racimo F, Mallick S, Schraiber JG, Jay F, Prüfer K, de Filippo C et al. 2012. A high-coverage genome sequence from an archaic Denisovan individual. Science 338: 222-226 Journal
Prüfer K, de Filippo C, Grote S, Mafessoni F, Korlevic P, Hajdinjak M, Vernot B, Skov L, Hsieh P, Peyregne S et al. 2017. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science 358: 655-658. Journal
Prüfer K, Racimo F, Patterson N, Jay F, Sankararaman S, Sawyer S, Heinze A, Renaud G, Sudmant PH, de Filippo C et al. 2014. The complete genome sequence of a Neanderthal from the Altai Mountains. Nature 505: 43-49. Journal
Rohland N, Glocke I, Aximu-Petri A, Meyer M. 2018. Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing. Nature protocols 13: 2447-2461. Journal
Slon V, Hopfe C, Weiss CL, Mafessoni F, de la Rasilla M, Lalueza-Fox C, Rosas A, Soressi M, Knul MV, Miller R et al. 2017. Neandertal and Denisovan DNA from Pleistocene sediments. Science 356: 605-608. Journal
Slon V, Glocke, I., Barkai, R., Gopher, A., Hershkovitz, I., Meyer, M. 2016. Mammalian mitochondrial capture, a tool for rapid screening of DNA preservation in faunal and undiagnostic remains, and its application to Middle Pleistocene specimens from Qesem Cave (Israel). Quaternary International 398: 210-218. Journal
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