Main

Palaeolithic assemblages typically contain a multitude of objects that may differ in age by hundreds or thousands of years, even when found in close proximity1. Thus, it can be challenging to associate human remains with specific objects. Recent advances in the retrieval of human DNA from sediments6,7,8 can be used to connect artefacts with genetic populations. However, precise identification of the specific makers or users of these objects would require the recovery of human DNA directly from the objects themselves, analogous to modern-day forensic investigations. In theory, such analyses are most promising for artefacts made from animal bones or teeth, not only because they are porous and thereby conducive to the penetration of body fluids (for example, sweat, blood or saliva) but also because they contain hydroxyapatite, which is known to adsorb DNA and reduce its degradation by hydrolysis and nuclease activity9,10. Ancient bones and teeth may therefore function as a trap not only for DNA that is released within an organism during its lifetime and subsequent decomposition but also for exogenous DNA that enters the matrix post-mortem through microbial colonization11 or handling by humans. However, DNA extraction from ancient skeletal material either requires destructive sampling, or risks alteration of specimens if they are directly submerged in extraction buffer12,13. Conservation is a primary concern because of the scarcity of bone and tooth artefacts at Pleistocene sites, especially of pendants and other ornaments that were extensively handled or worn in close body contact. We therefore set out to develop a method for DNA isolation from bones and teeth that preserves the integrity of the material, including surface microtopography, and to investigate the possibility of DNA retrieval from bone and tooth artefacts.

A non-destructive DNA isolation method

To identify reagents compatible with non-destructive DNA extraction, we selected ten unmodified faunal remains from the Palaeolithic sites of Quinçay and Les Cottés in France (Extended Data Table 1 and Supplementary Information 1), which were similar in size and shape to material typically used for osseous artefact production, and submerged them in several reagents previously used in ancient DNA extraction, as well as in water for comparison. These included (1) a guanidinium thiocyanate-containing reagent previously suggested for non-destructive DNA extraction12, (2) an ethylenediaminetetraacetate (EDTA) solution, which is a decalcifier commonly used in ancient DNA extraction14,15,16, (3) a sodium hypochlorite (bleach) solution, which is an oxidizing reagent used to remove surface-exposed contaminant DNA11,17, and (4) a sodium phosphate buffer supplemented with detergent11, which has been recently shown to enable temperature-controlled DNA release from powdered bone samples18.

Mapping of the microtopography using quantitative 3D surface texture analysis19,20 before and after the treatments revealed substantial surface alterations on all objects exposed to either the guanidinium thiocyanate reagent or EDTA (Extended Data Fig. 1 and Supplementary Information 2). By contrast, only sporadic and smaller alterations were detected with the other reagents, including sodium phosphate buffer (Fig. 1b), possibly due to the removal of traces of sediment and other small particles, as indicated by visible changes in coloration of some of the objects (Extended Data Fig. 2). On the basis of these results, we developed a non-destructive DNA isolation method for the stepwise release of DNA from the bone or tooth matrix using serial incubations in sodium phosphate buffer at 21, 37, 60 and 90 °C, with three incubations per temperature (Fig. 1a).

Fig. 1: Overview of the non-destructive DNA extraction method.
figure 1

a, Workflow of the gradual, non-destructive DNA extraction method using sodium phosphate buffer at elevated temperatures. b, Four 3D surface texture measurements (1–4) indicated on the outline of a tooth used for testing (SP6649) before and after non-destructive DNA extraction showing no substantial surface alterations. c, Photographs of DCP1 before and after cleaning and non-destructive DNA extraction.

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We then applied this method to 11 osseous objects, labelled Q10 to Q19 as well as Q27, that were excavated several decades ago in the Châtelperronian layers of Quinçay Cave in France and that had potentially been used as tools some 35–45 thousand years ago (ka)21 (Extended Data Table 1 and Extended Data Fig. 3). We prepared single-stranded DNA libraries22,23 from the first DNA fraction recovered at each temperature, and enriched the libraries for mammalian mitochondrial (mt) DNA24. A metagenomic pipeline for assigning sequenced mtDNA fragments to mammalian taxa on the biological family level6 identified 1,628 cervid mtDNA fragments in the 60 °C and 90 °C fractions of object Q10, a reindeer bone (109 and 1,519 fragments, respectively; Fig. 2 and Supplementary Data 1). These fragments showed elevated frequencies of cytosine (C)-to-thymine (T) substitutions at their ends, consistent with deamination of cytosine residues seen in ancient DNA6,25 (Supplementary Data 1). Another object, Q15, which was made of ivory, yielded 2,004 elephantid mtDNA sequences with elevated frequencies of C-to-T substitutions in the 37 °C, 60 °C and 90 °C fractions (248, 325 and 1,431 fragments, respectively). In addition, we identified hominid and suid mtDNA fragments with no evidence for ancient DNA base damage in every DNA fraction from the 11 objects, thus resulting from contamination with human and pig DNA after excavation. Human DNA contamination was particularly severe, amounting to between 70.9% and 98.3% of the identified mtDNA fragments (between 293 and 92,949 fragments in total; 17,627 on average), thereby potentially masking traces of ancient human or other mammalian DNA.

Fig. 2: Number and taxonomic composition of mammalian and human mtDNA fragments recovered from six artefacts during stepwise, non-destructive DNA extraction.
figure 2

DNA fractions are denoted S (attached sediment), P (sediment pellet recovered during water wash), and 21, 37, 60 and 90 (three incubations in phosphate buffer at the indicated temperatures in °C). Low library preparation efficiencies indicate reduced DNA recovery due to the co-extraction of inhibitory substances. Assignments to ‘ancient’ and ‘other’ taxa were performed independently for each family based on the significance of evidence for cytosine deamination. The bottom right chart shows the number of deaminated hominin mtDNA fragments recovered in each fraction after hominin-specific mtDNA capture (only positive fractions). Bov, Bovidae; Can, Canidae; Cer, Cervidae; Ele, Elephantidae; Equ, Equidae; Fel, Felidae; Hom, Hominidae; Hya, Hyaenidae; Oth, other; Mur, Muridae; Mus, Mustelidae; Rhi, Rhinocerotidae; Spa, Spalacidae; Sui, Suidae; Urs, Ursidae.

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Studying freshly excavated artefacts

As present-day human DNA contamination seemed to be ubiquitous on surfaces of objects that were handled with bare hands during and after excavation, we collected artefacts from ongoing excavations at two Palaeolithic sites, using gloves and facemasks as soon as they became partly exposed to prevent contamination. At Bacho Kiro Cave in Bulgaria, we recovered three Upper Palaeolithic tooth pendants (henceforth ‘BKP1–BKP3’) from layers I, H/I and I/J of niche 1 (Extended Data Fig. 3). At Denisova Cave, a tooth pendant (‘DCP1’) was recovered from layer 11 of the south chamber (Fig. 1c and Extended Data Fig. 4).

Larger clumps of sediment adhering to the artefacts were manually removed, and the artefacts were subsequently cleaned by three successive water washes. DNA was then extracted from the sediment clumps, the water was used for washing and the sediment particles were collected in this process (‘sediment pellets’), as well as directly from the artefacts using the non-destructive method described above. Ancient mammalian mtDNA was detected in all fractions that were analysed, except for some of the water washes and the associated sediment pellets (Fig. 2). The trajectories of DNA released from the four artefacts were similar in that the highest yield of mammalian mtDNA was obtained at 90 °C in the phosphate-based DNA extraction (up to 734, 6,614, 456 and 77,910 mtDNA fragments for BKP1, BKP2, BKP3 and DCP1, respectively; Supplementary Data 1). However, library preparation efficiencies were low for the Bacho Kiro Cave material (less than 10% for many fractions), presumably due to the co-extraction of inhibitory substances (Fig. 2), indicating that more DNA was released than could be recovered and sequenced.

The phosphate DNA fractions obtained at 37 °C, 60 °C and 90 °C are dominated by ancient ursid mtDNA fragments for BKP2 and BKP3, and cervid mtDNA fragments for DCP1, in agreement with their morphological identification (Extended Data Table 1). For BKP1, which is morphologically indeterminate, the phosphate fractions are dominated by bovid mtDNA fragments. In contrast to the phosphate fractions, DNA recovered from the sediment adhering to the artefacts is taxonomically more heterogeneous (Fig. 2). In addition, substantially lower numbers of human mtDNA fragments (between 0 and 2,969 per fraction, 246 on average; Supplementary Data 1) were recovered from the freshly excavated artefacts than from the Quinçay material. Similarly, very few suid mtDNA fragments (15 or less) were recovered, indicating that little post-excavation contamination had occurred. Of note, significant signals of cytosine deamination were observed among the human mtDNA fragments recovered in one of the 90 °C fractions from DCP1.

To increase human DNA recovery, we enriched all libraries again using a capture probe set specifically targeting human mtDNA. For the Bacho Kiro Cave material, this enabled the detection of small traces of ancient human DNA in a sediment pellet from BKP3 (29 deaminated mtDNA fragments), but none of the other fractions. For DCP1, human mtDNA fragments with significant evidence for ancient DNA base damage were identified in the first two sediment pellets recovered from the water washes, the first 37 °C and 60 °C fractions, and all three 90 °C fractions (Fig. 2). The largest numbers of deaminated human mtDNA fragments were obtained in the second and third 90 °C fractions (971 and 1,096, respectively), indicating that extended incubation at high temperature enabled ancient human DNA release from the pendant.

Preparation of additional libraries from the second 90 °C fraction of DCP1, the fraction with the lowest estimate of present-day human contamination (0.1%, 95% CI: 0.0–2.8%), yielded 62-fold average coverage of the human mtDNA genome and a near-complete consensus sequence (Supplementary Information 5). This sequence, which falls together with mtDNA sequences assigned to haplogroup U in a phylogenetic tree (Fig. 3a), contains seven ‘diagnostic’ positions that distinguish it from the mtDNA sequences of other human individuals (Extended Data Table 2). Among the mtDNA fragments overlapping these positions, 86.6% (95% CI: 82.2–90.5%) match the state of DCP1 (Extended Data Table 3), suggesting that the mtDNA fragments recovered in this fraction originate predominantly, but not exclusively, from a single ancient human individual, presumably the user or the maker of the pendant. Support for the DCP1 consensus sequence is slightly lower in the first (77.8%, 95% CI: 40.0–97.2%) and third (82.8%, 95% CI: 78.9–86.2%) 90 °C phosphate fractions, consistent with the slightly higher estimates of present-day human contamination in these fractions (12.8%, 95% CI: 1.0–24.6% and 6.6%, 95% CI: 4.3–8.9%, respectively). By contrast, support for the DCP1 consensus is low in the preceding 60 °C fraction (37.5%, 95% CI: 8.5–75.5%), indeterminate in the 37 °C fraction and low in the first (20.0%, 95% CI: 5.7–43.7%) and second (9.5%, 95% CI: 1.2–30.4%) sediment pellets. These results indicate that the initial water washes and incubations in phosphate buffer at below 90 °C primarily released ancient human DNA from one or more other individuals, which were present in smaller quantities in the surrounding sediment or had adsorbed directly to the surface of DCP1.

Fig. 3: Ancient human mtDNA and nuclear DNA isolated from DCP1.
figure 3

a, The position of DCP1 in a Bayesian tree reconstructed from modern31,32 and ancient33 human mtDNA sequences (see Supplementary Information 5 for the full tree). Nodes are labelled with the corresponding posterior probabilities, and the x axis represents years from the present. Identified haplogroups are outlined by the bars on the right. rCRS, revised Cambridge reference sequence. b, X–autosome proportion in DCP1 (using all and deaminated molecules only) in comparison to data from six other ancient hominin individuals34,35. Circles correspond to the calculated values of the ratios for the number of X to (X + autosomal) fragments for each individual (n (of single-nucleotide polymorphisms (SNPs)) = 20,526, 3,734, 124,862, 85,901, 34,756, 41,632, 34,677 and 72,992 SNPs for each calculation, as ordered on the x axis). The error bars represent 95% binomial CIs of the measurement in each individual. c, Principal component (PC) analysis of non-African modern human genomes36 (grey) with ancient human genomes (coloured) projected on top. DCP1 was analysed twice, using all data versus deaminated fragments only. AG3, Afontova Gora 3; ANA, ancient Native Americans; MA1, Mal’ta 1; Russia_HG, Russian hunter–gatherers; UKY001, Ust Kyakhta; WSHG, West Siberian hunter–gatherers.

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Phylogenetic analyses and dating

On the basis of the branch length of the DCP1 consensus sequence in a tree with other present-day and ancient human mtDNA genomes (Fig. 3a), we estimated its age to 18.5 thousand years (kyr), with a 95% highest posterior density interval ranging from 4.6 to 31.6 kyr (Supplementary Information 5). Furthermore, we used cervid mtDNA probes to reconstruct the complete mtDNA genome of the tooth, identified as wapiti (Cervus canadensis), at 635-fold coverage. A tree with eight additional ancient wapiti mtDNA genomes of known age generated in this study (Supplementary Information 6), estimates the age of DCP1 at 24.7 kyr (highest posterior density interval of 12.8–39.0 kyr). Both genetic ages are consistent with each other and with the younger of two radiocarbon dates that we obtained from charcoal discovered in the proximity of DCP1 in layer 11 (OxA-X-3089-11: 24,200–23,830 calibrated years before present (cal bp) and OxA-X-3089-12: 39,180–37,560 cal bp) at 95.4% probability (Supplementary Information 1). We therefore suggest that genetic dating obviates the need for direct radiocarbon dating of the pendant, although this remains technically possible after non-destructive DNA extraction (Supplementary Information 3).

For nuclear DNA analysis, hybridization capture was performed using libraries from the second and third 90 °C phosphate fractions, targeting sites in the human genome that are known to be polymorphic in modern or archaic humans and that are located in regions of high sequence divergence between humans and other mammals8. Sequence information was obtained for 336,429 of these sites (71.5% of the sites targeted), with estimates of present-day human and faunal contamination both below 1%. Comparisons with present-day human populations26 using ƒ3-statistics and D-statistics27,28 show high affinities to Native Americans (Extended Data Fig. 5). When projected into a principal component analysis with other ancient human individuals (Fig. 3c), DCP1 falls within a group of Ancient North Eurasian individuals from further east in Siberia, which includes the approximately 24 ka Mal’ta 1 and the approximately 17 ka Afontova Gora 3 individuals29,30. Both of these individuals are genetically closer to DCP1 than non-Ancient North Eurasian individuals when tested with D-statistics (Extended Data Fig. 6b), and all three show similar affinities to ancient Siberians and Native Americans with ƒ3-statistics and D-statistics (Extended Data Fig. 6a,c). In addition, shotgun data were produced from one of the libraries to allow a comparison of sequence coverage for the X chromosome and the autosomes, which is compatible with the human DNA in the 90 °C fraction originating predominantly from a female individual (Fig. 3b and Supplementary Information 7).

Conclusions

In summary, our work highlights that artefacts made from bones or teeth are a previously untapped source of ancient human DNA that can provide insights about the ancestry and biological sex of the individuals who handled, carried or wore these objects in the deep past. The non-destructive DNA extraction method reported here allows a stepwise release of this DNA, making it possible to distinguish DNA that penetrated deeply into an object during its manufacture or use from DNA that may originate from the surrounding sediment. Of note, the coverage depth of targeted sites in the human nuclear genome achieved from DCP1 is similar to what has been obtained with hybridization capture from well-preserved Pleistocene human remains30. Furthermore, the recovery of both human and faunal DNA enabled two independent genetic estimates of its age.

Further work is needed to determine how often human DNA can be recovered from Palaeolithic osseous artefacts. As surface DNA contamination can hamper these analyses, we urge archaeologists to apply protocols for minimizing handling during and after excavation. If this is done, it might become possible to systematically combine genetic and cultural analyses to study Pleistocene artefact use and uncover possible task specialization by individuals of a particular biological sex or genetic ancestry.

Methods

Sample collection

For reagent testing, we collected ten unmodified faunal remains, similar in and size and shape to material used in artefact production, from the late Middle Palaeolithic and early Upper Palaeolithic deposits of the French sites Quinçay (seven) and Les Cottés (three) (Extended Data Table 1 and Extended Data Fig. 2). The estimated ages of these specimens range from 55 to 35 kyr21,37,38. Non-destructive DNA extraction was then applied to 15 osseous specimens excavated at Quinçay Cave, all from layers attributed to the Châtelperronian technocomplex and probably dating to 45–35 ka21,37, to three tooth pendants excavated in the Initial Upper Palaeolithic (45–43 ka)39 layers in the niche 1 area of Bacho Kiro Cave40,41, as well as on one tooth pendant excavated in 2019 in layer 11 (39–24 ka), square E-3, of the south chamber of Denisova Cave (Extended Data Table 1 and Extended Data Fig. 3). Samples from Bacho Kiro and Denisova Cave were excavated and handled using sterile gloves and additional precautions were taken to minimize the introduction of modern DNA contamination. More information about the samples and the archaeological context in which they were recovered is provided in Supplementary Information 1.

Testing reagents for non-destructive DNA extraction using 3D surface texture measurements

We evaluated four reagents for their potential use in non-destructive DNA extraction by applying each to two Pleistocene samples comparable with the ones usually transformed into bone or tooth tools and ornaments, preferably choosing one bone and one tooth fragment for each reagent (Extended Data Table 1). As none of the objects was perfectly clean and sediment microparticles may be released upon exposure to liquids, we also performed treatments with water to obtain baseline measurements of changes in the microtopography that are independent of the chemical compositions of the reagents. Reagents and incubation conditions were as follows:

  1. (1)

    Following a protocol42 further detailing the non-destructive DNA extraction method for museum specimens by Rohland et al. (2004)12, samples were completely submerged in 10–15 ml guanidinium thiocyanate buffer (5 M guanidinium isothiocyanate, 50 mM Tris-HCl, pH 8.0, 25 mM NaCl, 1.3% Triton X-100, 20 mM EDTA, pH 8.0, and 50 mM DTT) and incubated for 5 days in the dark.

  2. (2)

    Incubation in EDTA, supplemented with a detergent (0.45 M EDTA, pH 8.0, and 0.05% Tween-20), was performed for 15 min at room temperature. EDTA is widely used in bone extraction protocols as a decalcifying agent to dissolve hydroxyapatite, the main mineral component of bones14,15,16. It is therefore expected to cause severe alterations to the sample material and was included to demonstrate the effect of such alterations on the quantitative 3D-surface texture analysis (3DST) measurements.

  3. (3)

    Two samples were submerged for 15 min in 0.5% sodium hypochlorite (bleach) solution, a reagent previously used for decontamination of ancient skeletal remains11,17. Bleach treatment destroys surface-bound DNA and is not suitable for DNA extraction. However, it may be used in later implementations of the method to remove contaminant DNA before non-destructive DNA extraction.

  4. (4)

    Temperature-controlled release of DNA was performed following a modified version of the method by Essel et al. (2021)18 by submerging the samples in sodium phosphate buffer (0.5 M sodium phosphate, pH 7.0, and 0.1% Tween-20). Serial incubations were performed at 21 (room temperature), 37, 60 and 90 °C, each with incubation times of 30 min for a total of three incubations per temperature.

All treatments were performed in 50-ml Falcon tubes without agitation to avoid any mechanical damage to the sample. Reagent volumes were chosen individually for each sample (ranging from 5 ml to 17.5 ml) to ensure complete submergence. Incubations above room temperature were performed in a Heating-ThermoMixer MHR 11 (Hettich Benelux) equipped with inserts for 50-ml Falcon tubes. For the 90 °C incubation in phosphate buffer, the temperature of the device was set to 99 °C to ensure that 90 °C was reached inside the tube by the end of the incubation time. After the treatments, all samples were placed in fresh 50-ml Falcon tubes and incubated in water for 1 h at room temperature to remove residual reagents. Samples were dried at room temperature for 5 days before they were returned to their storage containers.

Changes in the microtopography of the bone or tooth objects were tracked using quantitative 3DST19 before and after extraction following established protocols19,43. Meshed axiomatic 3D models were generated and the following ISO 25178 parameters were used for statistical testing (paired Student’s t-test, before and after the treatment, α ≤ 0.01): mean roughness (Sa), void volume (Vvv), peak curvature (Spc) and peak density (Spd). Further details regarding the 3DST measurements are provided in Supplementary Information 2.

In addition, we explored the compatibility of phosphate-based non-destructive DNA extraction with subsequent 14C dating (Supplementary Information 3).

Non-destructive DNA isolation from artefacts

As sodium phosphate buffer did not cause substantial alterations of ancient bones and teeth in 3DST measurements, we modified a previously described method for temperature-controlled gradual DNA release from bone and tooth powder using this reagent18 for non-destructive DNA extraction from complete bones and teeth. Stepwise extraction of DNA makes it possible to closely monitor the release of different DNA components during the extraction process (endogenous DNA, environmental DNA from the surrounding sediment, ancient human DNA and present-day contamination), potentially allowing inferences to be drawn about whether these components originate from traces of sediment that may still be adherent to the object, from its surface or its interior. We then applied this protocol to a total of 15 specimens from Quinçay, Bacho Kiro Cave and Denisova Cave. Temperature-controlled non-destructive DNA extraction was performed in an ancient DNA clean-room at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, according to the four steps below (see Fig. 1a for a schematic overview):

(1) Removal of sediment

This step was only performed for the freshly excavated material. First, clumps of sediment attached to the specimen were carefully removed by hand using a flexible disposable plastic microspatula. The specimen was then put into a 50-ml Falcon tube, rinsed by pouring between 20 ml and 50 ml of water into the tube, and transferred to a fresh Falcon tube. This procedure was repeated two to three times until no more sediment was released into the water. The tubes with water containing the sediment that had been washed off were then centrifuged at 16,400g for 5 min to pellet the sediment, and the clear supernatants were transferred to fresh tubes. All three types of material collected in this procedure (the manually removed sediment, the sediment pellet collected by centrifugation and the clear water) were subsequently subjected to DNA purification (see below).

(2) Temperature-controlled DNA release using sodium phosphate buffer

Each cleaned specimen was put into a 50-ml Falcon tube to which sodium phosphate buffer (0.5 M sodium phosphate, pH 7.0, and 0.1% Tween-20) was added until the specimen was completely submerged in the reagent (between 5 ml and 50 ml). After 30 min of incubation at room temperature (without agitation), the buffer was transferred to a fresh 50-ml Falcon tube. This step was repeated twice at room temperature (21 °C, for a total of three incubations) and then three times each at 37 °C, 60 °C and 90 °C (see above for details on the device and temperature settings used). A final incubation in water was performed at room temperature to remove residual reagent and the specimens were dried at room temperature for 5 days.

(3) DNA concentration

To facilitate subsequent DNA purification, half of the volumes of the water and phosphate buffer DNA fractions generated in steps (1) and (2) were reduced to between 50 µl and 75 µl by concentrating the DNA using Amicon Ultra-4 Centrifugal Filter Units with Ultracel-3 membranes (Millipore). For this, up to 4 ml or 15 ml of the respective sample was added to a filter unit, which was spun for 90 min at 4,000g in a centrifuge with an active cooling unit set to 21 °C. In cases in which the sample volume exceeded 4 ml or 15 ml, the flow-through was discarded and the filter unit was reloaded with remaining sample. Finally, a buffer exchange was performed by adding 4 ml or 15 ml TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) to the concentrated sample on top of the filter unit and spinning for 30 min at 4,000g. The supernatant was filled up to 300 µl with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and then transferred into a fresh 1.5-ml Eppendorf low-bind tube and stored at −20 °C until further processing.

(4) DNA purification

DNA was isolated from the concentrated DNA fractions prepared in step (3) using a column-based method for silica-based ancient DNA extraction detailed elsewhere44. For this, 300 µl concentrated DNA was used as input for DNA purification using binding buffer ‘D’ and the purified DNA was recovered in 50 µl elution buffer. DNA extraction from the sediment samples removed from the artefacts manually or through water washes (sediment pellets) was performed using the same method but different input volumes. In brief, lysates were prepared by transferring up to 130 mg sediment to a 2-ml low-bind Eppendorf tube, adding up to 2 ml lysis buffer (0.45 M EDTA, pH 8.0, 0.05% Tween-20 and 0.25 mg ml−1 proteinase K) (1 ml for samples of less than 100 mg or 0.5 ml for samples of less than 25 mg) and incubating overnight at 37 °C under rotation. DNA purification was performed using 500 µl or 1,000 µl lysate.

Negative controls containing sodium phosphate buffer or lysis buffer without sample material were carried alongside the samples through all steps of subsequent sample preparation and sequencing. For a subset of samples, DNA was extracted from only the first phosphate buffer fraction produced at each of the four incubation temperatures. Supplementary Data 1 provides an overview of the DNA extracts generated in this study.

Library preparation

Of the extract, 10 µl was then converted into single-stranded DNA libraries using the automated protocol described in Gansauge et al. (2020)23. The number of unique library molecules obtained and the efficiency of library preparation were determined using two quantitative PCR assays45. Libraries were then amplified and double-indexed by PCR46. One library was prepared from each DNA fraction, except for the second and third 90 °C phosphate fractions from the DCP1 from which five libraries were prepared, each to maximize the yield of sequence data. Negative controls containing no sample material were carried along each set of extraction and library preparation. Note that there was substantial variation in library preparation efficiency across samples (Fig. 2 and Supplementary Data 1), ranging from 21.8% on average for the phosphate DNA fractions obtained from Bacho Kiro Cave specimens to 60.8% for DCP1 and 62.0% for the Quinçay specimens.

Enrichment of mitochondrial and nuclear DNA by hybridization capture

To determine the taxonomic composition of the DNA recovered from the artefacts, libraries prepared from nearly all extracts generated in this study (all samples and controls, with the exception of the second and third phosphate fractions from BKP3) were enriched for mammalian mtDNA by hybridization capture with a probe set (‘AA75’) encompassing the mtDNA genomes of 242 mammalian species24. In cases in which several libraries were prepared from the same DNA extracts (that is, the 90 °C phosphate fractions from DCP1), only the first library was enriched for mammalian mtDNA. In addition, all libraries were enriched specifically for hominin mtDNA using a probe set (‘AA163’) designed in 1-bp tiling based on the revised Cambridge reference sequence of the human mitochondrial genome32. Both types of mtDNA captures were performed using two consecutive rounds of automated capture as detailed in Slon et al. (2017)6 or Zavala et al. (2022)47. An overview of the capture reactions performed is provided in Supplementary Data 1.

In addition to mtDNA captures, 11 libraries prepared from the first, second and third 90 °C phosphate fractions of DCP1 were enriched for human nuclear DNA by two consecutive rounds of in-solution capture48. This enrichment was performed using a subset of a previously designed capture probe panel8, named AA204, targeting two groups of SNPs: (1) 59,232 SNPs, randomly selected from set 6 ‘hominin diagnostic sites’8, which represent positions in the genome where primates differ from other mammals and are used to quantify faunal mis-alignments; and (2) 411,492 SNPs selected from the ‘1240k’ panel30,49 (set 2 in Vernot et al. (2021)8). This set of SNPs is informative for investigating modern human population histories. Both SNP groups are located in regions of large evolutionary sequence divergence between humans and other mammals8.

Sequencing and raw sequence processing

Libraries enriched for mtDNA were combined into pools and sequenced on multiple lanes of a MiSeq sequencer (Illumina Technologies) in paired-end configuration with two index reads (2× 76 + 2× 8 cycles). Libraries enriched for human nuclear DNA were sequenced either on a HiSeq 2500 using the same configuration or on a HiSeq4000 (both Illumina Technologies) in single-read configuration with two index reads (1× 76 + 2× 8 cycles). In addition, one library prepared from the second 90 °C phosphate fraction of the DCP1 was sequenced directly (shotgun sequencing, without hybridization capture) on one lane of a HiSeq 4000 sequencer (Illumina Technologies) in single-read configuration. Base calling was performed using Illumina’s Bustard tool and sequences were assigned to the library that they originated from, requiring perfect matches to the expected index combinations. LeeHom (https://github.com/mpieva/leeHom/tree/v.1.1.5)50 was used to trim adapters and, for paired-end data, to merge overlapping paired-end reads.

Taxonomic assignment of mtDNA sequences

Sequences resulting from mammalian or human mtDNA capture were assigned to mammalian taxa on the biology family level using a previously published computational pipeline6 based on BLAST and MEGAN (version 0.0.12)51, with modifications in data filtering detailed in Vernot et al. (2021)8 (Supplementary Data 1 and Supplementary Information 4). In brief, as described in the latter study, false identifications of taxa were minimized by requiring at least three unique sequences (covering at least 105 positions in the reference genome) to be assigned to a family, and by requiring these sequences to represent at least 1% of all taxonomically identified sequences. The presence of ancient DNA was determined individually for the sequences from each family (and each library) by computing the frequency of terminal C-to-T substitutions as a proxy for the deamination rate at the molecule ends. Substitution frequencies significantly higher than 10% on both molecule ends (based on 95% binomial CIs) were then taken as evidence for the presence of ancient DNA from the respective family.

Human and cervid mtDNA analysis

Present-day human contamination in the fractions from DCP1 that yielded ancient human mtDNA was estimated using the software tool AuthentiCT (version 1.0.0)52 and a near-complete consensus sequence called for the second 90 °C fraction from DCP1 using positions with at least tenfold coverage of the mtDNA genome. This consensus sequence was then used for haplogroup assignment with Haplogrep 2 (version2.4.0)53 and to identify ‘diagnostic’ positions in the mtDNA genome, which were used to determine the support for the consensus sequence in each of the DNA fractions recovered from DCP1 (Extended Data Table 3). Tree building and genetic dating were performed using BEAST2 (version 2.6.6)54. Further information on the mtDNA analyses are provided in Supplementary Information 5. For genetic dating of the deer DNA component, near-complete wapiti mtDNA genomes were reconstructed from the first 60 °C phosphate fraction obtained from DCP1, as well as eight ancient wapiti samples as described in detail in Supplementary Information 6.

Human nuclear DNA analysis

Human DNA capture data were processed as previously described8 and data from the libraries with the lowest estimates of modern human contamination (based on AuthentiCT) were merged for further analyses. Principal component analysis including sequence data from present-day and other ancient human individuals was performed using smartpca (from EIGENSOFT package version 8.0.0)55. Using the R package admixr (version 0.7.1)28, ƒ3-statistics were calculated to determine shared genetic drift between DCP1 and a selection of modern and ancient human populations and their relationships were further evaluated using D-statistics. Sex determination for the human DNA component recovered from DCP1 was performed by comparing the coverage of the X chromosome and the autosomes in shotgun data obtained from the second 90 °C phosphate fraction after filtering against faunal mis-mappings8. Further details are provided in Supplementary Information 7.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.