A Complete Mtdna Genome of an Early Modern Human from Kostenki, Russia

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Johannes Krause,1,* Adrian W. Briggs,1 Martin Kircher,1 extracted from, e.g., Neandertal remains, are of a size less Tomislav Maricic,1 Nicolas Zwyns,1 Anatoli Derevianko,2 than 50 nt [7] and therefore not retrievable by PCR. By and Svante Paä¨bo1 contrast, modern DNA that contaminates a specimen may 1Max Planck Institute for Evolutionary Anthropology, often be of larger average size [7, 14–16]. Deutscher Platz 6, D-04103 Leipzig, Germany In order to test whether the extent of contamination differs 2Paleolithic Department, Institute of Archaeology & systematically between the longer molecules analyzed by Ethnography, Russian Academy of Sciences, Siberian Branch, PCR and the bulk of the molecules analyzed by direct high- Lavrentieva Prospekt, 17 Novosibirsk, 630090 Russia throughput sequencing, which also include shorter molecules, we analyzed 21 Neandertal DNA extracts by PCR-based contamination assays [7, 17] with up to three primer pairs Summary used to amplify regions of the mtDNA that differ between Neandertals and present-day humans. Between 30 and 2,118 The recovery of DNA sequences from early modern humans amplified fragments per extract were sequenced on the (EMHs) could shed light on their interactions with archaic 454 platform, and the ratios of Neandertal-like and modern- groups such as Neandertals and their relationships to cur- human-like mtDNA sequences were determined (Figure 1A). rent human populations. However, such experiments are Four extracts yielded less than 5% contaminating modern highly problematic because present-day human DNA fre- human sequences whereas the remaining 17 extracts con- quently contaminates bones [1, 2]. For example, in a recent tained between 9% and 100% contaminating modern human study of mitochondrial (mt) DNA from Neolithic European sequences. We then analyzed the same 21 Neandertal extracts skeletons, sequence variants were only taken as authentic with a recently published method for ancient DNA sequence if they were absent or rare in the present population, capture (PEC) [18]. This method uses 50-biotinylated oligonu- whereas others had to be discounted as possible contamina- cleotide primers that hybridize to sequences of interest. The tion [3, 4]. This limits analysis to EMH individuals carrying primers are then extended by a DNA polymerase to capture rare sequences and thus yields a biased view of the ancient ancient target sequences, allowing fragments of size w20 nt gene pool. Other approaches of identifying contaminating and above to be retrieved. We used up to six PEC primers DNA, such as genotyping all individuals who have come designed to anneal to conserved mtDNA regions adjacent into contact with a sample, restrict analyses to specimens to diagnostic differences between human and Neandertal where this is possible [5, 6] and do not exclude all possible mtDNA, followed by direct sequencing on the 454 platform. sources of contamination. By studying mtDNA in Neandertal This generated between 30 and 465 independent informative remains, where contamination and endogenous DNA can be fragments per extract (Figure 1B). In 14 of the 21 extracts, distinguished by sequence, we show that fragmentation exclusively Neandertal and no contaminating molecules were patterns and nucleotide misincorporations can be used to detected, and the remaining seven extracts had contamination gauge authenticity of ancient DNA sequences. We use these levels lower than or equal to 1%. A major reason for this features to determine a complete mtDNA sequence from discrepancy in the apparent extent of contamination is evident a w30,000-year-old EMH from the Kostenki 14 site in Russia. from the size distributions of ancient DNA fragments retrieved by PEC (see Figure S2, available online), which shows that only Results and Discussion 27% of the fragments are larger than the smallest size amplified by PCR (67 nt). Thus, the direct sequencing taps into DNA extracted from fossil remains is invariably degraded to a majority of short molecules that cannot be analyzed by a low average fragment size [1, 7–10]. Until recently, the only PCR. In this population, endogenous Neandertal molecules way to gauge the length of DNA molecules from the organism dominate quantitatively over contaminating molecules to such of interest has been to compare the success rates of poly- an extent that in many cases contaminating molecules are not merase chain reactions (PCRs) performed with primer pairs even seen among the molecules sequenced. that target templates of different lengths [11]. However, In addition to a reduction in size, cytosine deamination is the because PCR requires two primers of length w20 nt and most common modification readily detectable in ancient DNA usually targets a segment of > 10 nt between the primers, the [19]. With sequencing on high-throughput platforms, it has abundance of template fragments below a length of w50 bp been shown to occur particularly toward the ends of the mole- 0 remain unknown. This situation has changed with the applica- cules and to result in C-to-T substitutions close to 5 -ends and 0 tion of single primer extension-based approaches [12] and in the complementary G-to-A substitutions at 3 -ends as high-throughput DNA sequencing to ancient DNA, e.g., the a result of end polishing before sequencing adaptor ligation lllumina Genome Analyzer II (GA2) [13] and Roche 454 plat- [12, 20]. In order to directly study this feature as well as frag- forms [9]. In particular, the adaptor-ligation and direct- ment size in large numbers of not only endogenous but also sequencing approach of the two latter technologies allow contaminating modern human DNA, we captured informative DNA fragments of short lengths to be completely sequenced. mtDNA molecules [7, 14] by PEC from libraries prepared This has shown that more than half of DNA fragments from Neandertal individuals that had earlier been found to be highly contaminated and had therefore been excluded from further study. They come from individual 2 [21] from Feldhofer, *Correspondence: [email protected] Germany; individual 2 [14] from Mezmaiskaya, Russia; and Current Biology Vol 20 No 3 232

PCR contamination estimate Figure 1. PCR and PEC Contamination Estimate in A 100% Neandertal Extracts Estimates of the level of modern human DNA contamination (red) compared to endogenous Neandertal DNA 80% (yellow) in 21 Neandertal extracts (see also Table S1). (A) For each extract the proportion of human to Nean- dertal clones for the PCR contamination assay is shown. 60% (B) For each extract, the numbers of human and Nean- dertal DNA fragments retrieved with the PEC are shown (see also Figure S2). 40%

20% It has been shown that fragmentation of ancient DNA occurs predominantly at purine bases [20]. In order to study whether this 0% feature differs between endogenous and con- 1 3 5 7 9 111315171921 PEC contamination estimate taminating DNA, we analyzed the base compo- B Contamination Neandertal sition close to 50- and 30-ends of mtDNA 500 sequences from the Feldhofer, Mezmaiskaya, 450 and Teshik Tash libraries by aligning each frag- 400 ment to a reference mtDNA and gauging the base composition at the ﬁrst 10 bp outside 350 the terminal base of the fragment. As previ- 300 ously described, for the endogenous Nean- 250 dertal fragments an elevation of G and A in frequency was found at the 50-end, whereas 200 C and T were found elevated at the 30-end 150 (Figure S3), consistent with a preferential 100 fragmentation of ancient DNA at purine bases [20]. In contrast, the human contaminants in independent informative fragments 50 the Feldhofer and Mezmaiskaya libraries as 0 well as the fragments in the highly contami- 1 3 5 7 9 111315171921 nated Teshik Tash library show no increase Neandertal extract in frequency of purines or pyrimidines on either side of the fragment (Figure S3). In conclu- sion, fragment length, deamination-induced from Teshik Tash, Uzbekistan [22]. For the two former libraries, sequence errors at ends of molecules, and purine-associated 1,492 and 534 contaminating molecules were found among fragmentation represent features by which endogenous and 1,865 and 1,063 informative molecules sequenced, respec- contaminating populations of DNA molecules can be distin- tively, and for the latter library 220 informative molecules guished in at least some late Pleistocene specimens. were retrieved of which only ﬁve were of Neandertal origin Given that sequencing of shorter molecules appears to (Table S2). reduce the problem of contamination and that these features In the two former cases, the mode of the size distributions of may help distinguish endogenous from contaminating DNA, contaminating molecules seems to be higher than 81 nt, which we attempted to sequence the mitochondrial genome of an is the maximum length of molecules sequenced. In contrast, EMH. To do this, we extracted DNA from the Markina Gora the mode of the endogenous DNA fragments is w60 and skeleton found in the Kostenki 14 site in Russia, which was w45 nt, respectively (Figure 2). In Teshik Tash, where practi- excavated in 1954 [24]. Based on stratigraphic evidence, it is cally all mtDNA molecules captured represent contamination, older than 30,000 years but younger than 33,000 years no direct comparison to the endogenous DNA can be made, (Supplemental Information). All attempts to produce direct but it should be noted that the mode of the size distribution radiocarbon dates failed or gave unreliable results, probably of the contaminating molecules is similar to the endogenous because of previous treatment of the bones with organic DNA found in the Feldhofer specimen (w60 bp, Figure 2), sug- compounds. We used a total of 574 PEC primers in four multi- gesting that fragment size alone cannot be used to gauge the plex mixes to retrieve the complete mtDNA from a sequencing authenticity of ancient DNA [23]. In the endogenous DNA mole- library produced from the DNA extract. The primers were cules from the Feldhofer and Mezmaiskaya specimens, cyto- chosen to match Neandertal mtDNA sequences so that the sine residues at the 50-ends of DNA fragments appear as capture would be equally biased for all modern human mtDNA, thymine residues in w28% and w35% of cases, respectively, given that Neandertal mtDNAs fall outside the variation of and similar levels of G-to-A substitutions are seen at the modern human mtDNAs [7]. From the retrieved molecules 30-ends. By contrast, in contaminating DNA molecules cyto- a total of 4,066,269 sequences were produced on the Illumina sine residues at the 50-ends appear as thymine residues in GA2 platform and processed with a custom mapping assem- w4% and w2% of cases, respectively (Figure 2). In the Teshik bler. Approximately 30% of the raw sequences represented Tash specimen, where contamination greatly dominates, mtDNA fragments. Sequences with the same start and end C-to-T misincorporations are seen at less than 3% of 50-ends coordinates were collapsed because they may stem from (Figure 2). the same ancient molecules, yielding 10,664 distinct mtDNA A Complete mtDNA Genome of an Early Modern Human 233

Neandertal DNA contamination length distribution fragments: 529 fragments: 534 0.40 0.40 100 0.35 0.35 90 80 0.30 0.30 70 0.25 0.25 T 60 0.20 G 0.20 50 0.15 C 0.15 40 fragments base frequency A 30 0.10 0.10 20 0.05 0.05 10 0.00 0.00 0 0 2 4 6 8 10 12 14 -14 -12 -10 -2-4-6-8 0 02468 10 12 14 -14 -12 -10 -8 -6 -2-4 0 20 25 30 35 40 45 50 55 60 65 70 75 80 5’ position along DNA fragment 3’ 5’ position along DNA fragment 3’ length bin Neandertal DNA contamination fragments: 403 fragments: 1,492 length distribution 0.40 0.40 250 0.35 0.35 200 0.30 0.30 0.25 0.25 T 150 0.20 G 0.20

C fragments 0.15 0.15 100

base frequency A 0.10 0.10

Feldhofer 2Feldhofer 2 Mezmaiskaya 50 0.05 0.05 0.00 0.00 02468 10 12 14 -14 -12 -10 -2-4-6-8 0 0 2 4 6 8 10 12 14 -14 -12 -10 -8 -2-4-6 0 0 5’ position along DNA fragment 3’ 5’ position along DNA fragment 3’ 20 25 30 35 40 45 50 55 60 65 70 75 80 length bin contamination fragments: 3,274 length distribution 0.40 0.40 180 0.35 0.35 160 140 0.30 0.30 120 0.25 0.25 100 0.20 0.20 80 0.15 0.15 fragments base frequency 60 0.10 0.10 40 Teshik Tash Tash Teshik 0.05 0.05 20 0.00 0.00 0 02468 10 12 14-14 -12 -10 -4-6-8 -2 0 20 25 30 35 40 45 50 55 60 65 70 75 80 85 5’ position along DNA fragment 3’ length bin Neandertal DNA contaminating DNA

Figure 2. Misincorporation Patterns and Length Distribution of Neandertal DNA and Contaminating DNA Nucleotide misincorporation patterns and length distributions for Neandertal and contaminating mtDNA from Feldhofer 2, Mezmaiskaya 2, and Teshik Tash specimens. The maximum sequence lengths determined were 83 bp for Mezmaiskaya and Feldhofer and 87 bp for Teshik Tash (see also Figure S3). sequences. The average length of these sequences is 54 bp likely to differ from the consensus sequence [25]. Among the (Figure 3A). They were used to construct a consensus mtDNA 77 distinct DNA fragments that overlap these positions, only sequence where the average coverage is 31.2 and over 99% of one shows a base different from the consensus sequence positions have 4-fold and more coverage (Figure 3E). Cytosine (Figure 4), indicating that the vast majority 98.7% (95% C.I. residues in the consensus sequence appear as thymine resi- 93.1%–99.9%) of DNA comes from one single individual. dues at 50-ends of sequences, and guanidine residues appear Hence, if the consensus sequence would derive from as adenine residues at 30-ends in w38% of cases (Figure 3B). a contaminating DNA source, the Kostenki 14 specimen would Furthermore, purines are substantially elevated at the base be contaminated by DNA from a single individual that carries 50 to the mtDNA sequences and pyrimidines are elevated at an mtDNA sequence that is unusual in the current human the base 30 to sequences (Figure 3C), indicating preferential gene pool. Furthermore, the DNA from that individual would breakage at purines [20]. Thus, the mtDNA sequences deter- be degraded and chemically modified to an extent and in mined from the Kostenki 14 individual are similar to Neandertal a pattern typical of endogenous mtDNA sequences in Nean- molecules of a similar or slightly older age with respect to dertals rather than contaminating DNA in such remains. length, nucleotide misincorporations, and fragmentation Although this is possible, we suggest that the more plausible patterns. alternative is that the consensus sequence is endogenous to Next, we investigated whether mtDNA sequences from one the Kostenki specimen. or more individuals are present in the Kostenki 14 DNA library. We next analyzed how the Kostenki 14 mtDNA sequence In order to maximize the power to detect the presence of relates to Neandertal and current human mtDNAs. It is clearly multiple mtDNA sequences, we identified three positions distinct from Neandertal mtDNAs and carries five diagnostic where the Kostenki 14 consensus sequence differs from substitutions that define mtDNA haplogroup U2 (Figure S4) a world-wide sample of 311 complete mtDNAs [7] (542C > T, (rCRS positions 11,467, 12,308, 12,372, 1,811, and 16,051, 711T > C, and 15,262T > C) as well as one position (13,269A based on Phylotree.org-mtDNA, build 5). In a phylogenetic > G) that differs from more than 99% of these mtDNAs. At tree relating all 63 mtDNAs from the 311 present-day mtDNA these positions, mtDNA from another individual is particularly dataset in haplogroup U as well as the Kostenki mtDNA, the ssrnl orltdwt Ccneta a hw before shown was also (see as shown content is U GC haplogroup with into correlated fall that strongly mtDNAs Kostenki is 63 the and from sequence PEC consensus by 14 captured Kostenki fragments the S5 mtDNA of of tree frequency parsimony base Maximum nucleotide (D) (C) library. and 14 patterns, misincorporation nucleotide mtDNA (B) distribution, 14 Length Kostenki (A) of Features Sequence 3. Figure 234 Biology Current .()Rdnatcvrg fKsek 4(e,atrcutrn nqemlcls n Ccnet(le ln h opeemDAaeson Coverage shown. are mtDNA complete the along (blue) content GC and molecules) unique clustering after (red, 14 Kostenki of coverage Redundant (E) ). Kostenki 14 fragments: 10,664 D AB fragment length distribution Misincorporation patterns 0.40

1400 0.35 3 No 20 Vol 1200 0.30

1000 0.25 base frequency

800 0.20

fragments 600 0.15 400 0.10

Kostenki 200 0.05

U-root 0 0.00 20 25 30 35 40 45 50 55 60 65 70 75 80 02468 10 12 14-14 -12 -10 -4-6-8 -2 0 length bin 5’ position along DNA fragment 3’ C

7 14] [7, fragmentation pattern 5’ fragmentation pattern 3’ 0.45 0.45

0.40 0.40 base frequency sealso (see 0.35 0.35 0.30 T 0.30 G iueS6 Figure 0.25 C 0.25 0.20 A 0.20 base frequency 0.15 0.15 ). 0.10 0.10 -10 -5 0 5 10 -10 -5 0 5 10 position along DNA fragment from 5’ position along DNA fragment from 3’ E coverage + GC content Kostenki mtDNA 0.80

0.70

0.60 100 coverage 0.50

0.40

GC content 10 0.30

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0.10 1 0 2000 4000 6000 8000 10000 12000 14000 16000 iue 4and S4 Figures position along mtDNA GC content coverage A Complete mtDNA Genome of an Early Modern Human 235

rCRS - 711 rCRS - 13,269 Europe that fall into haplogroup U [27, 28], although in our opinion it is hard to exclude contamination in such PCR-based rCRS T rCRS A rCRS T studies [1]. The fact that haplogroup U2 is present in the Euro- Consensus C Consensus G Consensus C pean gene pool today argues for at least some level of genetic Run2:1:43:147:1123 G Run2:1:42:1191:1304 C Run1:1:10:146:1062 C continuity between early modern humans and present-day Run2:1:44:1155:1085 G Run1:1:2:538:151 C Run1:1:3:807:1961 C Europeans. However, there is an obvious need to study more Run2:1:40:1646:897 G Run2:1:44:1048:1264 C Run1:1:10:255:842 C early modern humans as well as other parts of the genome in Run1:1:3:1527:1893 C Run2:1:39:12:1034 G Run2:1:43:769:1455 C order to arrive at a comprehensive view of the population Run1:1:5:1125:254 C Run2:1:41:1213:1287 G Run1:1:19:221:1517 C history of modern humans in Europe. Run2:1:41:1211:558 C Run1:1:6:93:1070 G Run2:1:41:1285:1149 C We note that we have earlier claimed that modern human Run1:1:6:840:1932 C Run1:1:14:839:492 G Run2:1:42:1255:1065 C Run1:1:22:1048:931 C Run2:1:40:1160:556 G Run2:1:44:738:411 C DNA sequences cannot be reliably determined because of Run2:1:40:1688:1032 C Run2:1:41:1268:591 G Run1:1:2:352:1341 C the inability to distinguish endogenous from contaminating Run1:1:21:1727:1631 C Run1:1:4:223:727 G Run1:1:5:750:1630 C molecules on the basis of their sequences [1, 2]. We now Run1:1:15:773:1819 C Run1:1:2:10:592 G Run1:1:10:785:441 C believe that this is possible as the result of several advances Run2:1:42:1398:864 C Run2:1:39:1230:694 G Run2:1:40:1462:1869 C brought about by library sequencing approaches. First, the Run1:1:2:517:124 G Run2:1:40:1476:1676 C access to short fragments allowed by sequencing from Run1:1:7:463:1535 G Run2:1:39:984:1250 C rCRS - 542 adaptors greatly improves the ratio of endogenous DNA to Run2:1:38:1716:811 G Run2:1:40:939:576 C contaminant DNA. Second, deep sequencing of libraries rCRS C Run1:1:2:920:153 G Run1:1:6:1630:792 C allows nucleotide positions that carry private or rare alleles Consensus T Run1:1:6:624:1027 G in an individual to be sequenced to deep coverage to ascertain Run1:1:20:1261:1275 T Run1:1:3:910:996 G that the human DNA in the library comes from a single indi- Run2:1:42:966:260 T Run2:1:44:827:446 G vidual. Third, if the DNA comes from a single individual, the Run1:1:5:1066:901 G Run1:1:10:211:699 T overall patterns of DNA degradation, such as fragment length, Run1:1:21:1245:1938 G Run2:1:44:818:2022 T nucleotide misincorporations, and fragmentation patterns, Run1:1:3:212:325 T Run1:1:2:133:1763 G can be used to identify datasets that are likely to be authentic. Run1:1:10:890:1989 T Run2:1:41:1214:533 G With this approach, it may even become possible to analyze Run2:1:39:1436:720 T Run2:1:42:1331:1100 G the nuclear genomes of early modern humans [23]. However, Run1:1:21:759:188 T Run2:1:40:575:601 G Run1:1:14:551:1801 T Run1:1:2:1049:1890 G it should not be overlooked that this is still very challenging. Run2:1:41:991:502 T Run2:1:43:1524:960 G For example, if more than one mtDNA sequence had been Run1:1:2:1616:559 C Run1:1:2:1392:1939 G present in the Kostenki 14 bone, or if coverage had been lower, Run1:1:29:81:266 T Run1:1:2:722:955 G our conﬁdence in the results would have been greatly dimin- Run2:1:42:1315:818 T Run2:1:42:1483:1299 G ished. Thus, to an even greater extent than is the case for Run1:1:5:620:181 T Run2:1:43:703:485 G Neandertals, determination of early modern human DNA Run2:1:43:1303:1843 T Run2:1:39:1353:796 G sequences will be conﬁned to exceptionally well-preserved Run2:1:42:794:815 T Run2:1:44:1195:655 G specimens.

Figure 4. Contamination Estimate of Kostenki 14 mtDNA Accession Numbers Nucleotide state for unique fragments overlapping each of the four positions private to Kostenki 14 (542, 711, 13,269, and 15,262) or in less than 1% The mtDNA sequence of the Kostenki 14 EMH is deposited at the European frequency in a data set of 311 worldwide sampled mtDNAs (13,269) (see Bioinformatics Institute nucleotide database under the accession number also Table S2). FM 600416. nucleotide distance of the Kostenki consensus sequence to Supplemental Information the most common recent ancestor (MCRA) of haplogroup U Supplemental Information includes Supplemental Data, four tables, seven is significantly shorter than all other 63 mtDNAs (Figure 3E, figures, and Supplemental Experimental Procedures can be found with Figure S5). This may reflect the age of the Kostenki bone. If this article online at doi:10.1016/j.cub.2009.11.068. we assume that the nucleotide sequence differences to the MCRA of the 63 mtDNAs are Poisson distributed and an age Acknowledgments of the MCRA of 51,700 years [26], we estimated a minimum age of 10,400 years for the Kostenki 14 mtDNA with a best The authors thank Vladimir B. Doronichev, Ilia Gokhman, Liubov V. Golova- age estimate of 32,730 years. However, this is at best a rough nova, Jeffrey M. Good, Richard E. Green, Jean-Jacques Hublin, Michael estimate given many uncertainties associated with this calcu- Lachmann, Matthias Meyer, Bernd H. Northoff, Michael P. Richards, Ralf Schmitz, Andrei A. Sinitsyn, Udo Stenzel, Christine Verna, and the anony- lation. We note that when multiple well-dated EMH mtDNAs mous reviewers for helpful comments and samples, as well as the Max have been determined, it may be possible to more accurately Planck Society for financial support. estimate the rate and mode of evolution of human mtDNAs and thus to use nucleotide sequence differences more reliably Received: October 14, 2009 for dating. Revised: November 23, 2009 Today, haplogroup U2 exists in North Africa, western Asia, Accepted: November 23, 2009 and Europe [10]. The presence of haplogroup U in an early Published online: December 31, 2009 modern human in Europe is in agreement with analyses of present-day mtDNA, which suggested that haplogroup U has References a deep coalescence and may thus be older than other hap- 1. Pa¨ a¨ bo, S., Poinar, H., Serre, D., Jaenicke-Despres, V., Hebler, J., Roh- logroups in Europe [10, 26]. It is also in agreement with recent land, N., Kuch, M., Krause, J., Vigilant, L., and Hofreiter, M. (2004). analyses of up to 13,400-year-old preagricultural skeletons in Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645–679. Current Biology Vol 20 No 3 236

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