Letter

https://doi.org/10.1038/s41586-018-0455-x

The genome of the offspring of a Neanderthal
mother and a Denisovan father
Viviane Slon1,7*, Fabrizio Mafessoni1,7, Benjamin Vernot1,7, Cesare de Filippo1, Steffi Grote1, Bence Viola2,3, Mateja Hajdinjak1,
Stéphane Peyrégne1, Sarah Nagel1, Samantha Brown4, Katerina Douka4,5, Tom Higham5, Maxim B. Kozlikin3,
Michael V. Shunkov3,6, Anatoly P. Derevianko3, Janet Kelso1, Matthias Meyer1, Kay Prüfer1 & Svante Pääbo1*

Neanderthals and Denisovans are extinct groups of homininsthat separated from each other more than 390,000 years ago1,2.
Here we present the genome of ‘Denisova 11’, a bone fragmentfrom Denisova Cave (Russia)3 and show that it comes from anindividual who had a Neanderthal mother and a Denisovan father.
The father, whose genome bears traces of Neanderthal ancestry,came from a population related to a later Denisovan found in the
cave4–6. The mother came from a population more closely related
to Neanderthals who lived later in Europe2,7 than to an earlier
Neanderthal found in Denisova Cave8, suggesting that migrationsof Neanderthals between eastern and western Eurasia occurred
sometime after 120,000 years ago. The finding of a first-generation
Neanderthal–Denisovan offspring among the small number ofarchaic specimens sequenced to date suggests that mixing between
Late Pleistocene hominin groups was common when they met.
Neanderthals and Denisovans inhabited Eurasia until they were
replaced by modern humans around 40,000 years ago (40 ka) 9.
Neanderthal remains have been found in western Eurasia10, whereas
physical remains of Denisovans have thus far been found only inDenisova Cave4–6,11,12, where Neanderthal remains have also been
recovered8. Although little is known about the morphology ofDenisovans, their molars lack the derived traits that are typical of
Neanderthals5,11.
DNA recovered from individuals of both groups suggests that they
diverged from each other more than 390 ka1,2. The presence of small
amounts of Neanderthal DNA in the genome of ‘Denisova 3’, thefirst Denisovan individual to be identified4–6, indicates that the twogroups mixed with each other at least once8. It has also been shownthat Neanderthals mixed with the ancestors of present-day nonAfricans around 60 ka2,8,13, and possibly with earlier ancestors of modern
humans1,14,15; and that Denisovans mixed with the ancestors of presentday Oceanians and Asians5,16,17. Denisovans may furthermore havereceived gene flow from an archaic hominin that diverged more than amillion years ago from the ancestors of modern humans8.
A fragment of a long bone, ‘Denisova 11’ (Fig. 1), was identified among over 2,000 undiagnostic bone fragments excavated in
Denisova Cave as being of hominin origin using collagen peptide
mass fingerprinting3. Its mitochondrial (mt)DNA was found to be of
the Neanderthal type and direct radiocarbon dating showed it to be
more than 50,000 years old3. From its cortical thickness, we infer that
Denisova 11 was at least 13 years old at death (Extended Data Fig. 1 andSupplementary Information 1). We performed six DNA extractions18,19
from bone powder collected from the specimen, produced ten DNA
libraries20 from the extracts (Extended Data Table 1 and Supplementary
Information 2, 3) and sequenced the Denisova 11 genome to an averagecoverage of 2.6-fold. The coverage of the X chromosome was similar
to that of the autosomes, indicating that Denisova 11 was a female.
Using three different methods, we estimate that contaminatingpresent-day human DNA fragments constitute at most 1.7% of the data
(Supplementary Information 2).
To determine from which hominin group Denisova 11 originated,
we compared the proportions of DNA fragments that match derived
alleles from a Neanderthal genome (‘Altai Neanderthal’, also known as‘Denisova 5’) or a Denisovan genome (Denisova 3), both determinedfrom bones discovered in Denisova Cave6,8, as well as from a presentday African genome (Mbuti)6 (Supplementary Information 4). At
informative sites1, 38.6% of fragments from Denisova 11 carried alleles
matching the Neanderthal genome and 42.3% carried alleles matching
the Denisovan genome (Fig. 2a), suggesting that both archaic groups
contributed to the ancestry of Denisova 11 to approximately equal
extents (Supplementary Information 4). Approximately equal proportions of Neanderthal-like and Denisovan-like alleles are found in eachof the ten DNA libraries originating from Denisova 11 but not in libraries from other projects that were prepared, sequenced and processed inparallel, which excludes an accidental mixing of DNA in the laboratory
or a systematic error in data processing (Supplementary Information 3).
To estimate the heterozygosity of Denisova 11, we restrict the analysis to transversion polymorphisms to prevent deamination-derived
substitutions from inflating the estimates, and find 3.7 transversions
per 10,000 autosomal base pairs. This is over four times higher than theheterozygosity of the two Neanderthal (Altai Neanderthal and ‘Vindija
33.19’) and one Denisovan (Denisova 3) genomes sequenced to date,and similar to the heterozygosity seen in present-day Africans. In fact,
the heterozygosity of Denisova 11 is similar to what would be expected
if this individual carried one set of chromosomes of Neanderthal
origin and one of Denisovan origin, as estimated from the number ofdifferences between randomly sampled DNA fragments from either
the Vindija 33.19 or the Altai Neanderthal genome and the Denisova
3 genome (Fig. 2b and Supplementary Information 5).
Denisova 11 could have had approximately equal amounts of
Neanderthal and Denisovan ancestry because she belonged to a population with mixed Neanderthal and Denisovan ancestry, or because
her parents were each from one of these two groups. To determine
which of these two scenarios fits the data best, we considered sites atwhich the genomes of the Altai Neanderthal and Denisova 3 carry atransversion difference in a homozygous form. At each of these sites,
we recorded the alleles carried by two randomly drawn DNA fragmentsfrom Denisova 11. Note that in 50% of cases, both fragments will comefrom the same chromosome, making 50% of heterozygous sites appear
homozygous. As a consequence, the expected proportion of apparent
heterozygous sites is 50% for a first-generation (F1) offspring, whereas
it is 25% in a population at Hardy–Weinberg equilibrium with mixed
ancestry in equal proportions (Supplementary Information 6). We findthat in 43.5% of cases, one fragment from Denisova 11 matches the
Neanderthal genome and the other matches the Denisovan genome,
whereas in 27.3% and 29.2% of cases both fragments match the state1

Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. 2Department of Anthropology, University of Toronto, Toronto, Ontario, Canada.
Institute of Archaeology and Ethnography, Russian Academy of Sciences, Novosibirsk, Russia. 4Max Planck Institute for the Science of Human History, Jena, Germany. 5Oxford Radiocarbon
Accelerator Unit, RLAHA, University of Oxford, Oxford, UK. 6Novosibirsk State University, Novosibirsk, Russia. 7These authors contributed equally: Viviane Slon, Fabrizio Mafessoni, Benjamin Vernot.
*e-mail: viviane_slon@eva.mpg.de; paabo@eva.mpg.de
3

6 S E P TE M B ER 2 0 1 8 | V O L 5 6 1 | N A T U RE | 1 1 3© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter
Goyet
Feldhofer
Ust’-Ishim*?

Goyet Q56-1

Feld1 Feld2

Spy

Vindija

Ust’-Ishim 1*

Spy 94aEl Sidrón

Denisova cave

33.16 33.19 33.25 33.26

Les Cottés*

Mezmaiskaya*

Tianyuan
Denisova 11

Oase
SD1253
Mez1

Mez2

Oase 1

Sima de los Huesos

Den2
AT-5431

Tianyuan

Altai Den3
(Den5)
1 cm

Z4-1514

Den4

Den8

AT-5482

Fig. 1 | Location of Neanderthals, Denisovans and ancient modern
humans dated to approximately 40 ka or earlier. Only individuals fromwhom sufficient nuclear DNA fragments have been recovered to enable
their attribution to a hominin group are shown. Full or abbreviated namesof specimens are shown near each individual. Blue, Neanderthals; red,
Denisovans; yellow, ancient modern humans. Asterisks indicate that the

genome was sequenced to high coverage; individuals with an unknown sex
are marked with a question mark. Note that Oase 1 has recent Neanderthal
ancestry (blue dot) that is higher than the amount seen in non-Africans.
Denisova 3 has also been found to carry a small percentage of Neanderthal
ancestry. Data were obtained from previous publications1,2,5–8,11–13,21–24.

seen in the Neanderthal or the Denisovan genome, respectively(Fig. 2c). For comparison, when a low-coverage Neanderthal genome(‘Goyet Q56-1’)7 is analysed in the same way, the two fragments match
different states in 2.1% of cases, while they both match the Neanderthal
state in 90.3% of cases and the Denisovan state in 7.5% of cases (Fig. 2c).
Obviously, the Altai Neanderthal and Denisova 3 are unlikely to be
identical to the genomes of the individuals that contributed ancestry toDenisova 11. To take this into account, we used coalescent simulations
to estimate the expected proportions of DNA fragments matching a
Neanderthal or a Denisovan genome in populations with demographic
histories similar to those of the Altai Neanderthal and Denisova 3(Supplementary Information 6). The proportion of cases in which oneof the two DNA fragments sampled from Denisova 11 matches the
Neanderthal state and the other the Denisovan state fits the expectation for an F1 Neanderthal–Denisovan offspring, but not an offspringof two F1 individuals, an offspring of an F1 parent and a Neanderthal

or a Denisovan parent, nor an individual from a population of mixed
ancestry at Hardy–Weinberg equilibrium (Extended Data Fig. 2 andSupplementary Information 6). We conclude that Denisova 11 did not
originate from a population carrying equal proportions of Neanderthal
and Denisovan ancestry. Rather, she was the offspring of a Neanderthal
mother, who contributed her mtDNA, and a Denisovan father.
We next plotted the distribution of sites across the genome, forwhich Denisova 11 carries an allele matching the Altai Neanderthal
genome and a different allele matching the Denisova 3 genome. Suchsites are distributed largely uniformly (Fig. 3), as would be expected
for an F1 offspring of Neanderthal and Denisovan parents. To explore
the ancestry of the parents of Denisova 11, we looked for regions in
the genome that deviate from a pattern consistent with Denisova
11 being an F1 offspring (Extended Data Fig. 3). Using four tests
for enrichment of Denisovan or Neanderthal ancestry, we identify
at least five approximately 1-Mb long (0.72–0.95 Mb) regions, all ofcExpected

NN

60

ND
DD

40

1
20
0

D
1 ×

1

F

F

2

F

va

11

-1
56
tQ

nis
o
De

a3
dij
Vin

Fig. 2 | Denisova 11 has both Neanderthal and Denisovan ancestry.
a, Percentage of DNA fragments from Denisova 11 matching derived
alleles found on each branch of a tree relating a Neanderthal, a Denisovan
and a present-day human genome. b, Distribution of heterozygosity per
chromosome in two Neanderthals (blue), a Denisovan (red), Denisova
11 (purple) and present-day humans (n = 235 non-African individuals(yellow) and n = 44 African individuals (orange) from a previous
publication28), and the expectation for a Neanderthal–Denisovan F1
offspring (grey). The violins represent the distribution from the minimum
and maximum heterozygosity values for the autosomes of each archaic0yeModernhuman(Mbuti)

Sites (%)

Denisovan(Denisova 3)

2

Observed

Go

Neanderthal(Altai)

3

i
nis
ov
a3
nis
ov
a1
No
1
nAf
ric
an
s
Af
Vin
ric
dij
an
a−
s
De
nis
Alt
o
va
ai−
De
nis
ov
a

42.3%

80

3.1

38.6%

4

De

1.2%

100

9

95.2%

5

Alt
a

99.7%

Transversion heterozygotes
per 10,000 bps

b

De

a

hominin and of present-day humans (n = 5,170 pairs of chromosomes
for non-Africans and n = 968 for Africans). White squares represent
autosome-wide estimates for the archaic hominins, and the average of
estimates across individuals for present-day humans. c, Percentage of sites
at which two sampled DNA fragments both carry Neanderthal-like alleles(NN, blue), Denisovan-like alleles (DD, red), or one allele of each type
(ND, purple); and the expectations for an offspring of a Neanderthal anda Denisovan (F1), of two F1 parents (F2), and of an F1 and a Denisovan(F1×D). The expected proportions for simulated Neanderthal andDenisovan genomes are shown in Extended Data Fig. 2.

1 1 4 | N A T U RE | V O L 5 6 1 | 6 S E P TE M B ER 2 0 1 8© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH
Chr. 1
Chr. 2
Chr. 3
Chr. 4
Chr. 5
Chr. 6
Chr. 7
Chr. 8Chr. 9Chr. 10Chr. 11
Chr. 12

Denisova 11

Chr. 13
Chr. 14Goyet Q56-1

Chr. 15
Chr. 16
Chr. 17
Chr. 18
Chr. 19
Chr. 20
Chr. 21Chr. 22
Chr. X

0

50

100

150

200

250

Position (Mb)which are homozygous for Neanderthal ancestry. This suggests thatthe Denisovan father of Denisova 11 had some Neanderthal ancestry.
Given conservative estimates of the size and number of these regions,
it is likely that there was more than one Neanderthal ancestor in his
genealogy, possibly as far back as 300–600 generations before his lifetime (Supplementary Information 7). Notably, the heterozygosity in theregions of Neanderthal ancestry in Denisova 11 is higher than in thesame regions in the genomes of Vindija 33.19 or the Altai Neanderthal,suggesting that the Neanderthals that contributed to the ancestry ofDenisova 11’s father were from a different population than her mother(Supplementary Information 5).
To explore how the mother of Denisova 11 was related to the two
Neanderthals that have been sequenced to high coverage to date, we
evaluated the proportions of fragments from Denisova 11 that match
derived alleles from either of these two Neanderthal genomes. Denisova
11 shares derived alleles seen in the Altai Neanderthal genome in 12.4%
of cases and those present in the Vindija 33.19 genome in 19.6% ofcases, showing that the Neanderthal mother of Denisova 11 came froma population that was more closely related to Vindija 33.19 than to
the Altai Neanderthal (Supplementary Information 8). We estimate
the population split times of Denisova 11’s Neanderthal mother fromthe ancestors of the Altai Neanderthal to approximately 20,000 years(20 kyr) before the time when the Altai Neanderthal lived, and her
split time from the ancestors of Vindija 33.19 to around 40 kyr before
Vindija 33.19. The population split between the Denisovan father ofDenisova 11 and Denisova 3 is estimated to approximately 7 kyr before
the latter individual (Supplementary Information 8). In Fig. 4, we present a population scenario that is compatible with these observations aswell as with the population split times and molecular estimates of theages of the three high-coverage archaic genomes2. We caution that theage estimates are associated with uncertainties, for example, regarding
demography, mutation rates and generation times, and note that additional gene flow events are likely to have affected the population split
times. Nevertheless, that a Neanderthal in Siberia who lived approximately 90 ka shared more alleles with Neanderthals who lived at least
20 kyr later in Europe2,7 than with an earlier Neanderthal from the samecave8 suggests that eastern Neanderthals spread into Western Europe

match the Denisovan genome. Black lines indicate centromeres. The inset
shows one region out of five (green boxes) for which both chromosomescarry predominantly Neanderthal-like alleles. For comparison, thedistribution of alleles in this region is shown for a Neanderthal genome(Goyet Q56-1).
450
430
410
390

140

Time (ka)Fig. 3 | Distribution of Neanderthal-like and Denisovan-like alleles
across the Denisova 11 genome. Positions for which one randomly drawn
DNA fragment matches the Neanderthal genome and another matches the
Denisovan genome are marked in purple. Positions are marked in blue if
both DNA fragments match the Neanderthal genome and in red if both

130
120
110

Altai
Neanderthal

100
90
80

Denisova 11

Denisova 370

Vindija 33.19

60

and other late
Neanderthals

50

Fig. 4 | Relationships and gene flow events between Neanderthal andDenisovan populations inferred from genome sequences. Diamonds
indicate ages of specimens estimated via branch shortening2; circles
indicate population split times estimated from allele sharing between
Denisova 11 and the high-coverage Neanderthal and Denisovan genomes(blue and red) and among the three high-coverage genomes (yellow, froma previous publication2). Markers indicate the means of these estimates,
error bars indicate 95% confidence intervals based on block jackknife
resampling across the genome (n = 523 blocks). Note that the confidence
intervals do not take the uncertainty with respect to population size,
mutation rates or generation times into account. Ages before present are
based on a human–chimpanzee divergence of 13 million years22,29. The
arrow indicates Neanderthal gene flow into Denisovans.
6 S E P TE M B ER 2 0 1 8 | V O L 5 6 1 | N A T U RE | 1 1 5© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter
sometime after 90 ka or that western Neanderthals spread to Siberia
before that time and partially replaced the local population. These two
non-mutually exclusive hypotheses could be tested by sequencing the
genomes of early Neanderthals from Western Europe.
In conclusion, the genome of Denisova 11 provides direct evidence for genetic mixture between Neanderthals and Denisovans
on at least two occasions: once between her Neanderthal motherand her Denisovan father, and at least once in the ancestry of her
Denisovan father. Therefore, of the six individuals from Denisova Cavefrom whom nuclear DNA is available5,6,8,11,12, two (Denisova 3 andDenisova 11) show evidence of gene flow between Neanderthals andDenisovans. We note that of the three genomes21–24 retrieved frommodern humans who lived at a time when Neanderthals were present
in Eurasia (that is, approximately 40 ka or earlier)9, one individual—‘Oase 1’—had a Neanderthal ancestor four to six generations back in
his family tree23.
It is notable that one direct offspring of a Neanderthal and a
Denisovan (Denisova 11) and one modern human with a close
Neanderthal relative (Oase 1) have been identified among the few
individuals from whom DNA has been retrieved and who lived at thetime of overlap of these groups (Fig. 1). In conjunction with the presence of Neanderthal and Denisovan DNA in ancient and present-day
people2,5,8,13,16,17,25–27, this suggests that mixing among archaic
and modern hominin groups may have been frequent when they
met. However, Neanderthals inhabited western Eurasia10 whereas
Denisovans inhabited yet unknown parts of eastern Eurasia5,17. Thus,
their zones of overlap may have been restricted in space and time. This,as well as possibly reduced fitness of individuals of mixed ancestry,
may explain why Neanderthals and Denisovans remained geneticallydistinct. By contrast, the spread of modern humans across Eurasia after
around 60,000 years ago may have allowed repeated interactions witharchaic groups over a wider spatial range. Admixture between them
may have resulted in archaic populations becoming partly absorbed
into what were probably larger modern human populations6,8.

Online content

Any methods, additional references, Nature Research reporting summaries, source
data, statements of data availability and associated accession codes are available athttps://doi.org/10.1038/s41586-018-0455-x.

Received: 20 February; Accepted: 18 June 2018;
Published online 22 August 2018.
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Acknowledgements We thank B. Schellbach and A. Weihmann for DNA
sequencing; G. Renaud and U. Stenzel for data processing; F. Brock for thecomputed tomography scans; R. Barr, P. Korlević and C. Zickert for graphics;
and M. Slatkin and L. Vigilant for comments on the manuscript. This work
was funded by the Max Planck Society; the Max Planck Foundation (grant31-12LMP Pääbo to S.Pä.); the European Research Council (grant agreements
694707 to S.Pä., 324139 (PalaeoChron) to T.H. and 715069 (FINDER) to
K.D.); and the Russian Science Foundation (project no. 14-50-00036 to M.B.K.,
M.V.S. and A.P.D.).
Reviewer information Nature thanks D. Lambert, R. Nielsen and C. Stringer for
their contribution to the peer review of this work.
Author contributions V.S. and S.N. performed the laboratory work; V.S., F.M.,
B.Ve., C.d.F., S.G., M.H., S.Pe., J.K., M.M., K.P. and S.Pä. analysed the genetic data;
B.Vi. carried out the morphological analysis; S.B., K.D., T.H., M.B.K., M.V.S. and
A.P.D. discovered Denisova 11 and provided archaeological data; V.S., K.P. and
S.Pä. wrote the manuscript with input from all authors.
Competing interests The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41586018-0455-x.
Supplementary information is available for this paper at https://doi.org/
10.1038/s41586-018-0455-x.
Reprints and permissions information is available at http://www.nature.com/
reprints.
Correspondence and requests for materials should be addressed to V.S. or S.Pä.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

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Letter RESEARCH
Methods

Sampling and pre-treatment of bone powder. An overview of the laboratory
experiments is shown in Extended Data Table 1. Bone powder was removed fromthe specimen using disposable sterile dentistry drills after the removal of a thinlayer of surface material. Six samples were collected, each consisting of approximately 30 mg of bone powder. Because a previous analysis of the bone revealed that
it is contaminated with present-day human DNA3, each sample of bone powder was
incubated with 1 ml 0.5% sodium hypochlorite solution as previously described19
and as indicated in Extended Data Table 1, to reduce the amounts of present-day
human and microbial DNA7,19. Residual sodium hypochlorite was removed bythree consecutive 3-min washes with 1 ml water19. One extraction negative control(no powder) was included in each set of extractions.
DNA extraction and DNA library preparation. DNA was extracted using silica
columns18 as previously described19, and eluted in 50 μl 10 mM Tris-HCl, 1 mM
EDTA, 0.05% Tween-20, pH 8.0. Subsequently, 10 μl of each DNA extract (including the extraction negative controls) were used to prepare single-stranded DNA
libraries as previously described19,20. A library preparation negative control was
included in every experiment. Two additional 5-μl aliquots from extracts E3652
and E3655 were used to generate additional libraries (library preparation setup
C in Extended Data Table 1), resulting in a total of 10 DNA libraries. The number of DNA molecules in the libraries was estimated by digital droplet PCR30 orquantitative PCR20. Each library was amplified to the plateau while incorporatinga pair of unique indexes31 using 1 μM primers19,31 and AccuPrime Pfx DNA poly­merase (Life Technologies)32. Amplification products were purified using the
MinElute PCR purification kit (Qiagen) or SPRI technology33 on a Bravo NGS
workstation (Agilent Technologies) as previously described34. Indexed DNA
libraries were pooled with libraries from other projects. Heteroduplices, which
confound DNA separation and concentration measurements in chromatography,
were removed from the pools by single cycle amplification using Herculase
II Fusion DNA polymerase (Agilent Technologies)32 with primers IS5 and IS635.
Prior to deeper sequencing of libraries R5507, R5509, R9880, R9881, R9882, R9883
and R9873, heteroduplices were removed from each library separately. The concentration of DNA in each pool or each individual library, respectively, was determinedusing the electrophoresis system implemented on the DNA-1000 chip (Agilent
Technologies).
Sequencing and data processing. Sequencing was performed on Illumina
platforms (MiSeq or HiSeq 2500) using 76-cycle paired-end runs adapted to
double-indexed libraries31. Bases were called using Bustard (Illumina). Adaptor

sequences were trimmed and overlapping paired-end reads were merged into single sequences using leeHom36. Demultiplexing was carried out using jivebunny7.
Sequences generated from a given library were merged using SAMtools37 andaligned to the human reference genome (hg19/GRCh37) with the decoy sequences
as previously described2 using BWA38 with parameters adjusted to ancient DNA6.
PCR duplicates were collapsed using bam-rmdup (https://bitbucket.org/ustenzel/
biohazard) and DNA fragments of length ≥35 bases that mapped within regions of
unique mappability (Map35_100% from a previous publication8) with a mapping
quality of 25 or higher7 were used for analyses. Further filtering criteria used forcertain analyses are described in the Supplementary Information.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this paper.
Code availability. The computer code used for simulations is included in
Supplementary Information 6.
Data availability. Sequences generated from Denisova 11 have been deposited in
the European Nucleotide Archive under study accession number PRJEB24663.
30. Slon, V. et al. 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). Quat. Int. 398,
210–218 (2016).
31. Kircher, M., Sawyer, S. & Meyer, M. Double indexing overcomes inaccuracies in
multiplex sequencing on the Illumina platform. Nucleic Acids Res. 40, e3 (2012).
32. Dabney, J. & Meyer, M. Length and GC-biases during sequencing library
amplification: a comparison of various polymerase-buffer systems withancient and modern DNA sequencing libraries. Biotechniques 52, 87–94(2012).
33. DeAngelis, M. M., Wang, D. G. & Hawkins, T. L. Solid-phase reversible
immobilization for the isolation of PCR products. Nucleic Acids Res. 23,
4742–4743 (1995).
34. Slon, V. et al. Neandertal and Denisovan DNA from Pleistocene sediments.
Science 356, 605–608 (2017).
35. Meyer, M. & Kircher, M. Illumina sequencing library preparation for highly
multiplexed target capture and sequencing. Cold Spring Harb. Protoc.
https://doi.org/10.1101/pdb.prot5448 (2010).
36. Renaud, G., Stenzel, U. & Kelso, J. leeHom: adaptor trimming and merging forIllumina sequencing reads. Nucleic Acids Res. 42, e141 (2014).
37. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics
25, 2078–2079 (2009).
38. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheelertransform. Bioinformatics 26, 589–595 (2010).

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RESEARCH Letter

Extended Data Fig. 1 | Comparison between cortical thickness of longbones from modern humans, Neanderthals and Denisova 11. Maximum
cortical thickness of femora, tibiae, humeri, radii and ulnae from humans

from the Bronze Age and two Neanderthals compared to the minimum
thickness of Denisova 11 (dashed line).

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH

Extended Data Fig. 2 | Comparison of the genome of Denisova 11
and simulated genomes. Percentage of sites at which Denisova 11
and genomes simulated under the demographic model described in
Supplementary Information 6 carry two Neanderthal alleles (NN, blue),
two Denisovan alleles (DD, red) or one allele of each type (ND, purple).

a, Percentages calculated for two random DNA fragments from Denisova
11 and from simulated F1, F2, Neanderthal (NF0) or Denisovan (DF0)genomes. b, Proportions of sites for the simulated genotypes, before
sampling two fragments.

© 2018 Springer Nature Limited. All rights reserved.

RESEARCH Letter

Extended Data Fig. 3 | Neanderthal and Denisovan allele proportions
from Denisova 11 in 1-Mb windows. The y axis shows −log(P) of
the deviation of Neanderthal and Denisovan allele counts from thegenome-wide average (χ2 test of goodness-of-fit; see Supplementary

Information 7). The colour shows the proportion of alleles matching
the Neanderthal state (%N) within each 1-Mb window (100-kb steps,n = 26,414 windows).

© 2018 Springer Nature Limited. All rights reserved.

Letter RESEARCH
Extended Data Table 1 | DNA extracts and DNA libraries prepared from the Denisova 11 specimen

Data are shown by DNA extraction set, and libraries prepared in the same setup are denoted with the same letter (A, B or C). Relevant negative controls are marked in grey. The number of moleculesin each library was quantified by digital droplet PCR or quantitative PCR (denoted by asterisk). The numbers of DNA fragments sequenced per library are indicated for the combined data from allsequencing runs. Mapped fragments were counted if they were at least 35 bases long and mapped to the human reference genome with a mapping quality of 25 or higher; and their percentage wascalculated out of sequenced fragments of length 35 bases or more. Following the removal of PCR duplicates, unique DNA fragments were retained if they mapped to the reference genome within theused mappability track. Such fragments were considered to contain a terminal cytosine (C) to thymine (T) substitution relative to the human reference genome if a putative cytosine deamination waswithin the first three or last three bases of the strand. bp, base pairs; ENC, extraction negative control; Extr., extraction; L, length; LNC, library preparation negative control; Map35_100%, previously
published mappability track8; MQ, mapping quality; Prep., preparation.

© 2018 Springer Nature Limited. All rights reserved.

Life Sciences Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life
science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list
items might not apply to an individual manuscript, but all fields must be completed for clarity.
For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Researchpolicies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.
Please do not complete any field with "not applicable" or n/a. Refer to the help text for what text to use if an item is not relevant to your study.
For final submission: please carefully check your responses for accuracy; you will not be able to make changes later.

`

Experimental design

1. Sample size
Describe how sample size was determined.

The sample size is 1, as we are determining the genome of a single individual. For
comparisons with other archaic genomes, all available high-coverage Neandertal andDenisovan genomes (n=3) were used.

nature research | life sciences reporting summary

Corresponding author(s): Viviane Slon; Svante Pääbo

2. Data exclusions
Describe any data exclusions.

Sequencing data excluded based on pre-established criteria: sequences that did not map to
the human genome, sequences that were shorter than 35 bases, sequences mapping with alow mapping quality or within regions of low mappability - all of which were excluded to
avoid using sequences that were not endogenous to the individual sequenced.

3. Replication
Describe the measures taken to verify the reproducibilityof the experimental findings.

The bone fragment was sampled on three different occasions, on different areas of thespecimen (total of six samples of bone powder). Six DNA extracts were prepared on threeoccasions, and ten DNA libraries were generated in three different experiments. We show in
SI 3 that the results are stable across all different libraries.
To allow the reproducibility of the analyses, all filtering steps and the comparative data used
are detailed in the Methods section and the supplementary information; and the sequencing
data generated here has been deposited in the European Nucleotide Archive.

4. Randomization
Describe how samples/organisms/participants wereallocated into experimental groups.

This is not relevant for our study as we determined the genome of a single individual,
therefore there were no experimental groups.

5. Blinding
Describe whether the investigators were blinded togroup allocation during data collection and/or analysis.

Blinding was not relevant for our study, as this is the genome of a single individual.

Note: all in vivo studies must report how sample size was determined and whether blinding and randomization were used.

November 20171

6. Statistical parameters

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)
A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same
sample was measured repeatedly
A statement indicating how many times each experiment was replicated
The statistical test(s) used and whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of any assumptions or corrections, such as an adjustment for multiple comparisons
Test values indicating whether an effect is present
Provide confidence intervals or give results of significance tests (e.g. P values) as exact values whenever appropriate and with effect sizes noted.

A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)
Clearly defined error bars in all relevant figure captions (with explicit mention of central tendency and variation)
See the web collection on statistics for biologists for further resources and guidance.

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For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the
Methods section if additional space is needed).

` Software
Policy information about availability of computer code

7. Software
Describe the software used to analyze the data in thisstudy.

Basecalling: Bustard; Adapter trimming: leeHom; Demultiplexing: jivebunny; Mapping: BWA;
Handling bam files: SAMtools; PCR duplicates removal: bam-rmdup; F3- and F4-statistics:
Admixtools; Identification of variable sites: heffalump; Heterozygosity estimates: snpAD;
Demography simulations: scrm; HMM: pomegranate; Statistics: R.

For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be madeavailable to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance forproviding algorithms and software for publication provides further information on this topic.

` Materials and reagents
Policy information about availability of materials

8. Materials availability
Indicate whether there are restrictions on availability of
unique materials or if these materials are only available
for distribution by a third party.

The sequencing data generated from the Denisova 11 bone fragment and the CT scans of thespecimen are publicly available. Requests for further sampling of the bone fragment should
be addressed to the Institute of Archaeology and Ethnography of the Russian Academy of
Sciences.

9. Antibodies
Describe the antibodies used and how they were validated No antibodies were used.
for use in the system under study (i.e. assay and species).

10. Eukaryotic cell lines
No eukaryotic cell lines were used.

b. Describe the method of cell line authentication used.

No eukaryotic cell lines were used.

c. Report whether the cell lines were tested for
mycoplasma contamination.

No eukaryotic cell lines were used.

d. If any of the cell lines used are listed in the databaseof commonly misidentified cell lines maintained by
ICLAC, provide a scientific rationale for their use.

No eukaryotic cell lines were used.
November 2017

a. State the source of each eukaryotic cell line used.

2

Animals and human research participants

Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines

11. Description of research animals
Provide all relevant details on animals and/or
animal-derived materials used in the study.

No animals were used.

Policy information about studies involving human research participants

12. Description of human research participants
Describe the covariate-relevant population
characteristics of the human research participants.

The research did not involve human participants.

nature research | life sciences reporting summary`

November 20173