NATURE Vol 444 16 November 2006 ARTICLES precise(see Supplementary Methods and Results). Using this line we humans carry a single nucleotide polymorphism(SNP). The latter can estimate the ancestral population size, given estimates about the case identifies SNPs that were present in the common ancestor of population split time from independent sources. If we use a split time Neanderthals and present-day humans. Using the SNPs that overlap of 400,000 years inferred from the fossil record ( J Hublin, with our data from two large genome-wide data sets(Hap Map",786 communication), then our point estimate of the ancestral p SNPs and Perlegen, 318 SNPs), we find that the Neanderthal sample size is -3,000. Given uncertainty in both the sequen has the derived allele in -30%of all SNPs. This number is presum- time and the population split time, our estimate of the ancestral ably an overestimate since the SNPs analysed were ascertained to be population size varies from 0 to 12,000 of high frequency in present-day humans and hence are more likely These results suggest that the population ancestral to present-day to be old. Nevertheless, this high level of derived alleles in the mans and Neanderthals was similar to present-day humans in Neanderthal is incompatible with the simple population split model having a small effective size and thus that the effective population estimated in the previous section, given split times inferred from the size on the hominid lineage had already decreased before the split fossil record. This may suggest gene flow between modern humans between humans and Neanderthals. Therefore the small effective and Neanderthals. Given that the Neanderthal X chromosome shows population size seen in present-day human samples may not be a higher level of divergence than the autosomes(REG, unpublished nique to modern humans, but was present also in the common observation), gene flow may have occurred predominantly from ancestor of Neanderthals and modern humans. We speculate that a modern human males into Neanderthals. More extensive sequencing small effective size, perhaps associated with numerous expansions of the Neanderthal genome is necessary to address this possibility from small groups, was typical not only of modern humans but of ny groups of the genus Homo. In fact, the origin of Homo erectus Rationale and prospects for a Neanderthal genome sequence nay have been associated with genetic or cultural adaptations that We demonstrate here that DNA sequences can be generated from the appearance outside Africa arour expansions as indicated by their Neanderthal nuclear genome by massive parallel sequencing on the resulted in drastic population 454 sequencing platform. It is thus feasible to determine large amounts of sequences from this extinct hominid. As a corollary, it Neanderthal sequences and human polymorphisms is possible to envision the determination of a Neanderthal genome Another question that can be addressed with these data is how sequence. For several reasons, we believe that this would represent a the Neanderthal has the ancestral allele (that is, the same allele se valuable genomic resource he chimpanzee)versus the derived(or novel) allele at sites First, a Neanderthal genome sequence would allow all nucleotide sequence differences as well as many copy-number differences between the human and chimpanzee genomes to be temporally resolved with respect to whether they occurred before the separation of humans from Neanderthals, or whether they occurred after or at the time of separation. The latter class of changes is of interest, because some of them will be associated with the emergence of mod before ern humans. A Neanderthal genome sequence would therefore alloy the research community to determine whether DNA sequence differ ences between humans and chimpanzees that are found to be fune tionally important represent recent changes on the human lineage No data other than a Neanderthal genome sequence can provide thi Neanderthal Human Ancestral effective population Divergence time for sequence one Second, the fact that Neanderthals carry the derived allele for a Split time into two grou substantial fraction of human SNPs suggests a method of identifying to the separation of human and Neanderthal populations. Such ∷" selective sweeps in the human genome will make the variation in 是x10.000 these regions younger than the separation of humans and Neanderthals. As we show above, in regions not affected by sweeps a substantial proportion of polymorphic sites in humans will carry N99 -.:.,4 derived alleles in the Neanderthal genome sequence, whereas no sites will do so in regions affected by sweeps. This represents an approach to identifying selective sweeps in humans that is not possible from 00,000 400.000 Third, once large amounts of Neanderthal genome sequence is generated, it will become possible to estimate the misincorporation probabilities for each class of nucleotide differences between th Neanderthal and chimpanzee genomes with high accuracy by ana- humans and Neanderthals. a, Schematic illustration of the model used to lysing regions covered by many reads such as mt DNA, repeated ge estimate ancestral effective population size By split time, we mean the time, ome regions of high sequence identity, as well as single-copy regions in the past, after which there was no more interbreeding between two groups. covered by multiple reads. Once this is done, the confidence that any By divergence, we mean the time, in the past, at which two genetic regions particular nucleotide position where the Neanderthal differs from separated and began to accumulate substitutions independently. Effective human as well as chimpanzee is correct can be reliably estimated. Go pulation size is the number of individuals needed under ideal conditions In combination with future knowledge about the function of genes produce the amount of observed genetic diversity within a population b, The likelihood estimates of population split times and ancestral and biological systems, comprehensive information from the population sizes. The likelihoods are grouped by colour. The red-yellow Neanderthal genome will then allow aspects of Neanderthal biology points are statistically equivalent based on th hood ratio test to be deciphered that are unavailable by any other means. approximation. The black line is the line of best fit to red-yellow points(see Are fossil and technical resources today sufficient to imagine the Supplementary Methods ). This graph is scaled assuming a determination of a Neanderthal genome sequence? The results pre an-chimpanzee average sequence divergence time of 6, 500,000 years. sented here are derived from approximately one fifteenth of an extract E2006 Nature Publishing Groupprecise (see Supplementary Methods and Results). Using this line we can estimate the ancestral population size, given estimates about the population split time from independent sources. If we use a split time of 400,000 years inferred from the fossil record (J. J. Hublin, personal communication), then our point estimate of the ancestral population size is ,3,000. Given uncertainty in both the sequence divergence time and the population split time, our estimate of the ancestral population size varies from 0 to 12,000. These results suggest that the population ancestral to present-day humans and Neanderthals was similar to present-day humans in having a small effective size and thus that the effective population size on the hominid lineage had already decreased before the split between humans and Neanderthals. Therefore, the small effective population size seen in present-day human samples may not be unique to modern humans, but was present also in the common ancestor of Neanderthals and modern humans. We speculate that a small effective size, perhaps associated with numerous expansions from small groups, was typical not only of modern humans but of many groups of the genus Homo. In fact, the origin of Homo erectus may have been associated with genetic or cultural adaptations that resulted in drastic population expansions as indicated by their appearance outside Africa around two million years ago. Neanderthal sequences and human polymorphisms Another question that can be addressed with these data is how often the Neanderthal has the ancestral allele (that is, the same allele seen in the chimpanzee) versus the derived (or novel) allele at sites where humans carry a single nucleotide polymorphism (SNP). The latter case identifies SNPs that were present in the common ancestor of Neanderthals and present-day humans. Using the SNPs that overlap with our data from two large genome-wide data sets (HapMap49, 786 SNPs and Perlegen50, 318 SNPs), we find that the Neanderthal sample has the derived allele in ,30% of all SNPs. This number is presum￾ably an overestimate since the SNPs analysed were ascertained to be of high frequency in present-day humans and hence are more likely to be old. Nevertheless, this high level of derived alleles in the Neanderthal is incompatible with the simple population split model estimated in the previous section, given split times inferred from the fossil record. This may suggest gene flow between modern humans and Neanderthals. Given that the Neanderthal X chromosome shows a higher level of divergence than the autosomes (R.E.G., unpublished observation), gene flow may have occurred predominantly from modern human males into Neanderthals. More extensive sequencing of the Neanderthal genome is necessary to address this possibility. Rationale and prospects for a Neanderthal genome sequence We demonstrate here that DNA sequences can be generated from the Neanderthal nuclear genome by massive parallel sequencing on the 454 sequencing platform. It is thus feasible to determine large amounts of sequences from this extinct hominid. As a corollary, it is possible to envision the determination of a Neanderthal genome sequence. For several reasons, we believe that this would represent a valuable genomic resource. First, a Neanderthal genome sequence would allow all nucleotide sequence differences as well as many copy-number differences between the human and chimpanzee genomes to be temporally resolved with respect to whether they occurred before the separation of humans from Neanderthals, or whether they occurred after or at the time of separation. The latter class of changes is of interest, because some of them will be associated with the emergence of mod￾ern humans. A Neanderthal genome sequence would therefore allow the research community to determine whether DNA sequence differ￾ences between humans and chimpanzees that are found to be func￾tionally important represent recent changes on the human lineage. No data other than a Neanderthal genome sequence can provide this information. Second, the fact that Neanderthals carry the derived allele for a substantial fraction of human SNPs suggests a method of identifying genomic regions that have experienced a selective sweep subsequent to the separation of human and Neanderthal populations. Such selective sweeps in the human genome will make the variation in these regions younger than the separation of humans and Neanderthals. As we show above, in regions not affected by sweeps a substantial proportion of polymorphic sites in humans will carry derived alleles in the Neanderthal genome sequence, whereas no sites will do so in regions affected by sweeps. This represents an approach to identifying selective sweeps in humans that is not possible from other data. Third, once large amounts of Neanderthal genome sequence is generated, it will become possible to estimate the misincorporation probabilities for each class of nucleotide differences between the Neanderthal and chimpanzee genomes with high accuracy by ana￾lysing regions covered by many reads such as mtDNA, repeated gen￾ome regions of high sequence identity, as well as single-copy regions covered by multiple reads. Once this is done, the confidence that any particular nucleotide position where the Neanderthal differs from human as well as chimpanzee is correct can be reliably estimated. In combination with future knowledge about the function of genes and biological systems, comprehensive information from the Neanderthal genome will then allow aspects of Neanderthal biology to be deciphered that are unavailable by any other means. Are fossil and technical resources today sufficient to imagine the determination of a Neanderthal genome sequence? The results pre￾sented here are derived from approximately one fifteenth of an extract Present Time before present Ancestral effective population size Split time into two groups Divergence time for sequence one Divergence time for sequence two Chimpanzee Neanderthal Human a 0 600,000 Most likely Least likely 200,000 400,000 Split time (years) 5,000 10,000 ~3,000 Effective population size of ancestor 0 b 15,000 Figure 6 | Estimate of the effective population size of the ancestor of humans and Neanderthals. a, Schematic illustration of the model used to estimate ancestral effective population size. By split time, we mean the time, in the past, after which there was no more interbreeding between two groups. By divergence, we mean the time, in the past, at which two genetic regions separated and began to accumulate substitutions independently. Effective population size is the number of individuals needed under ideal conditions to produce the amount of observed genetic diversity within a population. b, The likelihood estimates of population split times and ancestral population sizes. The likelihoods are grouped by colour. The red–yellow points are statistically equivalent based on the likelihood ratio test approximation. The black line is the line of best fit to red–yellow points (see Supplementary Methods). This graph is scaled assuming a human–chimpanzee average sequence divergence time of 6,500,000 years. NATURE| Vol 444| 16 November 2006 ARTICLES 335 ©2006 NaturePublishingGroup