The only part of the Neanderthal genome that was sequenced completely from multiple specimens until recently was the mitochondrial DNA (mtDNA). The major analysis that was made with the mtDNA sequence was to seek an answer to the question of interbreeding. The Neanderthal mtDNA sequence consistently falls outside the spectrum of variations observed in the modern human mtDNA sequence, indicating no interbreeding between the species [24-26, 45, 46]. Previously, besides detecting specific mutations in MCPH1 and FOXP2, the candidate gene approach also detected the presence of fragments of the MC1R gene that may indicate the red hair and pale skin of Neanderthals [47], segments of the ABO blood group locus [48], and a taste receptor gene [49]. Despite many critical technical challenges in sequencing ancient genomes ([26]; reviewed in [50]), the whole genome of Neanderthals has been sequenced recently by Green et al. [27], and it allowed this research group to perform a genome-wide sequence comparison with the modern human genome sequence, and the significant results from the initial analyses are discussed briefly below.

Green et al. [27] inferred that 10,535,445 substitutions and 479,863 indels have occurred in the modern human genome after the divergence from chimps. The vast majority of these occurred before Neanderthals and modern humans diverged. However, 78 non-synonymous nucleotide substitutions that are fixed for a derived state in modern humans are different in the Neanderthal counterpart, as Neanderthals carry the ancestral state of these polymorphic nucleotides. Only five genes were identified that have more than one fixed substitution in their coding regions, and one of them has an altered start or stop codon. It is particularly interesting that three of these genes are expressed in skin, including RPTN, which encodes the protein repetin, and TRPMI, which encodes melastatin. This might be indicative of a change in selection of skin physiology in the modern human lineage. When looking into differences in regulatory elements, a total of 132 substitutions and 36 indels were identified in the untranslated regions. One microRNA of unknown function, hsa-mir-1304, which was identified by parallel sequencing of human embryonic stem cells [51], was found to have one fixed substitution and one single nucleotide insertion. Since the substitution occurs in the seed region, it is not unlikely that this microRNA has different targets between these two species.

There are 212 positively selected regions identified in modern humans that occurred early during the history in conjunction with or shortly after their divergence from Neanderthals. The largest of the positively selected regions contains the gene THADA, single-nucleotide polymorphisms (SNPs) in the vicinity of which have been linked with type II diabetes [52]. This change may have affected the energy metabolism of early modern humans. A number of other genes that lie on these selectively swept regions have been associated with other genetic disorders, such as autism, schizophrenia, and Down syndrome. Since both autism and schizophrenia are related to cognitive development, it could be assumed that multiple genes involved in cognitive development in humans were positively selected early in the history of modern humans.

Even though previous studies with Neanderthal mtDNA [26, 33] and initial sequencing of nuclear DNA showed no evidence of interbreeding between Neanderthals and humans [21, 53], the most striking revelation after the comparative analyses between the whole-genome sequence of Neanderthals and multiple modern human individual genomic sequences was the demonstration of admixture between these two species. Green et al. [27] compared the Neanderthal genome with eight modern-day human genomes of European Americans, East Asians, and West African ancestry. Surprisingly, the Neanderthal genome appeared more similar to all non-African genomes than to African ones. They share significantly more derived alleles (alleles that are different from in chimp) with non-African populations than with the African, and when compared with European and Asian individual genomes, Neanderthals are found equally close to both populations. This and some other analyses made by the group only indicate an exchange of DNA between Neanderthals and the non-African population. With further comparative analysis, the same group also identified that gene flow occurred unidirectionally from Neanderthals to the modern non-African human population.

A couple of other findings from different experiments, along with a genome-wide comparison, provide strong evidence for exchange of genetic information. The first finding involves the MAPT locus in chromosome 17, which has two distinct haplotypes, H1 and H2. H1 is abundant in almost all populations of modern humans, while H2 is found only among Europeans and found to have entered into the Homo lineage approximately only 10,000 to 30,000 ya. However, a comparison between H1 and H2 in chimp suggests that the common founder of H1 and H2 is far older than 30,000 years. Even though the Neanderthal genome sequence has been found to carry the H1 haplotype, coinhabitation of the H2 chromosome carriers during the time period when modern humans coexisted with archaic humans can not be ruled out because of the scarcity of archaic genome data. Thus, one can still argue that the H2 haplotype found in modern humans could possibly be a result of horizontal gene transfer between modern humans and Neanderthals and remained in modern humans under selective pressure, possibly because the H1 haplotype has a role in neurodegenerative diseases [54]. In a similar scenario, haplotype D of the microcephalin gene is found to have originated 1.1 mya in a lineage other than modern humans but integrated into the modern human genome only about 37,000 ya. It has thus been speculated that this haplotype was horizontally transferred into modern humans from archaic humans, most likely Neanderthals [34]. However, in a more recent study, the microcephalin locus from a Neanderthal individual in Italy was sequenced and found to be homozygous for the ancestral non-D haplotype [55]. The whole-genome study by Green et al. [27] does not support these speculations, either, since the Neanderthals they analyzed do not carry these D-haplotypes.

One striking revelation from the whole-genome comparison by Green et al. [27] is the equal level of similarity of the Neanderthal genome with Papuan and Chinese and French, although fossil records show the existence of Neanderthals only in Europe and western Asia. The group explained this anomaly by arguing that the interbreeding between the species occurred earlier than previously expected, before the divergence of Europeans, East Asians, and Papuans. Archaeological evidence suggests that modern humans appeared in the Middle East before 100,000 ya, where Neanderthals were already present, and probably remained until 50,000 ya [56]; this makes the prediction by Green et al. [27] probable.

For a more comprehensive comparison of whole-genome sequences between Neanderthals and modern humans, the sequence coverage of Neanderthals has to be increased. The three approaches made so far to sequence the Neanderthal genome have resulted in the sequencing of only 65,000 bases [21], 1 million bases [19], and finally, the draft genome sequenced recently [27], consisting of only two-thirds of the whole genome with a mere 1.3× coverage. With such low coverage, it is hard to form meaningful contigs, and a number of important genetic entities will remain unnoticed. Even though it was beyond imagination to sequence a Pleistocene specimen a decade ago, the progress that has been made in the last 5 years is good enough to expect that more such specimens be sequenced in coming years. The more specimens from various geographic locations that are sequenced, the more likely it will be to construct a reference genome sequence for Neanderthals. As the human genome sequence varies considerably among different populations, it is expected that Neanderthals also have variation in their genomic sequences among different populations from different locations. Such variations can only be identified by sequencing a wide range of specimens, and these variations may again change insights into the Neanderthal-modern human relationship.

The sequencing of Neanderthals first started with its mitochondrial DNA in 1997 [25]. Comparisons between Neanderthal and modern human mitochondrial DNA have been made extensively, but these comparisons only reveal the maternally inherited difference between the species, as mitochondria DNA is transmitted maternally. The complete genomic sequence of Neanderthals published recently is also from a female specimen. Thus, the Y-chromosome of Neanderthals or paternal inheritance has yet to be examined. Comparisons of the Y chromosome sequence of Neanderthals with currently established Y-haplogroups for modern humans should provide some insights into the admixture hypothesis. With respect to the recent finding of admixture of Neanderthals with non-African populations, the Neanderthal Y chromosome should not match the Y haplogroups A or B, as these haplogroups are the oldest of the clades and almost restricted to Africans and their descendants [57, 58]. Since haplogroup E is found in Africa, the Middle East, Southern Europe, and Asia [59-61], the Neanderthal Y chromosome may match this haplogroup, but it should not match the haplogroups E1b1a^*, E2b1, or B2a1a, as they are specifically treated as Bantu expansion markers, while Neanderthals interbred only with non-Africans [62, 63].

Almost half of the human genome comprises retrotransposons. Although they were overlooked for a significant period of time in our genetic study, their importance in chromosome structure, gene regulation, and disease predisposition has now been well established. Retroelements are widely divided into two categories-one with long terminal repeats (LTRs) and another without the LTRs. Short and long interspersed repeat elements (SINEs and LINEs, respectively) are two of the major classes of non-LTR retroelements, while SINEs are the more abundant class. Among SINEs, Alu is the predominant type of retroelement. Among all Alus found in the entire human genome, only about 0.5% is found to be present in the human genome but absent in orthologous regions of other primates and are thus identified as human-specific. This 'young' group of Alus is composed of only about 5,000 Alu elements that are believed to have integrated in the human genome after the divergence of humans and great apes [64-68]. Studying the retrotransposon insertion loci in Neanderthals will identify truly modern human-specific retrotransposon insertion polymorphisms. A similar comparative analysis would reveal other transposable elements, such as L1, SVAs, and HERVs, that are specific to modern humans only, as well as those that are specific to Neanderthals. Retroelements are particularly important in population genetics. It is extremely rare that a newly inserted transposable element is completely excised; thus, they act as a genetic fossil that is homoplasy-free. This identical-by-descent nature of retroelements makes them better markers for population and evolutionary studies than SNPs, in the sense that SNPs can, though rarely, be mutated back to the previous state. SNPs are also very hard to detect while handling ancient genomes due to transformation and deamination [69], while retrotransposon insertion polymorphisms (RIPs) refer to the presence or absence of a retrotransposon. Once a retrotransposon is inserted at a new location in an individual, it is subject to genetic drift. Over a short period, it starts spreading in to the population. Depending on when a retroelement has integrated at a certain locus, it will be shared by different species or, if recently enough, by different populations of the same species. Thus, RIPs occurring before the divergence of chimps and humans are shared by humans and chimps, but those occurring after are present only in humans. RIPs that are even more recent are specific to certain human populations only [70, 71]. For instance, some RIPs are found only in Africans, some in Han Chinese, and so on. The detailed information about all polymorphic retroelements and their frequency in different populations is extensively cataloged in the dbRIP database [43]. The identical-by-descent and homoplasy-free nature of RIPs makes them useful genetic markers in population and evolutionary genetics. The specificity of RIPs can play a significant role in answering the question of admixture of Neanderthals and modern humans. Finding RIPs that are shared between Neanderthals and non-African populations but not present in African populations can be considered solid support for the proposed admixture between Neanderthals and non-African populations. In an ongoing study in our laboratory, over 500 RIPs were identified to be present in Khoisan and Bantu individuals, who represent the oldest lineage of modern humans from Southern Africa, but not in the reference human genome (unpublished). These oldest African lineage-specific RIPs theoretically should also be absent from Neanderthals.

Pseudogenization has always been an interesting topic that has not been explored much. Many pseudogenes have been identified lately that lost their functional capacity in human lineage after the divergence of humans and chimps, particularly related to immunological functions [72]. The "less is more" hypothesis states that gene loss may direct evolutionary changes, as these pseudogenized genes have impacts on adaptation of the species through evolution [73]. Even though pseudogenization does not initiate under selective pressure, the gene loss is retained and subsequently allows adaptation. Pseudogenes are found particularly important in humans, and some of them have even been identified as being responsible for certain human-specific phenotypes. For example, the sarcomeric myosin gene was lost at the time of emergence of the genus Homo and is thought to be responsible for marked reduction of hominin masticatory muscles, leading to expansion of brain size [74]. Analyzing pseudogenes in Neanderthals would be beneficial to identify genes that were lost after the divergence of Neanderthals and modern humans and determine what biological impact they may have.

HACNSs are sequences in the human genome that were conserved throughout vertebrate history but changed significantly after the divergence of humans and chimps. Human genomes have a number of such sequences that obtained surprisingly more mutations after the emergence of humans than before. These regions are typically rich in cis-regulatory transcriptional enhancers that render specific expression pattern of genes involved during development [75-77]. Identification of these cis-regulatory elements in human or other large genomes is done mostly by cross-species sequence comparison, primarily because the functional cis-regulatory elements are generally unique to the genome, which makes paralogy-based identification of such sequences nearly impossible [78]. Since it has long been proposed that phenotypic variation between humans and chimps is mostly brought about by regulatory elements than coding sequences [79], it would be particularly interesting to make an elaborated comparison between differences in conserved regions in modern humans and Neanderthals. Such a comparison between the human genome and initial Neanderthal draft genome sequence [27], involving a total of 2,613 human accelerated regions, revealed that the Neanderthal sequence carried 3,259 human-specific changes in these regions. The comparison revealed that 51 positions in 45 regions were different between these two species; Neanderthals carried the ancestral form while all modern humans carry the human-specific variant. In a recent study, it has been found that the Neanderthal genome retains the ancestral state of a polymorphic site in a conserved noncoding microRNA, which is involved in regulating two genes that are important for teeth formation [80]. This may explain the dental differences between modern humans and Neanderthals. These findings are interesting enough to initiate further studies to analyze the probable impact of the variations in conserved noncoding sequences (CNSs) that exist between these two species. Furthermore, previously in three different studies, the number of HACNSs was found to be between 202 and 1175 [10, 11, 15]. All these studies used different methods to identify CNSs and included more species during the comparison. Their data can also be included in the future for the comparison of CNSs between modern humans and Neanderthals. The most rapidly evolving HACNS identified so far, named HACNS1, has accumulated 16 human-specific changes out of its 546 bases since the divergence of humans and chimps [12]. HACNS1 functions in multiple structures early in the developing stages of the mouse embryo as a transcriptional enhancer, including the developing anterior limb [12]. But, this function is missing for the orthologous enhancers in chimpanzee and rhesus, suggesting that HACNS1 has a different function in humans. When the chimpanzee enhancers are humanized by introducing 13 of the 16 human-specific substitutions, gene expression was observed in the limb, indicating that the substitutions that were identified by comparative analysis were directly responsible for the functional modification. In a recent study, 16 human-specific mutations in HACNS1 were also found in Neanderthals, suggesting that the phenotypic function that is related to this region was also expressed in Neanderthals [81]. The comparison can be extended to other human accelerated CNS regions to identify any substitutions that occurred after the divergence of humans from Neanderthals.

Conserved regions have also been presumed to play important roles in developing certain functions in humans-for instance, noncoding regions of 150 presynaptic genes in humans are highly conserved and may have critical regulatory roles in the expression of these genes [82]. Comparative analyses of these regions between different populations of modern humans and Neanderthals may give directions to a better understanding of neurodevelopmental and psychiatric disorders. Involvement of noncoding sequences in the developing brain was also revealed by another experiment involving 49 human accelerated regions (HARs); the most accelerated region in the human genome, HAR1, is part of a noncoding RNA sequence expressed in the developing brain [83]. A study by Burbano et al. [83] identified that 8.3% of HAR substitutions are not shared between modern humans and Neanderthals, but the study did not focus on HAR1. A complete Neanderthal genomic sequence should provide an understanding of the evolutionary origin of these regulatory RNAs.