Udo Stenzel

A draft sequence of the Neandertal genome.

Kissing Cousins Neandertals, our closest relatives, ranged across Europe and Southwest Asia before their extinction approximately 30,000 years ago. Green et al. (p. 710) report a draft sequence of the Neandertal genome, created from three individuals, and compare it with genomes of five modern humans. The results suggest that ancient genomes of human relatives can be recovered with acceptably low contamination from modern human DNA. Because ancient DNA can be contaminated with microbial DNA, Burbano et al. (p. 723) developed a target sequence capture approach to obtain 14 kilobases of Neandertal DNA from a fairly poorly preserved sample with a high microbial load. A number of genomic regions and genes were revealed as candidates for positive selection early in modern human history. The genomic data suggest that Neandertals mixed with modern human ancestors some 120,000 years ago, leaving traces of Neandertal DNA in contemporary humans. Gene flow has occurred from Neandertals to humans of Eurasian descent, but not to Africans. Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe and western Asia before disappearing 30,000 years ago. We present a draft sequence of the Neandertal genome composed of more than 4 billion nucleotides from three individuals. Comparisons of the Neandertal genome to the genomes of five present-day humans from different parts of the world identify a number of genomic regions that may have been affected by positive selection in ancestral modern humans, including genes involved in metabolism and in cognitive and skeletal development. We show that Neandertals shared more genetic variants with present-day humans in Eurasia than with present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the ancestors of non-Africans occurred before the divergence of Eurasian groups from each other.

Genetic history of an archaic hominin group from Denisova Cave in Siberia

Using DNA extracted from a finger bone found in Denisova Cave in southern Siberia, we have sequenced the genome of an archaic hominin to about 1.9-fold coverage. This individual is from a group that shares a common origin with Neanderthals. This population was not involved in the putative gene flow from Neanderthals into Eurasians; however, the data suggest that it contributed 4–6% of its genetic material to the genomes of present-day Melanesians. We designate this hominin population ‘Denisovans’ and suggest that it may have been widespread in Asia during the Late Pleistocene epoch. A tooth found in Denisova Cave carries a mitochondrial genome highly similar to that of the finger bone. This tooth shares no derived morphological features with Neanderthals or modern humans, further indicating that Denisovans have an evolutionary history distinct from Neanderthals and modern humans.

A High-Coverage Genome Sequence from an Archaic Denisovan Individual

Ancient Genomics The Denisovans were archaic humans closely related to Neandertals, whose populations overlapped with the ancestors of modern-day humans. Using a single-stranded library preparation method, Meyer et al. (p. 222, published online 30 August) provide a detailed analysis of a high-quality Denisovan genome. The genomic sequence provides evidence for very low rates of heterozygosity in the Denisova, probably not because of recent inbreeding, but instead because of a small population size. The genome sequence also illuminates the relationships between humans and archaics, including Neandertals, and establishes a catalog of genetic changes within the human lineage. A close-up look provides clues to the relationships between modern humans, Denisovans, and Neandertals. We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.

Patterns of damage in genomic DNA sequences from a Neandertal

High-throughput direct sequencing techniques have recently opened the possibility to sequence genomes from Pleistocene organisms. Here we analyze DNA sequences determined from a Neandertal, a mammoth, and a cave bear. We show that purines are overrepresented at positions adjacent to the breaks in the ancient DNA, suggesting that depurination has contributed to its degradation. We furthermore show that substitutions resulting from miscoding cytosine residues are vastly overrepresented in the DNA sequences and drastically clustered in the ends of the molecules, whereas other substitutions are rare. We present a model where the observed substitution patterns are used to estimate the rate of deamination of cytosine residues in single- and double-stranded portions of the DNA, the length of single-stranded ends, and the frequency of nicks. The results suggest that reliable genome sequences can be obtained from Pleistocene organisms.

Targeted retrieval and analysis of five Neandertal mtDNA genomes

Economic Ancient DNA Sequencing Analysis of ancient DNA is often limited by the availability of ancient material for sequencing. Briggs et al. (p. 318; see the news story by Pennisi) describe a method of ancient DNA sequence retrieval that greatly reduces shotgun sequencing costs while avoiding the many difficulties associated with direct PCR-based approaches. They generated five complete and one near-complete Neandertal mitochondrial DNA genomes, which would have been economically impossible with a simple shotgun approach. Analysis of these genomes shows that Neandertal populations had a much smaller effective population size than modern humans or great apes. Targeted sequencing improves Neandertal mitochondrial DNA retrieval and reveals low diversity among individuals. Analysis of Neandertal DNA holds great potential for investigating the population history of this group of hominins, but progress has been limited due to the rarity of samples and damaged state of the DNA. We present a method of targeted ancient DNA sequence retrieval that greatly reduces sample destruction and sequencing demands and use this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. We find that mtDNA genetic diversity in Neandertals that lived 38,000 to 70,000 years ago was approximately one-third of that in contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

A Complete Neandertal Mitochondrial Genome Sequence Determined by High-Throughput Sequencing

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.

Parallel tagged sequencing on the 454 platform

Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplification or gel purification steps. The method relies on attaching sample-specific barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-five 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days.

Improved base calling for the Illumina Genome Analyzer using machine learning strategies

The Illumina Genome Analyzer generates millions of short sequencing reads. We present Ibis (Improved base identification system), an accurate, fast and easy-to-use base caller that significantly reduces the error rate and increases the output of usable reads. Ibis is faster and more robust with respect to chemistry and technology than other publicly available packages. Ibis is freely available under the GPL from http://bioinf.eva.mpg.de/Ibis/.

Targeted high-throughput sequencing of tagged nucleic acid samples

High-throughput 454 DNA sequencing technology allows much faster and more cost-effective sequencing than traditional Sanger sequencing. However, the technology imposes inherent limitations on the number of samples that can be processed in parallel. Here we introduce parallel tagged sequencing (PTS), a simple, inexpensive and flexible barcoding technique that can be used for parallel sequencing any number and type of double-stranded nucleic acid samples. We demonstrate that PTS is particularly powerful for sequencing contiguous DNA fragments such as mtDNA genomes: in theory as many as 250 mammalian mtDNA genomes can be sequenced in a single GS FLX run. PTS dramatically increases the sequencing throughput of samples in parallel and thus fully mobilizes the resources of the 454 technology for targeted sequencing.

Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil–DNA–glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms.