Tomislav Maricic

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.

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.

Multiplexed DNA sequence capture of mitochondrial genomes using PCR products

Background To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length. Methodology/Principal Findings We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GAII instrument. Conclusions/Significance This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.

Targeted Investigation of the Neandertal Genome by Array-Based Sequence Capture.

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. Array capture of Neandertal DNA identifies amino acid substitutions that occurred after the split between humans and Neandertals. It is now possible to perform whole-genome shotgun sequencing as well as capture of specific genomic regions for extinct organisms. However, targeted resequencing of large parts of nuclear genomes has yet to be demonstrated for ancient DNA. Here we show that hybridization capture on microarrays can successfully recover more than a megabase of target regions from Neandertal DNA even in the presence of ~99.8% microbial DNA. Using this approach, we have sequenced ~14,000 protein-coding positions inferred to have changed on the human lineage since the last common ancestor shared with chimpanzees. By generating the sequence of one Neandertal and 50 present-day humans at these positions, we have identified 88 amino acid substitutions that have become fixed in humans since our divergence from the Neandertals.

A complete mtDNA genome of an early modern human from Kostenki, Russia

The recovery of DNA sequences from early modern humans (EMHs) could shed light on their interactions with archaic groups such as Neandertals and their relationships to current human populations. However, such experiments are highly problematic because present-day human DNA frequently contaminates bones [1, 2]. For example, in a recent study of mitochondrial (mt) DNA from Neolithic European skeletons, sequence variants were only taken as authentic if they were absent or rare in the present population, whereas others had to be discounted as possible contamination [3, 4]. This limits analysis to EMH individuals carrying rare sequences and thus yields a biased view of the ancient gene pool. Other approaches of identifying contaminating DNA, such as genotyping all individuals who have come into contact with a sample, restrict analyses to specimens where this is possible [5, 6] and do not exclude all possible sources of contamination. By studying mtDNA in Neandertal remains, where contamination and endogenous DNA can be distinguished by sequence, we show that fragmentation patterns and nucleotide misincorporations can be used to gauge authenticity of ancient DNA sequences. We use these features to determine a complete mtDNA sequence from a approximately 30,000-year-old EMH from the Kostenki 14 site in Russia.

A recent evolutionary change affects a regulatory element in the human FOXP2 gene

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands

To increase the yield of DNA sequence generated by the 454 technology from small amounts of starting DNA, we investigated the efficiency of each step in the 454 library preparation process. We find that the last step, when the single-stranded library is released by NaOH, is inefficient and highly variable. When this step is replaced with heat treatment, library amounts dramatically increase. Furthermore, when sequencing templates are first isolated by NaOH treatment and subsequently by heat treatment, the sequences of both strands of individual template DNA molecules can be determined. Using this approach, we confirm that C/G base pairs observed as T/A base pairs in Neanderthal DNA sequences are due to a modification of the cytosine rather than guanine residues.

deML: Robust demultiplexing of Illumina sequences using a likelihood-based approach

Motivation: Pooling multiple samples increases the efficiency and lowers the cost of DNA sequencing. One approach to multiplexing is to use short DNA indices to uniquely identify each sample. After sequencing, reads must be assigned in silico to the sample of origin, a process referred to as demultiplexing. Demultiplexing software typically identifies the sample of origin using a fixed number of mismatches between the read index and a reference index set. This approach may fail or misassign reads when the sequencing quality of the indices is poor. Results: We introduce deML, a maximum likelihood algorithm that demultiplexes Illumina sequences. deML computes the likelihood of an observed index sequence being derived from a specified sample. A quality score which reflects the probability of the assignment being correct is generated for each read. Using these quality scores, even very problematic datasets can be demultiplexed and an error threshold can be set. Availability and implementation: deML is freely available for use under the GPL (http://bioinf.eva.mpg.de/deml/). Contact: gabriel.reno@gmail.com or kelso@eva.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.