Human Population Genomics

Killer Immunoglobulin-like Receptors (KIR) comprise a diverse, highly polymorphic family of cell-surface glycoproteins that are principally expressed by Natural Killer (NK) cells. These innate immune lymphocytes fulfill vital functions in human reproduction and immune responses to viral infection. KIR3DL2 is an inhibitory NK cell receptor that recognizes a common epitope of the HLA-A3 and HLA-A11 class I glycoproteins of the major histocompatibility complex. KIR3DL2 also binds exogenous DNA containing the CpG motif. This interaction causes internalization of the KIR-DNA. Exogenous CpG-DNA typically activates NK cells, but the specificity of KIR3DL2-DNA binding and internalization is unclear. We hypothesized that KIR3DL2 binds exogenous DNA in a sequence-specific manner that differentiates pathogen DNA from self-DNA. In testing this hypothesis, we surveyed octameric CpG-DNA sequences in the human genome, and in reference genomes of all bacteria, fungi, viruses, and parasites, with focus on medically relevant species. Among all pathogens, the nucleotides flanking CpG motifs in the genomes of parasitic worms that infect humans are most divergent from those in the human genome. We cultured KIR3DL2+NKL cells with the commonest CpG-DNA sequences in either human or pathogen genomes. DNA uptake was negatively correlated with the most common CpG-DNA sequences in the human genome. These CpG-DNA sequences induced inhibitory signaling in KIR3DL2+NKL cells. In contrast, KIR3DL2+NKL cells lysed more malignant targets and produced more IFNγ after culture with CpG-DNA sequences prevalent in parasitic worms. By applying functional immunology to evolutionary genomics, we conclude that KIR3DL2 allows NK cells to differentiate self-DNA from pathogen DNA.

Chromosome 21 is the smallest human chromosome and represents a model for physical mapping of the entire human genome. Three copies of this autosome cause Down syndrome, the most frequent genetic disorder associated with significant mental retardation. Five years ago, in the framework of the international human genome project, and as an integrated part of the chromosome 21 community, a consortium, of academic groups from Japan and Germany was formed to map and sequence this chromosome. Several whole-genome and chromosome-specific DNA libraries were constructed for mapping purposes. Using a combination of nested-deletion [1] and shotgun sequencing approaches, our center determined and analyzed over 17 million bases of high-quality data. In total, over 33.5 million basepairs of DNA, distributed over four contigs, were sequenced with very high accuracy [2]. The largest of these contigs is nearly 25.5Mb long. Only three small clone gaps remain, which together comprise approximately 100kb. Thus, we achieved a coverage of 99.7% of 21q. In addition, 211,116 bp from the short arm were also sequenced. Analysis of the chromosome revealed far fewer genes than previously estimated. Instead of 800-1000 expected genes, we found only 225 genes, of which 127 are known genes and 98 are predicted genes. In addition, 59 pseudogenes were identified. The completion of the 21q sequence provides a unique resource for understanding the molecular pathophysiology of Down syndrome, as well as all other monogenic and complex disorders that map to this chromosome, including Alzheimer’s disease, leukemia, autoimmune disease, epilepsy and manic-depressive psychosis. It also stands as a structural framework from which the complete molecular architecture of the chromosome can be determined. With the announcement of the first “working draft” of the human genome by the international human genome sequencing consortium this past June, there has been a great interest in the total number of genes in the human genome[3]. Some of the current estimates range from as low as 34,000 genes to as high as 140,000 genes. The low gene-density of chromosome 21 was quite unexpected. Most striking is a 7-Mb region near the region near the centromere that contains only one gene. This region is much larger than the whole genome of some species, such as Escherichia coli, yet evolutionary processes permitted the existence of such a gene-poor SNA segment. This finding leads us to propose that there are additional large gene-less regions in other human/mammalian chromosomes. By combining the gene numbers of chromosomes 21 and 22 [4] (770genes; 2-3% of human genome) and assuming that together they represent the average gene content of the human genome, we estimate that the total number of human genes may be close to 40,000. As part of the international human genome sequencing project, we are also sequencing parts of chromosomes 11 and 18. Our high-throughput sequence production line generates about 10 Mb per day. In order to handle such a high volume of output, we have developed a system for automated data assembly, annotation and release. All the data is released through our website and through the DNA Database of Japan immediately according to the policies established by the international consortium. In addition to chromosomes 11, 18 and 21, we are also interested in the structure of the entire human genome. In collaboration with the University of Tokyo, we have developed a database called HGREP [5] (Human Genome Reconstruction Project) that contains working draft and finished sequences which cover more than 85% of the human genomic sequences. Sequence entries are aligned along the chromosomes based on sequence similarities to STS markers, BAC-end and other entry sequences. Further, biological features, such as genes, gene functions, repeats and CpG islands, are fully annotated on these entries. To identify evolutionally conserved and biologically important information in the genome, we are taking the strategy of comparative genomic sequence analysis between human and other related genomes such as those of rodents and primates. We are currently sequencing several regions of the mouse, including some regions that are counterpart to human chromosome 21, and are preparing genomic resources for sequencing of the chimpanzee. Through comparative methods, we are also trying to understand telomeric regions, structures at the end of chromosomes that play an important role in several biological functions and have been associated with human diseases like an oncogenesis and a cellular aging. The structure and function of all genes and their regulatory regions can only be fully understood by looking at how they interact under different circumstances and in several different backgrounds. Determining which genes and elements are human-specific will lead to a deeper understanding of who we are and will advance medical sciences drastically.

At the First (1996) and Second (1997) International Strategy Meetings on Human DNA Sequencing, the attendees discussed and strongly endorsed the concept that human genomic DNA sequence produced by large-scale DNA sequencing centers funded by the Human Genome Project should release data as rapidly as possible. In the case of unfinished data, it was suggested that sequence assemblies of 1–2 kb in size should be released within 24 hr of generation. Finished data should be submitted to the public sequence databases on a similarly rapid time scale. Most of the funding agencies engaged in supporting the Human Genome Project have adopted policies that reflect the importance of rapid dissemination of genomic sequence data. Similarly the nucleotide sequence databases have made provision for the acceptance of the unfinished data as well as, of course, finished sequence data. At the 1998 meeting, the question was raised about similar data dissemination practices for genomic sequence data from other organisms. The attendees unanimously agreed that this was also critical and adopted the following statement as individual scientists. ‘‘As extensive determination of the genomic DNA sequence of several organisms proceeds, it is increasingly clear that sequence information has enormous and immediate scientific value, even prior to its final assembly and completion. Delaying the release of either unfinished or finished genomic DNA sequence data serves no useful purpose and actually has the effect of slowing the progress of research. Therefore, the attendees at the Third International Strategy Meeting on Human Genome Sequencing (Bermuda, Feb. 27–28, 1998) agreed unanimously to support, as individual scientists, the view that all publicly funded large-scale DNA sequencing projects, regardless of the organism, should deposit data immediately into the public domain, following the same guidelines that have previously been adopted by this group for human genomic sequence [see D.R. Bentley, Science 274: 533–534 (1996); http:// hugo.gdb.org/bermuda.htm]. The scientists attending this meeting will continue to adhere to these principles and urge all other scientists and policymaking groups involved in large-scale sequencing to adopt them as well.’’ This statement does not yet reflect, nor is it binding on, the policy of any funding agency. The attendees at the Third International Strategy Meeting on Human Genome Sequencing were Mark Adams, David Bentley, Bruce Birren, Martin Bobrow, Elbert Branscomb, Graham Cameron, Anthony V. Carrano, Ellson Chen, Francis Collins, David Cox, Mike Davies, Pieter de Jong, Richard Durbin, Glen Evans, Asao Fujiyama, Richard Gibbs, Eric Green, Phil Green, Mark Guyer, LaDeana Hillier, Ursula Hurtenbach, Elke Jordan, Jill Kent, Fumihiko Kikuchi, Steven Koonin, Jane Lamerdin, Eric Lander, Chris Martin, Richard McCombie, John McPherson, Michael Morgan, Richard Myers, James Ostell, Ari Patrinos, Jane Peterson, Bruce Roe, Jane Rogers, Andre Rosenthal, Yoshiyuki Sakaki, Barbara Skene, Edwin Southern, Jilly Steward, John Sulston, Mark Vaudin, Jeffrey Wadsworth, Robert Waterson, James Watson, Jean Weissenbach, Richard Wilson, and MarieLaure Yaspo.

As the cost of human full genome sequencing continues to fall, we will soon witness a prodigious amount of human genomic data in the public cloud. To protect the confidentiality of the genetic information of individuals, the data has to be encrypted at rest. On the other hand, encryption severely hinders the use of this valuable information, such as Genome-wide Range Query (GRQ), in medical/genomic research. While the problem of secure range query on outsourced encrypted data has been extensively studied, the current schemes are far from practical deployment in terms of efficiency and scalability due to the data volume in human genome sequencing. In this paper, we investigate the problem of secure GRQ over human raw aligned genomic data in a third-party outsourcing model. Our solution contains a novel secure range query scheme based on multi-keyword symmetric searchable encryption (MSSE). The proposed scheme incurs minimal ciphertext expansion and computation overhead. We also present a hierarchical GRQ-oriented secure index structure tailored for efficient and large-scale genomic data lookup in the cloud while preserving the query privacy. Our experiment on real human genomic data shows that a secure GRQ request with range size 100,000 over more than 300 million encrypted short reads takes less than 3 minutes, which is orders of magnitude faster than existing solutions.

The past year has brought unprecedented public attention to biomedical research, with a particularly intense focus on the Human Genome Project and the completion of a first-generation ∼3-billion-basepair human genome sequence. Much of this attention related to the competition between the two parallel, yet separate, efforts of the publicly-funded International Human Genome Sequencing Consortium and the private company Celera Genomics. Despite the apparent rancor between the groups, two celebratory events notably punctuate the past year: the joint media announcement in late June 2000 that both groups had generated a “working draft” sequence of the human genome and the two landmark scientific publications in February 2001 that describe the efforts of each project (International Human Genome Sequencing Consortium 2001; Venter et al. 2001). Numerous grandiose cliches and metaphors have been used to convey the magnitude of these accomplishments and their associated implications for biomedical research and clinical medicine. Here we add one more to this list. Our choice for capturing the essence of contemporary human genome analysis is an analogy to a mountain climbing expedition, one where significant progress has been made to provide a spectacular view of the genetic landscape. But this is not an expedition that is complete, with uncertain— yet exciting—genomic terrain ahead. Indeed, the Human Genome Project is now firmly at the “base camp” of the expedition to elucidate the human genetic blueprint and to begin to understand its content. Nevertheless, this is a milestone of tremendous significance and excitement. Here we outline some of the key lessons learned during the initial analysis of the human genome sequence. We highlight a few of the many remaining questions in understanding the genome’s structure and function, with most answers likely becoming available later in the expedition. Finally, we preview the anticipated climb to the final summit and the ascent to a complete and finished sequence.

Three sets of cosmid clones--containing the HLA-DR alpha chain gene and two additional related genes--were isolated from human genomic DNA libraries by using a cDNA probe for the HLA-DR alpha chain. Southern blot analysis using DNA from somatic cell hybrids indicated that all of the clones mapped to chromosome 6. Partial sequence analysis showed that the two additional related genes were highly homologous to each other, and to the HLA-DR alpha chain, in parts of the exon that encoded the alpha 2 domain but were more divergent in intron sequences. One of the genes corresponds to the HLA-DR-related DC series. DNA probes made from this gene revealed marked restriction enzyme polymorphism when hybridized to genomic DNA from HLA-DR typed homozygous cell lines. The patterns obtained from a number of homozygous and heterozygous cell lines correlated with the HLA-DR crossreactive serotypes and also indicated that there is a further sequence in the haploid human genome that is closely homologous with the DC alpha chain sequence. One family was studied and showed the expected HLA-DR-associated inheritance of restriction enzyme patterns. No polymorphism has yet been demonstrated in restriction enzyme fragments that include the other cloned sequence, which may correspond to the SB alpha chain gene or to a novel HLA-DR-related gene. These experiments indicate that there are at least three sequences in the human genome related, but not identical, to the HLA-DR alpha chain gene.

The incompleteness of partial human mitochondrial genome sequences makes it difficult to perform relevant comparisons among multiple resources. To deal with this issue, we propose a computational framework for deducing missing nucleotides in the human mitochondrial genome. We applied it to worldwide mitochondrial haplogroup lineages and assessed its performance. Our approach can deduce the missing nucleotides with a precision of 0.99 or higher in most human mitochondrial DNA lineages. Furthermore, although low-coverage mitochondrial genome sequences often lead to a blurred relationship in the multidimensional scaling analysis, our approach can correct this positional arrangement according to the corresponding mitochondrial DNA lineages. Therefore, our framework will provide a practical solution to compensate for the lack of genome coverage in partial and fragmented human mitochondrial genome sequences. In this study, we developed an open-source computer program, MitoIMP, implementing our imputation procedure. MitoIMP is freely available from https://github.com/omics-tools/mitoimp.

Although whole human genome sequencing can be done with readily available technical and financial resources, the need for detailed analyses of genomes of certain populations still exists. Here we present, for the first time, sequencing and analysis of a Turkish human genome. We have performed 35x coverage using paired-end sequencing, where over 95% of sequencing reads are mapped to the reference genome covering more than 99% of the bases. The assembly of unmapped reads rendered 11,654 contigs, 2,168 of which did not reveal any homology to known sequences, resulting in ∼1 Mbp of unmapped sequence. Single nucleotide polymorphism (SNP) discovery resulted in 3,537,794 SNP calls with 29,184 SNPs identified in coding regions, where 106 were nonsense and 259 were categorized as having a high-impact effect. The homo/hetero zygosity (1,415,123∶2,122,671 or 1∶1.5) and transition/transversion ratios (2,383,204∶1,154,590 or 2.06∶1) were within expected limits. Of the identified SNPs, 480,396 were potentially novel with 2,925 in coding regions, including 48 nonsense and 95 high-impact SNPs. Functional analysis of novel high-impact SNPs revealed various interaction networks, notably involving hereditary and neurological disorders or diseases. Assembly results indicated 713,640 indels (1∶1.09 insertion/deletion ratio), ranging from −52 bp to 34 bp in length and causing about 180 codon insertion/deletions and 246 frame shifts. Using paired-end- and read-depth-based methods, we discovered 9,109 structural variants and compared our variant findings with other populations. Our results suggest that whole genome sequencing is a valuable tool for understanding variations in the human genome across different populations. Detailed analyses of genomes of diverse origins greatly benefits research in genetics and medicine and should be conducted on a larger scale.

Expression profiling: Opportunities and pitfalls and impact on the study and management of allergic diseases

LINE-1s (Long interspersed elements or L1s) and Alus are highly successful non-long terminal repeat retrotransposons with copy numbers of ~520,000 and >1 million within the human genome, respectively. They are associated with human genetic variation and genomic rearrangement. Although they are abundant throughout primate genomes, their propagation strategy remains poorly understood. The recently released human and chimpanzee draft genome sequences provide the opportunity to compare the human genome with the chimpanzee genome. Thus, we were able to assess how these elements expanded in primate genomes and how they create genomic instability during their integration into the host genome as well as subsequent post-insertion recombination between elements. To understand the expansion of Alu elements, we first analyzed the evolutionary history of the AluYb lineage which is one of most active Alu lineages in the human genome. We suggest that the evolutionary success of Alu elements is driven at least in part by “stealth driver” elements that maintain low retrotransposition activity over extended periods of time and occasionally produce short-lived hyperactive copies responsible for the formation and remarkable expansion of Alu elements within the genome. Second, we conducted a detailed characterization of chimpanzee-specific L1 subfamily diversity. Our results showed that L1 elements have experienced different evolutionary fates in humans and chimpanzees lineages. These differential evolutionary paths may be the result of random variation or the product of competition between L1 subfamily lineages. Third, we report 50 deletion events in human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of human genomic sequence and ~15 kb of chimpanzee genomic sequence. This study provides the basis for developing models of the mechanisms for small and large L1 insertion-mediated deletions. Fourth, we analyzed the magnitude of Alu recombination-mediated deletions in the human lineage subsequent to the human-chimpanzee divergence. We identified 492 human-specific deletions (for a total of ~400 kb of sequence) attributable to this process. The majority of the deletions coincide with known or predicted genes, which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees.

On February 12, 2001, an unprecedented collection of papers describing the initial sequencing and analysis of the human genome was published in Nature and Science (International Human Genome Sequencing Consortium 2001; Venter et al. 2001). Although much celebration and press attention had surrounded the earlier announcement in June 2000 of the coverage of the vast majority of the human genome sequence in working draft form, the publications in February 2001 carried with them the kind of satisfying scientific significance that laborers in the genome fields had longed for—the full description of the methods used to determine the letters of over 90% of the human instruction book, and a host of surprising revelations from the analysis of its contents. This brief essay represents a personal reflection on how we got here, and where we are going. The achievement of these landmarks, coming years ahead of the original schedule, was only possible because of the advances begun early in the preceding decade, reflecting the polyphonic set of interconnected goals that the planners of the Human Genome Project (HGP) wisely included as part of the original master plan. Science traditionally operates by the process of researchers standing on the shoulders of those who came before, and that has certainly been true for the HGP. Building detailed genetic and physical maps, developing better, cheaper, and faster technologies for handling DNA, and mapping and sequencing the more modest-sized genomes of model organisms were all critical stepping stones on the path to initiating the large-scale sequencing of the human genome. Pilot efforts to sequence the human genome began in the mid-1990s. When the International Human Genome Sequencing Consortium met for the first time in Bermuda in 1996, there was a sense of excitement, but the magnitude of the task at hand was sobering– throughput was too low, costs were too high, technology was still immature. Despite that anxiety, the assembled scientific leaders from several countries at that meeting endorsed the importance of high quality sequence, and made one of the most crucial decisions of the genome era–immediate data release. Led by John Sulston and BobWaterston, whohad adopted this same policy for the sequence of C. elegans, the assembled sequencing center directors unanimously adopted a statement that all assembled contigs greater than 1 or 2 kb would be placed in public databases within 24 hours. The argument was simple: The sequence would only benefit the public fully if it could be understood, and that required making it immediately available so that all the creative minds of the planet could work on it. The establishment of this principle was one of the defining moments of the HGP. Over the next three years the rate-limiting steps of large-scale sequencing began to yield to creative innovations. The genome centers implemented major improvements in library production, template preparation, and laboratory information management, so that less and less human intervention was required in the main production pipelines. The advent of capillary sequencing machines from Amersham and ABD provided a much-needed boost in efficiency. Much has also been made of the appearance of a commercial entity on the scene in May 1998 (Celera Genomics) as an additional nudge to the HGP. Whereas it is fair to say that the resulting sense of competition provided an additional incentive to the genome centers, it would be misguided to say that the HGP was previously operating in a relaxed fashion, or that the significant advances in throughput would otherwise not have happened. After all, most of those advances were born of previous accomplishments of the HGP itself. From my perspective, a major turning point occurred in Houston in February 1999. The largest NIHfunded centers (at the Whitehead Institute, Washington University, and Baylor College of Medicine) had just undergone rigorous peer review of their proposals to scale up sequencing throughput and were about to receive a significant increase in funding. The Sanger Centre in Hinxton (UK) and the Joint Genome Institute (Walnut Creek, CA) of the Department of Energy were also scaling up production rapidly. An experiment carried out the preceding summer had documented the high degree of utility of a “working draft” human genome sequence; the half-dozen labs that compared draft and finished sequences found that the draft could answer most of the scientific queries they posed (although it was harder to work with), and suggested that the majority of the HGP’s efforts might well be devoted to obtaining working-draft coverage of the genome as quickly as possible, as long as the commitment to finishing was not diminished. Accordingly, the sequencing plans for the NIH and DOE that were E-MAIL: fc23a@nih.gov; FAX (301) 402-0837. Article and publication are at http://www.genome.org/cgi/doi/10.1101/ gr.1898. Commentary

BackgroundA population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets.ResultsWe developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences.ConclusionsThe described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing.

The hundreds of mitochondrial pseudogenes in the human nuclear genome sequence (numts) constitute an excellent system for studying and dating DNA duplications and insertions. These pseudogenes are associated with many complete mitochondrial genome sequences and through those with a good fossil record. By comparing individual numts with primate and other mammalian mitochondrial genome sequences, we estimate that these numts arose continuously over the last 58 million years. Our pairwise comparisons between numts suggest that most human numts arose from different mitochondrial insertion events and not by DNA duplication within the nuclear genome. The nuclear genome appears to accumulate mtDNA insertions at a rate high enough to predict within-population polymorphism for the presence/absence of many recent mtDNA insertions. Pairwise analysis of numts and their flanking DNA produces an estimate for the DNA duplication rate in humans of 2.2 x 10(-9) per numt per year. Thus, a nucleotide site is about as likely to be involved in a duplication event as it is to change by point substitution. This estimate of the rate of DNA duplication of noncoding DNA is based on sequences that are not in duplication hotspots, and is close to the rate reported for functional genes in other species.

Perioperative genomics

2025 marks the twenty-fifth anniversary of the completion of a working draft of the 3-Gb human genome sequence and its availability in public databases to promote research into human health and disease for the benefit of all. The sequence was produced by the International Human Genome Sequencing Consortium, which comprised sequencing centers from six countries who together undertook the largest collaborative biological project to date. Under the leadership of Sir John Sulston, the United Kingdom played a significant role in the project through the Sanger Centre (now the Wellcome Sanger Institute), which was founded in 1992 with support from the Wellcome Trust, a charitable foundation funding medical research. The Sanger Centre contributed approximately one-third of the final human genome sequence generated by the Human Genome Project and, along with the European Bioinformatics Institute, developed Ensembl, one of the major databases providing free access to genomic data and annotation for biomedical research. As a result of a chance meeting, I came to work at the Sanger Centre (and later the Wellcome Sanger Institute) from 1992 to 2007, initially as a scientific administrator and later as the Human Genome Project manager and head of sequencing. Over that period, the Sanger Centre became one of the largest genome sequencing centers in the world and began its transition to become a world-leading center in genomics research to advance biology and health.