Orchid v2.0.0 Released
The newest version of our paired end assembly viewer for use with Ace files has been released. This version adds the ability to specify read name interpretation, rings and ring locations and library size ranges in a configuration file so no modification of Orchid is needed to customize for your local environment. It also has completely changed drawing routines that allow for infinite zooming for large projects. Download it from the Orchid page.
Widespread Parallel Evolution in Sticklebacks by Repeated Fixation of Ectodysplasin Alleles
Major phenotypic changes evolve in parallel in nature by molecular mechanisms that are largely unknown. In the March 25 Issue of Science (Colosimo PF et al. Science 307, 1928-33) we describe the use of positional cloning methods to identify the major chromosome locus controlling armor plate patterning in wild threespine sticklebacks. Mapping, sequencing, and transgenic studies show that the Ectodysplasin (EDA) signaling pathway plays a key role in evolutionary change in natural populations and that parallel evolution of stickleback low-plated phenotypes at most freshwater locations around the world has occurred by repeated selection of Eda alleles derived from an ancestral low-plated haplotype that first appeared more than two million years ago. Members of this clade of low-plated alleles are present at low frequencies in marine fish, which suggests that standing genetic variation can provide a molecular basis for rapid, parallel evolution of dramatic phenotypic change in nature.
Human Chromosome 16 Sequence Details Extensive Duplication
JGI researchers, in collaboration with scientists from six other institutions, have completed the sequencing and analysis of human chromosome 16. (The sequence and analysis of duplication-rich human chromosome 16) The last of the three chromosomes making up the U.S. Department of Energy's share of the Human Genome Project, chromosome 16 includes genes for metallothionein (a protein involved in regulating and detoxifying heavy metals), cadherin and iroquois gene families (which take part in DNA repair), and sites for diseases such as polycystic kidney disease and acute myelomonocytic leukemia. The work revealed 30 novel genes and 79 putative novel genes, 341 pseudogenes or pseudogene fragments, and some of the most extensive segmental duplication seen in any human chromosome.
Several large structural polymorphisms were found on chromosome 16. These tended to be associated with the many segmental duplications that make up 9.89% of the chromosome's sequence. (The average across the entire genome is 5.3%.) The large structural polymorphisms appear to lead to variations among humans that affect phenotype or disease susceptibility. For example, a 450-kb inversion was found to exist between two haplotypes of one of the most extensively duplicated regions, containing genes for a subunit of eukaryotic translation initiation factor 3 (EIF3S8), sulphotransferase 1A, and Batten disease. Another large polymorphism (360 kb), the human homolog of the hydrocephalus-inducing gene, is a recently duplicated gene sometimes found on chromosome 1. Over all, 91 genes were found in regions of segmental duplication. There appears to have been a recent expansion of duplication on the chromosome. By comparing substitution rates in great apes, the researchers estimated that up to 7% of the chromosome's mass is accounted for by segmental duplications that have arisen in the last 10 million years.
The analysis included comparisons between human chromosome 16 and homologous chimpanzee, dog, mouse, rat, chicken, and fish (Fugu) sequences. Segmental maps were used to analyze homologous relationships across the vertebrates, and fine-scale DNA comparisons were used to identify slowly evolving regions. In comparisons of the density of conserved noncoding regions across vertebrates, the densities for human/mouse/rat and human/mouse/dog/chicken were only slightly higher than the genome-wide average. Surprisingly, the density for human/mouse/fish was about 2.4 times the genome-wide average. Thus, while chromosome 16 has maintained expected levels of noncoding sequence conservation since the split between mammals and birds, it has retained a surprising amount of the more ancient noncoding sequence shared with fish.
In agreement with previous studies showing the association of human/fish conservation with developmental genes, the longest human/mouse/dog/chicken synteny segment contains a 5-Mb subregion on which 59% of the human/mouse/fish noncoding elements are clustered—along with at least six developmental transcription factors. In contrast, the second longest human/mouse/dog/chicken synteny block, nearly equal in length, showed no noncoding conservation between human/mouse/fish. This result suggests that the functions of the shorter sequence are more diverged in distant species than they are in mammals.
The sequence and initial analysis of chromosome 16 should provide a useful foundation for future analytical efforts. Indeed, one such effort, the Encyclopedia of DNA Elements (ENCODE) project, has already selected three sites on chromosome 16 for further study and deep annotation. One of the chosen sites (one of two selected randomly) lies within the gene desert in the second longest human/mouse/dog/chicken synteny segment and contains no genes. Its inclusion bodes well for further understanding of the role of noncoding sequence in human biology.
Coelacanth genome sequence reveals the evolutionary history of vertebrate genes
The coelacanth is one of the nearest living relatives of tetrapods. However, a teleost species such as zebrafish or Fugu is typically used as the outgroup in current tetrapod comparative sequence analyses. Such studies are complicated by the fact that teleost genomes have undergone a whole-genome duplication event, as well as individual gene-duplication events. In the December 14 issue of Genome Research (Noonan JP et al Genome Research 14, 2397-405) we demonstrate the value of coelacanth genome sequence by complete sequencing and analysis of the protocadherin gene cluster of the Indonesian coelacanth, Latimeria menadoensis. The coelacanth was shown to have 49 protocadherin cluster genes organized in the same three ordered subclusters, alpha, beta, and gamma, as the 54 protocadherin cluster genes in human. In contrast, whole-genome and tandem duplications have generated two zebrafish protocadherin clusters comprised of at least 97 genes. Additionally, zebrafish protocadherins are far more prone to homogenizing gene conversion events than coelacanth protocadherins, suggesting that recombination- and duplication-driven plasticity may be a feature of teleost genomes. Our results indicate that coelacanth provides the ideal outgroup sequence against which tetrapod genomes can be measured.
International Human Genome Sequencing Consortium Describes Finished Human Genome Sequence
The International Human Genome Sequencing Consortium, led in the United States by the National Human Genome Research Institute (NHGRI) and the Department of Energy (DOE), today published its scientific description of the finished human genome sequence, reducing the estimated number of human protein-coding genes from 35,000 to only 20,000-25,000, a surprisingly low number for our species.
The paper appears in the Oct. 21 issue of the journal Nature (Finishing the euchromatic sequence of the human genome ). In the paper, researchers describe the final product of the Human Genome Project, which was the 13-year effort to read the information encoded in the human chromosomes that reached its culmination in 2003. The Nature publication provides rigorous scientific evidence that the genome sequence produced by the Human Genome Project has both the high coverage and accuracy needed to perform sensitive analyses, such as focusing on the number of genes, the segmental duplications involved in disease and the "birth" and "death" of genes over the course of evolution.
"Only a decade ago, most scientists thought humans had about 100,000 genes. When we analyzed the working draft of the human genome sequence three years ago, we estimated there were about 30,000 to 35,000 genes, which surprised many. This new analysis reduces that number even further and provides us with the clearest picture yet of our genome," said NHGRI Director Francis S. Collins, M.D., Ph.D. "The availability of the highly accurate human genome sequence in free public databases enables researchers around the world to conduct even more precise studies of our genetic instruction book and how it influences health and disease."
One of the central goals of the effort to analyze the human genome is the identification of all genes, which are generally defined as stretches of DNA that code for particular proteins. According to the new findings, researchers have confirmed the existence of 19,599 protein-coding genes in the human genome and identified another 2,188 DNA segments that are predicted to be protein-coding genes.
"The analysis found that some of the earlier gene models were erroneous due to defects in the unfinished, draft sequence of the human genome," said Jane Rogers, Ph.D., head of sequencing at the Wellcome Trust Sanger Institute in Hinxton, England. "The task of identifying genes remains challenging, but has been greatly assisted by the finished human genome sequence, as well as by the availability of genome sequences from other organisms, better computational models and other improved resources."
The Nature paper also provides the scientific community with a peer-reviewed description of the finishing process, and an assessment of the quality of the finished human genome sequence, which was deposited into public databases in April 2003. The assessment confirms that the finished sequence now covers more than 99 percent of the euchromatic (or gene-containing) portion of the human genome and was sequenced to an accuracy of 99.999 percent, which translates to an error rate of only 1 base per 100,000 base pairs - 10 times more accurate than the original goal.
The contiguity of the sequence is also massively improved. The average DNA letter now sits on a stretch of 38.5 million base pairs of uninterrupted, high-quality sequence - about 475 times longer than the 81,500 base-pair stretch that was available in the working draft. Access to uninterrupted stretches of sequenced DNA can greatly assist researchers hunting for genes and the neighboring DNA sequences that may regulate their activity, dramatically cutting the effort and expense required to find regions of the human genome that may contain small and often rare variants involved in disease.
"Finished" doesn't mean that the human genome sequence is perfect. There still remain 341 gaps in the finished human genome sequence, in contrast to the 150,000 gaps in the working draft announced in June 2000. The technology now available cannot readily close these recalcitrant gaps in the human genome sequence. Closing those gaps will require more research and new technologies, rather than industrial-scale efforts like those employed by the Human Genome Project.
"The human genome sequence far exceeds our expectations in terms of accuracy, completeness and continuity. It reflects the dedication of hundreds of scientists working together toward a common goal - creating a solid foundation for biomedicine in the 21st century," said Eric Lander, Ph.D., director of the Broad Institute of MIT and Harvard in Cambridge, Mass.
In addition to reducing the count of human genes, scientists reported that the improved quality of the finished human genome sequence, compared with earlier drafts, provides a much clearer picture of certain phenomena such as duplication of DNA segments and the birth and death of genes.
Segmental duplications are large, almost identical copies of DNA, which are present in at least two locations in the human genome. A number of human diseases are known to be associated with mutations in segmentally duplicated regions, including Williams syndrome, Charcot-Marie-Tooth and DiGeorge syndrome. "Segmental duplications were almost impossible to study in the draft sequence. Now, through the unstinting efforts of groups around the world, this important and rapidly evolving part of our genome is open for scientific exploration," said Robert H. Waterston, M.D., Ph.D., former director of the Genome Sequencing Center at Washington University in St. Louis and now chair of the Department of Genome Sciences at the University of Washington in Seattle.
Segmental duplications cover 5.3 percent of the human genome, significantly more than in the rat genome, which has about 3 percent, or the mouse genome, which has between 1 and 2 percent. Segmental duplications provide a window into understanding how our genome evolved and is still changing. The high proportion of segmental duplication in the human genome shows our genetic material has undergone rapid functional innovation and structural change during the last 40 million years, presumably contributing to unique characteristics that separate us from our non-human primate ancestors.
The consortium's analysis found the distribution of segmental duplications varies widely across human chromosomes. The Y chromosome is the most extreme case, with segmental duplications occurring along more than 25 percent of its length. Some segmental duplications tend to be clustered near the middle (centromeres) and ends (telomeres) of each chromosome, where, researchers postulate, they may be used by the genome as an evolutionary laboratory for creating genes with new functions.
The accuracy of the finished human genome sequence produced by the Human Genome Project has also given scientists some initial insights into the birth and death of genes in the human genome. Scientists have identified more than 1,000 new genes that arose in the human genome after our divergence with rodents some 75 million years ago. Most of these arose through recent gene duplications and are involved with immune, olfactory and reproductive functions. For example, there are two families of genes recently duplicated in the human genome that encode sets of proteins (pregnancy-specific beta-1 glycoprotein and choriogonadotropin beta proteins) that may be involved in the extended period of pregnancy unique to humans.
Additionally, researchers used the finished human genome to identify and characterize 33 nearly intact genes that have recently acquired one or more mutations, causing them to stop functioning, or "die." Scientists pinpointed these non-functioning genes, referred to as pseudogenes, in the human genome by aligning them with the mouse and rat genomes, in which the corresponding genes have maintained their functionality. Interestingly, researchers determined that 10 of these pseudogenes in the human genome sequence appear to have coded for proteins involved in olfactory reception, which helps to explain why humans have fewer functional olfactory receptors and, consequently, a poorer sense of smell than rodents. The molecular biology of the sense of smell was just recognized by the awarding of a Nobel Prize in Physiology or Medicine to Richard Axel and Linda B. Buck.
Next, the researchers aligned the 33 pseudogenes with the draft sequence of the chimpanzee genome to determine whether they were still functional before Homo sapiens' divergence from great apes about 5 million years ago. The analysis revealed that 27 of the pseudogenes were non-functional in both humans and chimps. However, five of the genes that were inactive in humans were found to be still functional in chimpanzees. "The identification of these pseudogenes and their functional counterparts in chimpanzee provides fertile ground for future research projects," said Richard Gibbs, Ph.D., director of Baylor College of Medicine's Human Genome Sequencing Center in Houston, which currently is sequencing the genome of another non-human primate, the rhesus macaque (Macaca mulatta).
More than 2,800 researchers who took part in the International Human Genome Sequencing Consortium share authorship on today's Nature paper, which expands upon the group's initial analysis published in Feb. 2001. Even more detailed annotations and analyses have already been published for chromosomes 5, 6, 7, 9, 10, 13, 14, 19, 20, 21, 22 and Y. Publications describing the remaining 12 chromosomes are forthcoming.
The finished human genome sequence and its annotations can be accessed through the following public genome browsers: GenBank (www.ncbi.nih.gov/Genbank) at NIH's National Center for Biotechnology Information (NCBI); the UCSC Genome Browser (www.genome.ucsc.edu) at the University of California at Santa Cruz; the Ensembl Genome Browser (www.ensembl.org) at the Wellcome Trust Sanger Institute and the EMBL-European Bioinformatics Institute; the DNA Data Bank of Japan (www.ddbj.nih.ac.jp); and EMBL-Bank (www.ebi.ac.uk/embl/index.html) at the European Molecular Biology Laboratory's Nucleotide Sequence Database.
The International Human Genome Sequencing Consortium includes scientists at 20 institutions located in France, Germany, Japan, China, Great Britain and the United States. The five largest sequencing centers are located at: Baylor College of Medicine; the Broad Institute of MIT and Harvard; DOE's Joint Genome Institute, Walnut Creek, Calif.; Washington University School of Medicine; and the Wellcome Trust Sanger Institute.
Human Chromosome 5 Completed
Four years after publicly revealing the official draft human genetic sequence, researchers have reached the halfway point in dotting the i's and crossing the t's of the genetic sentences describing how to build a human. The newly finalized chromosome 5 is the 12th chromosome polished off, with 12 more to go. As the new sequence reveals, this chromosome is a genetic behemoth containing key disease genes and a wealth of information about how humans evolved.
Chromosome 5 is the second of three chromosomes that the Department of Energy Joint Genome Institute (JGI) has finalized in collaboration with colleagues at the Stanford Human Genome Center (SHGC). The final sequence analysis will be published in the Sept. 16 issue of Nature.
"This extremely accurate sequence will be a powerful tool for scientists trying to understand human disease," said Secretary of Energy Spencer Abraham. "I'm pleased that the Department of Energy, which launched the human genome project in the mid-1980s, could help make this important contribution."
Lawrence Berkeley, Lawrence Livermore and Los Alamos national laboratory scientists and staff comprise the JGI, one of the world's largest and most productive public genome sequencing centers. JGI, in partnership with SHGC, completed the sequencing of three of the human genome's chromosomes--numbers 5, 16 and 19--which together contain some 3,000 genes, including those implicated in forms of kidney disease, prostate and colorectal cancer, leukemia, hypertension, diabetes and atherosclerosis. The chromosome 19 sequence was published in the April 1, 2004, issue of Nature.
"I am confident that the interesting features that we have identified from this sequence information are data that the research community can trust and put to good use," said Richard M. Myers, Professor and Chair of Genetics, who is also the director of the Stanford Human Genome Center.
Chromosome 5, the largest to be completed thus far, is made up of 180.9 million genetic letters--the As, Ts, Gs, and Cs that compose the genetic alphabet. Those letters spell out the chromosome's 923 genes, including 66 genes that are known to be involved in human disease. Another 14 diseases seem to be caused by chromosome 5 genes, but they haven't yet been linked to a specific gene. Other chromosome 5 genes include a cluster that codes for interleukins, molecules that are involved in immune signalling and maturation and are also implicated in asthma.
The spaces between the genes are as important as the genes themselves, said Eddy Rubin, JGI's director. "In addition to disease genes, other important genetic motifs gleaned from vast stretches of noncoding sequence have been found on Chromosome 5. Comparative studies conducted by our scientists of the vast gene deserts, where it was thought there was little of value have shown that these regions, conserved across many mammals, actually have powerful regulatory influence."
These gene-free stretches were previously considered "junk DNA," but in recent years those seemingly barren regions have taken on greater prominence as researchers have learned that they can control the activity of distant genes. Some of the noncoding regions have also stayed remarkably consistent compared with those in mice or fish rather than accumulating mutations over the course of evolution.
"If you have such large human regions that stay conserved over vast evolutionary distances, it strongly supports the idea that they must contain something important," said Jeremy Schmutz, the informatics group leader at SHGC. Any mutation that appeared in those conserved regions was likely to have either killed the animal or made it less able to reproduce, preventing the mutation from making it to the next generation. So far, nobody has shown what role the conserved regions play. "What this says is that we don't know as much about this conserved stuff as we think we do," Schmutz said.
Hidden in the chromosome 5 sequence are clues to how humans evolved after branching away from chimpanzees. On average, the chromosome is more than 99 percent similar between chimpanzees and humans, with the greatest similarity found in genes that cause diseases when mutated.
Despite similarities in the overall sequence, the human and chimpanzee chromosomes compared have some structural differences, including one large section that is flipped backward in humans compared to chimps. Such an inversion makes it impossible for the two chromosomes to pair up when the cell divides to create sperm and eggs. Over time, that incompatibility could have driven a reproductive wedge between the evolving populations.
Moving evolutionarily further away, about one-third of chromosome 5 is similar to a chicken chromosome that determines the chicken's sex, much like the X and Y chromosomes in humans. This finding backs up previous research suggesting that before mammals and birds split 300 million years ago, the sex chromosomes had not yet evolved. After the split, mammals and birds developed their own methods of creating males and females.
One duplicated region on chromosome 5 could eventually help explain how spinal muscular dystrophy is inherited. Researchers had known that deletions in the gene for survival of motor neurons, (SMN) caused the disease, but people with the same deletion can have much more or less severe forms of the disease. It turns out that the region contains many duplications and other rearrangements and varies considerably between people. Schmutz said that, with the sequence for this region in hand, researchers can now study how variations in the number of deletions or repetitions influences the disease severity.
For the chromosome 5 effort at JGI, Susan Lucas led the sequencing and Joel Martin the mapping and analysis efforts. Additional Stanford contributors included Jane Grimwood, the finishing group leader, and Mark Dickson, the production sequence group leader.
Human QA Published
We are pleased to announce the publication of the human genome sequence quality assessment in the journal Nature. The SHGC was funded by the National Human Genome Research Institute to assess the quality of the finished human genome sequence. We assessed, by resequencing and refinishing 35 Mb of sequence, the quality of the underlying sequencing and finishing of the Human Genome Consortium. You can read the full text of the article at Nature.
Gene-rich Human Chromosome 19 Sequence Completed
The United States Department of Energy (DOE) Joint Genome Institute (JGI) and Stanford University report today the completion of the sequencing of human chromosome 19, the most gene-rich of all the human chromosomes. This achievement is described in the April 1, 2004 edition of the journal Nature (The DNA sequence and Biology of Human Chromosome 19)
“Culminating 18 years of research, this partnership exemplifies DOE’s commitment to advancing our understanding of the complex interplay between our human health and the environment,” said Energy Secretary Spencer Abraham, whose agency funded the work through its Office of Science.
Embedded in this sequence information are critical regulatory networks of genes tasked with controlling such functions as repairing DNA damage caused by exposure to radiation and to other environmental pollutants. Studies of DNA-repair genes, initiated at the DOE National Laboratories, are yielding insights into the development of certain cancers, many of which appear to be caused by defects in DNA-repair pathways. Also, new insights are being gleaned about other gene families implicated in detoxifying and excreting chemicals foreign to the body.
“With this high-quality sequence now made freely available to the scientific community, more light will be shed on individual responses to medicines,” Abraham said. “This will enable the development of more sensitive diagnostics for susceptibility to a wide array of important diseases. In time, with this information in hand, physicians will be able to tailor more effective individualized therapeutic strategies.”
Chromosome 19, at 55.8 million bases or letters of genetic code, although representing only about 2% of the human genome, features nearly 1,500 genes. They include genes that code for such diseases as insulin-dependent diabetes, myotonic dystrophy, migraines, and familial hypercholesterolemia (an inherited form of elevated blood cholesterol), which increases the risk of cardiovascular disease. “Beyond the significant revelation that chromosome 19 has more than twice the gene density of the genome-wide average, it also offers a fertile landscape for exploring evolutionary motifs,” said JGI Director Eddy Rubin. “An intriguing picture has emerged regarding conservation and divergence, revealing large blocks of gene conservation with rodents as well as segments of coding and noncoding conservation with more distant species such as the pufferfish, Fugu rubripes, which was also sequenced here at the JGI. While not long ago these noncoding regions were considered nonsense, now they are actually proving to have powerful regulatory influence over the genes that they bracket.”
The DOE originally selected chromosome 19 as a sequencing target because of the agency’s abiding mission of investigating the link between DNA damage from radiation exposure and human cancer. Initial work conducted by Lawrence Livermore National Laboratory in the mid 1990s led to the mapping of multiple DNA-repair genes on chromosome 19. In 1999, the sequencing and finishing projects were transferred to the JGI and the Stanford Human Genome Center, respectively.
“Unlike earlier draft human genome sequences, this version is 500 times better in terms of contiguity and accuracy—which makes a huge difference if you are trying to do biology with that sequence,” said Richard Myers, Director, Stanford Human Genome Center. “It gives you a sense of the chromosome’s topography—one filled with such biologically interesting features as transcription factors, olfactory receptor genes, and zinc finger genes.”
Olfactory receptors represent the largest multigene family in higher organisms. They have evolved in response to the need for animals to recognize millions of odors—both threatening and attractive—in their environment. Transcription factors are proteins that need to be recognized by RNA polymerase in order to initiate the elaboration of nucleotides along the DNA molecule. Zinc finger proteins are chains of amino acids that capture a zinc ion and bind to RNA or DNA and play a critical role in a cell’s life cycle. These proteins regulate the expression of genes as well as nucleic acid recognition, reverse transcription, and virus assembly. Drug development efforts seek to disrupt these zinc finger structures to prevent viruses from functioning.
Chromosome 19, however, was not without its challenges, Myers added. “The sequence was harder to work through than expected. It was highly repetitive, with high GC content. It’s a real tribute to this team that they could get the sequence finished.”
Stanford’s role in the collaboration is the critical one of “finishing” the DNA sequence. The finishing process ensures that the information made available through the public databases is completely contiguous, with all ambiguities resolved. This painstaking process begins with the electronic transmission of draft data sets, some 20 billion bytes per week, and shipping of bacteria culture plates from the JGI’s Production Genomics Facility in Walnut Creek, California, to Stanford.
“To get this level of confidence several iterations of the genome sequence is required, typically at six to eight times coverage,” Myers said. In areas that fail to meet the required quality standard, directed finishing reactions of many different types are performed and the resulting data incorporated back into the draft assembly. Only after rigorous scrutiny, when all data has been extensively reviewed by a human finisher, and all gaps and low-quality areas have been resolved, will the sequence data be posted in the public databases. The quality of the finished chromosome 19 sequence far exceeds the 1 in 10,000 base pair error rate set by the International Human Genome Sequencing Consortium, with the error rate estimated to be much less than 1 in 100,000 base pairs.
“The JGI-Stanford partnership has been integral to the timely and economical completion of chromosome 19,” JGI Director Rubin said. “DOE’s contribution to sequencing the human genome totals some 11%, with chromosome 19 representing the first of the three chromosomes the team has tackled, together with the completion of Chromosomes 16 and 5 in the offing.” The magnitude of the accomplishment is further reflected in the nearly one hundred authors cited on the paper led by Jane Grimwood at Stanford and Susan Lucas at the JGI. Other authors include investigators at Lawrence Livermore and Los Alamos National Laboratories; University of California, Santa Cruz,;Children’s Hospital Oakland, California; the Howard Hughes Medical Institute at the University of Washington, Seattle; Case Western Reserve University; and the National Cancer Institute.
National DNA Day: April 30, 2004
National DNA Day commemorates the completion of the Human Genome Project in April 2003 and the discovery of DNA's double helix a half century ago. The day serves to recognize the sequencing of the human genome as one of the most significant scientific accomplishments of the past one hundred years and to inspire the next generation of scientists who will use the human genome sequence to benefit human health.
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