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New therapeutic targets for oral squamous cell carcinoma (OSCC) are urgently needed. We conducted genome-wide CRISPR-Cas9 screens in 21 OSCC cell lines, primarily derived from Asians, to identify genetic vulnerabilities that can be explored as therapeutic targets. We identify known and novel fitness genes and demonstrate that many previously identified OSCC-related cancer genes are non-essential and could have limited therapeutic value, while other fitness genes warrant further investigation for their potential as therapeutic targets. We validate a distinctive dependency on YAP1 and WWTR1 of the Hippo pathway, where the lost-of-fitness effect of one paralog can be compensated only in a subset of lines. We also discover that OSCCs with WWTR1 dependency signature are significantly associated with biomarkers of favorable response toward immunotherapy. In summary, we have delineated the genetic vulnerabilities of OSCC, enabling the prioritization of therapeutic targets for further exploration, including the targeting of YAP1 and WWTR1., eLife digest Many types of cancer now have 'targeted treatments', which specifically home in on genes cancer cells rely on for survival. But there are very few of these treatments available for the most common type of mouth cancer, oral squamous cell carcinoma, which around 350,000 people are diagnosed with each year. Designing targeted treatments relies on detailed knowledge of the genetic makeup of the cancer cells. But, little is known about which genes drive oral squamous cell carcinoma, especially among patients living in Asia, which is where over half of yearly cases are diagnosed. One way to resolve this is to use gene editing technology to find the genes that the cancer cells need to survive. Now, Chai et al. have used a gene editing tool known as CRISPR to examine 21 cell lines from patients diagnosed with oral squamous cell carcinoma. Most of these lines were from Asian patients, some of whom had a history of chewing betel quid which increases the risk of mouth cancer. By individually inactivating genes in these cell lines one by one, Chai et al. were able to identify 918 genes linked to the survival of the cancer cells. Some of these genes have already been associated with the spread of other types of cancer, whereas others are completely unique to oral squamous cell carcinoma. The screen also discovered that some cell lines could not survive without genes involved in a signalling pathway called Hippo, which is known to contribute to the progression of many other types of cancer. Uncovering the genes associated with oral squamous cell carcinoma opens the way for the development of new targeted treatments. Targeted therapies already exist for some of the genes identified in this study, and it may be possible to repurpose them as a treatment for this widespread mouth cancer. But, given that different cell lines relied on different genes to survive, the next step will be to identify which genes to inactivate in each patient.

In vitro cancer cell cultures are facile experimental models used widely for research and drug development. Many cancer cell lines are available and efforts are ongoing to derive new models representing the histopathological and molecular diversity of tumours. Cell models have been generated by multiple laboratories over decades and consequently their annotation is incomplete and inconsistent. Furthermore, the relationships between many patient-matched and derivative cell lines have been lost, and accessing information and datasets is time-consuming and difficult. Here, we describe the Cell Model Passports database; cellmodelpassports.sanger.ac.uk, which provides details of cell model relationships, patient and clinical information, as well as access to associated genetic and functional datasets. The Passports database currently contains curated details and standardized annotation for >1200 cell models, including cancer organoid cultures. The Passports will be updated with newly derived cell models and datasets as they are generated. Users can navigate the database via tissue, cancer-type, genetic feature and data availability to select a model most suitable for specific applications. A flexible REST-API provides programmatic data access and exploration. The Cell Model Passports are a valuable tool enabling access to high-dimensional genomic and phenotypic cancer cell model datasets empowering diverse research applications.

In vitro cancer cell cultures are facile experimental models used widely for research and drug development. Many cancer cell lines are available and efforts are ongoing to derive new models representing the histopathological and molecular diversity of tumours. Cell models have been generated by multiple laboratories over decades and consequently their annotation is incomplete and inconsistent. Furthermore, the relationships between many patient-matched and derivative cell lines have been lost, and accessing information and datasets is time-consuming and difficult. Here, we describe the Cell Model Passports database (cellmodelpassports.sanger.ac.uk) which provides details of cell model relationships, patient and clinical information, as well as access to associated genetic and functional datasets. The Passports database currently contains curated details and standardized annotation for >1600 cell models, including cancer organoid cultures. The Passports will be updated with newly derived cell models and datasets as they are generated. Users can navigate the database via tissue, cancer-type, genetic feature and data availability to select a model most suitable for specific applications. A flexible REST-API provides programmatic data access and exploration. The Cell Model Passports are a valuable tool enabling access to high-dimensional genomic and phenotypic cancer cell model datasets empowering diverse research applications. Citation Format: Dieudonne van der Meer, Syd Barthorpe, Wanjuan Yang, Howard Lightfoot, Caitlin Hall, James Gilbert, Hayley Francies, Mathew Garnett. Cell Model Passports—a hub for clinical, genetic and functional datasets of preclinical cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B069. doi:10.1158/1535-7163.TARG-19-B069

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like−/− mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development. Medical Research Council and the Wellcome Trust

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model., The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 249894 (SelSweep)

The Vertebrate Genome Annotation (Vega) database (http://vega.sanger.ac.uk) has been designed to be a community resource for browsing manual annotation of finished sequences from a variety of vertebrate genomes. Its core database is based on an Ensembl-style schema, extended to incorporate curation-specific metadata. In collaboration with the genome sequencing centres, Vega attempts to present consistent high-quality annotation of the published human chromosome sequences. In addition, it is also possible to view various finished regions from other vertebrates, including mouse and zebrafish. Vega displays only manually annotated gene structures built using transcriptional evidence, which can be examined in the browser. Attempts have been made to standardize the annotation procedure across each vertebrate genome, which should aid comparative analysis of orthologues across the different finished regions.

We describe the generation and analysis of an integrated sequence map of a 2.4-Mb region of pig chromosome 7, comprising the classical class I region, the extended and classical class II regions, and the class III region of the major histocompatibility complex (MHC), also known as swine leukocyte antigen (SLA) complex. We have identified and manually annotated 151 loci, of which 121 are known genes (predicted to be functional), 18 are pseudogenes, 8 are novel CDS loci, 3 are novel transcripts, and 1 is a putative gene. Nearly all of these loci have homologues in other mammalian genomes but orthologues could be identified with confidence for only 123 genes. The 28 genes (including all the SLA class I genes) for which unambiguous orthology to genes within the human reference MHC could not be established are of particular interest with respect to porcine-specific MHC function and evolution. We have compared the porcine MHC to other mammalian MHC regions and identified the differences between them. In comparison to the human MHC, the main differences include the absence of HLA-A and other class I-like loci, the absence of HLA-DP-like loci, and the separation of the extended and classical class II regions from the rest of the MHC by insertion of the centromere. We show that the centromere insertion has occurred within a cluster of BTNL genes located at the boundary of the class II and III regions, which might have resulted in the loss of an orthologue to human C6orf10 from this region.

We have generated an improved assembly and gene annotation of the pig X chromosome, and a first draft assembly of the pig Y chromosome, by sequencing BAC and fosmid clones, and incorporating information from optical mapping and fibre-FISH. The X chromosome carries 1,014 annotated genes, 689 of which are protein-coding. Gene order closely matches that found in Primates (including humans) and Carnivores (including cats and dogs), which is inferred to be ancestral. Nevertheless, several protein-coding genes present on the human X chromosome were absent from the pig (e.g. the cancer/testis antigen family) or inactive (e.g. AWAT1), and 38 pig-specific X-chromosomal genes were annotated, 22 of which were olfactory receptors. The pig Y chromosome assembly focussed on two clusters of male-specific low-copy number genes, separated by an ampliconic region including the HSFY gene family, which together make up most of the short arm. Both clusters contain palindromes with high sequence identity, presumably maintained by gene conversion. The long arm of the chromosome is almost entirely repetitive, containing previously characterised sequences. Many of the ancestral X-related genes previously reported in at least one mammalian Y chromosome are represented either as active genes or partial sequences. This sequencing project has allowed us to identify genes - both single copy and amplified - on the pig Y, to compare the pig X and Y chromosomes for homologous sequences, and thereby to reveal mechanisms underlying pig X and Y chromosome evolution.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

The Del(13)Svea36H mutation (referred to hereafter as Del36H) is a microscopically visible deletion of ∼20% of mouse chromosome 13 (Arkell et al. 2001). Mice that are heterozygous for Del36H display a phenotype that varies with genetic background and that can involve reduced size, craniofacial malformation, eyes open at birth, and a mild tail kink. These mice may model some aspects of human genetic disease, because the Del36H region shows conserved synteny with regions of human chromosome 6p22.1-6p22.3 and 6p25 that are lost in some deletion syndromes (Davies et al. 1999). Furthermore, several disease loci map to this region in humans: two eye defects (iridogoniodysgenesis and Axenfeld-Rieger anomaly; Mears et al. 1998; Nishimura et al. 1998), haemochromatosis (Feder et al. 1996), dyslexia (Grigorenko et al. 2003), and schizophrenia susceptibility (Straub et al. 2002). Mice with interstitial chromosome deletions like Del36H are potent experimental tools for functional genomics. In particular, they can be used to reveal recessive phenotypes due to mutations that map to a specific chromosomal region. However, the positional candidate approach to identifying mutations in genes underlying mutant phenotypes remains nontrivial, especially for point mutations such as those induced by ENU (Brown and Hardisty 2003). A prerequisite for effective mutation detection using this approach is a comprehensive gene list, with exhaustive annotation of exons and regulatory elements. A limited catalog of the genes deleted in Del36H can be found in genetic and radiation hybrid maps (Arkell et al. 2001; Avner et al. 2001; Hudson et al. 2001), and an automatically annotated genomic sequence is available (Waterston et al. 2002), but the current public mouse genome assembly is a mixture of draft and finished sequence and, by definition, draft genomic sequence contains gaps and regions of lower sequence quality. These artefacts can influence gene annotation and, therefore, the subsequent design of mutation detection assays. Manual annotation, in contrast, should provide a gold standard reference set. As well as being an invaluable resource for functional genomics, a large genomic region of this kind provides an opportunity to investigate the organization and evolution of a significant piece of the mouse genome. Such studies also rely on high-quality sequence and manual gene annotation to avoid errors in sequence alignment, identification of coding and pseudogenes, classification of repetitive elements, and so on. The accumulating information on genome sequences from a number of species raises many questions about genome evolution. Important among these are the relative roles of whole-genome, segmental, and individual gene duplication, and the mechanisms underlying these processes (Lynch and Conery 2000; Dehal et al. 2001; Eichler and Sankoff 2003; Friedman and Hughes 2004); the usefulness of inter-genome comparisons for identifying selectively conserved regions in genomes, including not only genes, but regulatory regions and functional RNA genes (Mallon et al. 2000; Dehal et al. 2001; Dermitzakis et al. 2002; Kondrashov and Shabalina 2002; Margulies et al. 2003; Frazer et al. 2004); the roles of repeated (transposable element-like) and repetitive (satellites, microsatellites, and minisatellites) sequences in genome evolution (Toth et al. 2000; Hancock 2002; Babcock et al. 2003; Alba and Guigo 2004; Han et al. 2004; Kazazian Jr. 2004); and the characteristics of sites of evolutionary chromosome breakpoints (Puttagunta et al. 2000; Dehal et al. 2001; Pevzner and Tesler 2003). Here, we describe the genomic architecture of Del36H based on 12.66 Mb of finished DNA sequence, annotated using a combination of manual annotation with synteny and comparative sequence analysis. We find that the region is gene rich, primarily as the result of high gene densities in regions containing gene families that are smaller or absent in the orthologous human regions, and which appear to contribute to the special requirements of the lifestyle of the mouse. We consider forces and processes that may have contributed to the expansion of these gene families during evolution. We also identify a segment of Del36H containing two nearby evolutionary breakpoints, and show that these lie in a gene desert, a potentially optimal site for chromosome breakage. Finally, we consider the evolutionary dynamics of Evolutionarily Conserved Regions (ECRs; Mallon et al. 2000) within Del36H and their potential application to the identification of regulatory, and potentially other functional sequences within noncoding regions of the mouse genome.

Chromosome 9 is highly structurally polymorphic. It contains the largest autosomal block of heterochromatin, which is heteromorphic in 6–8% of humans, whereas pericentric inversions occur in more than 1% of the population. The finished euchromatic sequence of chromosome 9 comprises 109,044,351 base pairs and represents >99.6% of the region. Analysis of the sequence reveals many intra- and interchromosomal duplications, including segmental duplications adjacent to both the centromere and the large heterochromatic block. We have annotated 1,149 genes, including genes implicated in male-to-female sex reversal, cancer and neurodegenerative disease, and 426 pseudogenes. The chromosome contains the largest interferon gene cluster in the human genome. There is also a region of exceptionally high gene and G + C content including genes paralogous to those in the major histocompatibility complex. We have also detected recently duplicated genes that exhibit different rates of sequence divergence, presumably reflecting natural selection.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of human, mouse and other genome sequences, available as either an interactive web site or as flat files. Ensembl also integrates manually annotated gene structures from external sources where available. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. These range from sequence analysis to data storage and visualisation and installations exist around the world in both companies and at academic sites. With both human and mouse genome sequences available and more vertebrate sequences to follow, many of the recent developments in Ensembl have focusing on developing automatic comparative genome analysis and visualisation.

The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hematopoiesis and vasculogenesis and a pattern of expression that is highly conserved between mammals and zebrafish. Here we report the isolation and characterization of the zebrafish SCL locus together with the identification of three neighboring genes, IER5,MAP17, and MUPP1. This region spans 68 kb and comprises the longest zebrafish genomic sequence currently available for comparison with mammalian, chicken, and pufferfish sequences. Our data show conserved synteny between zebrafish and mammalian SCL and MAP17 loci, thus suggesting the likely genomic domain necessary for the conserved pattern ofSCL expression. Long-range comparative sequence analysis/phylogenetic footprinting was used to identify noncoding conserved sequences representing candidate transcriptional regulatory elements. The SCL promoter/enhancer, exon 1, and the poly(A) region were highly conserved, but no homology to other known mouseSCL enhancers was detected in the zebrafish sequence. A combined homology/structure analysis of the poly(A) region predicted consistent structural features, suggesting a conserved functional role in mRNA regulation. Analysis of the SCL promoter/enhancer revealed five motifs, which were conserved from zebrafish to mammals, and each of which is essential for the appropriate pattern or level ofSCL transcription.[The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N. Tanese.]

INTRODUCTION Autoimmune hemolytic anemia (AIHA) is a rare autoimmune disorder in which auto-antibodies target red blood cell surface antigens, causing hemolysis. The incidence is estimated to be 0.8 per 100,000 (Lechner and Jager, Blood 2010). Depending on the temperature at which the auto-antibodies are most active, AIHA is classified as warm, cold, or mixed. Main risk factors include malignancy, viral infection, and rheumatologic disorders. Thromboembolism is an important complication of AIHA that has received increasing attention in case series and small observational reports. However, there has not yet been a study that compares the risk of thromboembolism in AIHA with that of matched, non-AIHA patients in a longitudinal fashion. OBJECTIVES 1) To assess the risk of arterial and venous thromboembolism in AIHA patients using a longitudinal, retrospective cohort study. 2) To define the contribution from usual thrombosis risk factors (defined in Methods section) in the development of thromboembolism in AIHA patients. METHODS We derived our cohorts from Stanford University's Standards-Based Translational Research Informatics Platform (STRIDE). The STRIDE database houses records since 2003 for over 2.1 million patients who receive their care at Stanford Hospital and Clinics. We identified 156 patients diagnosed with AIHA of any type and matched them with 312 non-AIHA patients (control) in a 1:2 ratio. To achieve stringent matching, patients in the control group were selected to have known risk factors for AIHA--malignancy, viral infections, and rheumatologic diseases--without developing AIHA itself. We assessed the incidence of arterial and venous thromboembolism in the AIHA and non-AIHA groups. Within each group, we assessed the association between thromboembolism and the presence of thrombosis risk factors, which we based on the PADUA criteria (Barbar et al, J Throm Haemost 2010). The PADUA risk factors comprise a weighted sum known as the PADUA score (max score of 20), and we compared the median PADUA score between AIHA and non-AIHA patients with thromboembolism using the Mann-Whitney rank sum test. Interquartile ranges (IQR) of PADUA scores were calculated. Finally, using inverse-probability weighting to achieve matching thromboembolism propensity scores between AIHA and non-AIHA patients, we derived an odds ratio for the development of thromboembolism given a diagnosis of AIHA. RESULTS A significantly higher proportion of AIHA patients developed arterial and venous thromboembolism than non-AIHA patients (29% vs. 19%, respectively; p < 0.05). Notably, the median PADUA score was not different between AIHA and non-AIHA patients with thromboembolism (4, IQR [2-7] vs 4.5, IQR [3-7], respectively, n.s.), despite the aforementioned difference in thromboembolism incidence. However, the distribution of PADUA risk factors in each group did differ: malignancy was seen in a smaller proportion of AIHA patients with thromboembolism than in non-AIHA counterparts (31% vs 57%, respectively; p < 0.05), while acute infection and/or rheumatologic disorders was seen in a larger proportion of AIHA patients with thromboembolism than non-AIHA counterparts (53% vs 25%, respectively; p < 0.05; see Table 1). After additional analysis to ensure propensity score matching, we found that AIHA confers an odds ratio of 2.44 (95% CI [1.16-5.10], p < 0.05) for the development of thromboembolism. CONCLUSION Different thrombosis risk factors contribute to the development of thromboembolism in AIHA patients than in non-AIHA patients. However, AIHA patients carry a significantly higher risk of thromboembolism than non-AIHA patients, and this risk is not attributable to the usual thrombosis risk factors considered in the PADUA criteria. Our finding suggests a need for clinical trials to study the role of thrombo-prevention in AIHA patients. Table 1 Percentage of PADUA risk factors in AIHA and non-AIHA patients with thromboembolism. Table 1. Percentage of PADUA risk factors in AIHA and non-AIHA patients with thromboembolism. Disclosures Chen: True North Therapeutics: Research Funding. Loftus:True North Therapeutics: Research Funding. Weber:True North Therapeutics: Research Funding. Hoang:True North Therapeutics: Research Funding. Gilbert:True North Therapeutics: Employment. Kummar:True North Therapeutics: Research Funding.

The Consensus Coding Sequence (CCDS) project (http://www.ncbi.nlm.nih.gov/CCDS/) is a collaborative effort to maintain a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assemblies by the National Center for Biotechnology Information (NCBI) and Ensembl genome annotation pipelines. Identical annotations that pass quality assurance tests are tracked with a stable identifier (CCDS ID). Members of the collaboration, who are from NCBI, the Wellcome Trust Sanger Institute and the University of California Santa Cruz, provide coordinated and continuous review of the dataset to ensure high-quality CCDS representations. We describe here the current status and recent growth in the CCDS dataset, as well as recent changes to the CCDS web and FTP sites. These changes include more explicit reporting about the NCBI and Ensembl annotation releases being compared, new search and display options, the addition of biologically descriptive information and our approach to representing genes for which support evidence is incomplete. We also present a summary of recent and future curation targets.

Acquired deletions of the long arm of chromosome 20 are the most common chromosomal abnormality seen in polycythemia vera and are also associated with other myeloid malignancies. Such deletions are believed to mark the site of one or more tumor suppressor genes, loss of which perturbs normal hematopoiesis. A common deleted region (CDR) has previously been identified on 20q. We have now constructed the most detailed physical map of this region to date—a YAC contig that encompasses the entire CDR and spans 23 cM (11 Mb). This contig contains 140 DNA markers and 65 unique expressed sequences. Our data represent a first step toward a complete transcriptional map of the CDR. The high marker density within the physical map permitted two complementary approaches to reducing the size of the CDR. Microsatellite PCR refined the centromeric boundary of the CDR to D20S465 and was used to search for homozygous deletions in 28 patients using 32 markers. No such deletions were detected. Genetic changes on the remaining chromosome 20 may therefore be too small to be detected or may occur in a subpopulation of cells.

The SCL/TAL-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor essential for the development of all hemopoietic lineages and also acts as a T-cell oncogene. Four related genes have been described in mammals (LYL-1, TAL-2, NSCL1, and NSCL2), all of which exhibit a high degree of sequence similarity to SCL/TAL-1 in the bHLH domain and two of which (LYL-1 and TAL-2) have also been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia. In this study we describe the identification and characterization of a pufferfish gene termed SLP-1, which represents a new member of this gene family. The genomic structure and sequence of SLP-1 suggests that it forms a subfamily with SCL/TAL-1 and LYL-1 and is most closely related to SCL/TAL-1. However, unlike SCL/TAL-1, SLP-1 is widely expressed. Sequence analysis of a whole cosmid containing SLP-1 shows that SLP-1 is flanked upstream by a zinc finger gene and a fork-head-domain gene and downstream by a heme-oxygenase and a RING finger gene.

Studies of X chromosome inactivation patterns are central to many aspects of our understanding of the pathogenesis of haematological malignancies. In patients with myeloproliferative disorders and myelodysplastic syndromes the demonstration of skewed X inactivation patterns in multiple haemopoietic lineages has been taken to indicate a stem cell origin for these groups of diseases. However, stem cell depletion or selection pressures can also produce skewed X inactivation patterns and might increase with age. We have therefore used the HUMARA assay to study X inactivation patterns of elderly patients with myeloproliferative disorders together with an age-matched control group of normal elderly women. A clonal pattern (clonal granulocytes and polyclonal T cells) was observed in 23.1% of normal women and 63.4% of patients with myeloproliferative disorders. This is the first report of X inactivation patterns in purified subpopulations of blood cells in normal elderly women. These results have three significant implications. Firstly, the finding of clonal granulocytes and polyclonal T cells in normal elderly women is likely to reflect age-related stem cell depletion or selection pressures. Secondly, the demonstration of clonal granulocytes and polyclonal T cells is not a useful diagnostic marker for myeloproliferative disorders or myelodysplastic syndromes in elderly women. Thirdly, our data raise the possibility that clonal blood cell patterns may precede rather than follow mutations which subsequently give rise to myelodysplastic or myeloproliferative phenotypes.

Background The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. Results The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. Conclusions This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

Model organisms are becoming increasingly important for the study of complex diseases such as type 1 diabetes (T1D). The non-obese diabetic (NOD) mouse is an experimental model for T1D having been bred to develop the disease spontaneously in a process that is similar to humans. Genetic analysis of the NOD mouse has identified around 50 disease loci, which have the nomenclature Idd for insulin-dependent diabetes, distributed across at least 11 different chromosomes. In total, 21 Idd regions across 6 chromosomes, that are major contributors to T1D susceptibility or resistance, were selected for finished sequencing and annotation at the Wellcome Trust Sanger Institute. Here we describe the generation of 40.4 mega base-pairs of finished sequence from 289 bacterial artificial chromosomes for the NOD mouse. Manual annotation has identified 738 genes in the diabetes sensitive NOD mouse and 765 genes in homologous regions of the diabetes resistant C57BL/6J reference mouse across 19 candidate Idd regions. This has allowed us to call variation consequences between homologous exonic sequences for all annotated regions in the two mouse strains. We demonstrate the importance of this resource further by illustrating the technical difficulties that regions of inter-strain structural variation between the NOD mouse and the C57BL/6J reference mouse can cause for current next generation sequencing and assembly techniques. Furthermore, we have established that the variation rate in the Idd regions is 2.3 times higher than the mean found for the whole genome assembly for the NOD/ShiLtJ genome, which we suggest reflects the fact that positive selection for functional variation in immune genes is beneficial in regard to host defence. In summary, we provide an important resource, which aids the analysis of potential causative genes involved in T1D susceptibility. Database URLs: http://www.sanger.ac.uk/resources/mouse/nod/; http://vega-previous.sanger.ac.uk/info/data/mouse_regions.html#Idd

An acquired deletion of the long arm of chromosome 20 is a recurrent abnormality in myeloproliferative disorders, particularly polycythemia vera and myelodysplastic syndromes. The association of 20q deletions with myeloid “stem cell” disorders suggests that the deletions mark the site of one or more genes, loss or inactivation of which plays a role in the regulation of normal hematopoietic progenitors. We have recently performed a detailed molecular analysis of 20q deletions in peripheral blood (PB) granulocytes and defined a commonly deleted region of 16 to 21 centimorgan (cM). To further reduce the size of the common deleted region we have searched for small deletions or mitotic recombination events, neither of which would be detected by conventional cytogenetics. We have studied 48 patients with polycythemia vera and four patients with idiopathic myelofibrosis. In each case, cytogenetic analysis had either failed or had shown no abnormalities of chromosome 20. Seventeen microsatellite markers that span the common deleted region were used to search for loss of heterozygosity in granulocyte DNA. No instance of microsatellite instability was observed in a total of 880 comparisons of granulocyte and T-cell DNA. Granulocyte DNA from four patients exhibited allele loss on 20q. In each case the allele loss was caused by an interstitial deletion because heterozygosity at distal markers was retained and because quantitative Southern blotting demonstrated hemizygosity. Loss of heterozygosity in PB granulocytes would be masked by the presence of significant numbers of normal granulocytes not derived from the malignant clone. Therefore, the human androgen receptor assay (HUMARA) was used to determine granulocyte clonality. In 21 of 27 informative female patients the majority of the granulocytes were clonally derived. In 5 patients the granulocytes appeared polyclonal and in 1 patient unilateral X inactivation was observed in both granulocytes and T cells. These results show that, in the vast majority of patients presented here, the failure to detect loss of heterozygosity cannot be attributed to the presence of normal polyclonal granulocytes. Our results therefore show that allele loss on chromosome 20q in polycythemia vera does not commonly involve mitotic recombination or chromosome loss and that microsatellite instability is a rare event in this disorder.

Manual annotation of genomic data is extremely valuable to produce an accurate reference gene set but is expensive compared with automatic methods and so has been limited to model organisms. Annotation tools that have been developed at the Wellcome Trust Sanger Institute (WTSI, http://www.sanger.ac.uk/.) are being used to fill that gap, as they can be used remotely and so open up viable community annotation collaborations. We introduce the ‘Blessed’ annotator and ‘Gatekeeper’ approach to Community Annotation using the Otterlace/ZMap genome annotation tool. We also describe the strategies adopted for annotation consistency, quality control and viewing of the annotation. Database URL: http://vega.sanger.ac.uk/index.html

The Human and Vertebrate Analysis and Annotation (HAVANA) group at the Wellcome Trust Sanger Institute produced the manually annotated geneset for the Encyclopedia of DNA Elements (ENCODE) pilot project and, as part of the Gencode subgroup, are reprising this role in the scale up to cover the whole human genome. Our manual annotation is checked computationally and validated experimentally. Loci and transcripts predicted to be absent from the initial annotation are identified by comparison with a number of state-of-the-art algorithms for identifying exons, splice sites, transcripts and pseudogenes. Where novel features are confirmed the annotation is updated. Annotated coding transcripts are analysed to assess their coding potential by investigating patterns of conservation within the coding sequence (CDS) and comparing predicted secondary structures of annotated CDSs to similar proteins with solved structures. Annotated coding transcripts are also checked against the current set of human Consensus CDSs (CCDS) to check agreement with other participating centres (EBI, NCBI, & UCSC).An initial round of annotation and analysis of chromosomes 21 and 22 has shown that while HAVANA annotation is both comprehensive and robust, it has benefitted from computational review. 13 novel non-coding loci, 27 novel splice variants and 6 extensions to existing variants were identified, many of which were found using supporting EST/mRNA sequences that were not present at the time of initial annotation. Fewer than 10 annotated CDSs required reclassification, no CCDS sequences required updating and 26 novel pseudogene were added. The annotation of human chromosome 2 is complete and we are currently annotating chromosomes 3 and 7. Data from all members of Gencode is distributed via DAS and is now visible in our Zmap annotation interface, allowing assessment of computational predictions contemporaneous with first-pass gene annotation.

Manual annotation‭ (‬the‭ "‬museum‭" ‬model of annotation‭) ‬relies on a small group of specialized curators to catalogue and classify genes according to their functional roles.‭ This‬ is both costly and time consuming and therefore is used only for model organisms with sufficient funding.‭ ‬Smaller research communities often have to rely on other models of annotation,‭ ‬mainly automated annotation‭ (‬the‭ "‬factory‭" ‬model,‭ ‬e.g.‭ ‬Ensembl‭)‬,‭ ‬and the‭ "‬jamboree‭" ‬model‭ (‬in which a group of leading biologists from the community and bioinformaticians come together for a short intensive annotation workshop‭)‬.‭ ‬At the Wellcome Trust Sanger Institute‭ (‬WTSI‭)‬,‭ ‬the Havana team provides high quality manual annotation of finished vertebrate genome sequences,‭ ‬namely human,‭ ‬mouse and zebrafish.‭ ‬We also perform the curation of specific finished regions such as the MHC in dog,‭ ‬cow and pig,‭ ‬whose whole genomes have been‭ ‬assembled from unfinished BACs or from whole genome shotgun sequences.‭ ‬In addition,‭ ‬we at Havana have also hosted annotation jamborees for the cow‭ (‬Bos taurus‭) ‬and pig‭ (‬Sus scrofa‭) ‬genomes.‭ ‬During those sessions,‭ ‬the research community had the opportunity to annotate their genes of interest under expert guidance using the custom written publicly available Otterlace annotation system,‭ ‬and the unified manual annotation guidelines.‭ ‬By making use of the tools and skills acquired during the cow and pig jamborees,‭ ‬the delegates can continue annotating their genomes remotely.‭ ‬For the pig genome,‭ ‬a highly contiguous physical map has been generated by an international effort of four laboratories (available in Pre!Ensembl) and‭ ‬is being used as a substrate for the swine genome sequencing project.‭ ‬Upcoming vertebrate genomes will be sequenced to a high depth coverage with the next generation sequencing technologies‭ (‬e.g.‭ ‬Illumina,‭ ‬454,‭ ‬SOLiD‭) ‬but will have the drawback of not being manually finished.‭ ‬Manual annotation will be more accurate than the automated predictions at coping with any assembly problems derived from these high coverage but unfinished‭ (‬or automatic pre-finished‭) ‬genomes.‭ ‬Once these inherent assembly errors are corrected and the gene structures are accurately identified with manual annotation,‭ ‬the curated genes will be incorporated and merged with the predicted gene models in Ensembl to provide a unified view of the landscape of vertebrate genomes.‭ ‬I will present an introduction to our manual annotation system and our experience using it for annotation jamborees at the WTSI.

The zebrafish genome, which consists of 25 linkage groups and is ~1.4Gb in size, is being sequenced, finished and analysed in its entirety at the Wellcome Trust Sanger Institute. The manual annotation is provided by the Human and Vertebrate Analysis and Annotation (HAVANA) group and is released at regular intervals onto the Vertebrate Genome Annotation (Vega) database ("http://vega.sanger.ac.uk":http://vega.sanger.ac.uk) and may be viewed as a DAS source in Ensembl ("http://www.ensembl.org/Danio_rerio":http://www.ensembl.org/Danio_rerio). Our annotation is compiled in close collaboration with the Zebrafish Information Network (ZFIN) ("http://zfin.org/":http://zfin.org/), which has enabled us to provide an accurate, dynamic and distinct resource for the zebrafish community as a whole.Annotation is based on the reference genome sequence, which is derived from a minimal tile path assembly composed of clones that have been mapped, sequenced and meticulously finished to a sequence accuracy of over 99.9% per 100Kb. We expect to have 90% of the zebrafish genome to a finished standard by the end of 2009. Our approach to annotation uses two strategies. Firstly, the generation and annotation of gene lists comprising of cDNA (8995 in total) found in ZFIN that maps to our current reference assembly. And, secondly, by using clone by clone annotation, where we have annotated over 3200 genes, 1100 transcripts and 130 pseudogenes across 11 linkage groups and 3530 clones. As well as our on-going genome annotation we also welcome external annotation requests for specific genes and regions, which already include the annotation of 93 genes associated with human obesity and the scheduled annotation of the Major Histocompatability Complex, which will utilise reference sequence taken from libraries of a double haploid fish and complement our previous work on the human and mouse MHC already published. External requests and any feedback, questions or requests can be sent to zfish-help [at] sanger.ac.uk.

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production. Fil: Bovine Genome Sequencing and Analysis Consortium. Bovine Genome Sequencing And Analysis Consortium; Estados Unidos Fil: Amadio, Ariel Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Poli, Mario Andres. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. © 2007 The Author(s). Fil: Horton, Roger. Wellcome Trust Sanger Institute; Reino Unido Fil: Gibson, Richard. Wellcome Trust Sanger Institute; Reino Unido Fil: Coggill, Penny. Wellcome Trust Sanger Institute; Reino Unido Fil: Miretti, Marcos Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; Argentina. Wellcome Trust Sanger Institute; Reino Unido Fil: Allcock, Richard J.. University of Western Australia; Australia Fil: Almeida, Jeff. Wellcome Trust Sanger Institute; Reino Unido Fil: Forbes, Simon. Wellcome Trust Sanger Institute; Reino Unido Fil: Gilbert, James G. R.. Wellcome Trust Sanger Institute; Reino Unido Fil: Halls, Karen. Wellcome Trust Sanger Institute; Reino Unido. University of Cambridge; Reino Unido Fil: Harrow, Jennifer L.. Wellcome Trust Sanger Institute; Reino Unido Fil: Hart, Elizabeth. Wellcome Trust Sanger Institute; Reino Unido Fil: Howe, Kevin. Cruk Cambridge Research Institute; Reino Unido Fil: Jackson, David K.. Wellcome Trust Sanger Institute; Reino Unido Fil: Palmer, Sophie. Wellcome Trust Sanger Institute; Reino Unido Fil: Roberts, Anne N.. University of Cambridge; Reino Unido Fil: Sims, Sarah. Wellcome Trust Sanger Institute; Reino Unido Fil: Stewart, C. Andrew. National Cancer Institute At Frederick; Estados Unidos Fil: Traherne, James A.. University of Cambridge; Reino Unido Fil: Trevanion, Steve. Wellcome Trust Sanger Institute; Reino Unido Fil: Wilming, Laurens. Wellcome Trust Sanger Institute; Reino Unido Fil: Rogers, Jane. Wellcome Trust Sanger Institute; Reino Unido Fil: De Jong, Pieter J.. Children's Hospital Oakland Research Institute; Estados Unidos Fil: Elliott, John F.. University of Alberta; Canadá Fil: Sawcer, Stephen. University of Cambridge; Reino Unido Fil: Todd, John A.. University of Cambridge; Reino Unido Fil: Trowsdale, John. University of Cambridge; Reino Unido Fil: Beck, Stephan G.. Wellcome Trust Sanger Institute; Reino Unido

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

The sequencing, annotation and comparative analysis of an 8Mb region of pig chromosome 17 allows the coverage and quality of the pig genome sequencing project to be assessed, Background We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage. Results Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs. Conclusion We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS.

Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome1, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome2,3. It is also enriched in segmental duplications, ranking third in density among the autosomes4. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution5,6, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.

With the completion of the human genome sequence and genome sequence available for other vertebrate genomes, the task of manual annotation at the large genome scale has become a priority. Possibly even more important, is the requirement to curate and improve this annotation in the light of future data. For this to be possible, there is a need for tools to access and manage the annotation. Ensembl provides an excellent means for storing gene structures, genome features, and sequence, but it does not support the extra textual data necessary for manual annotation. We have extended Ensembl to create the Otter manual annotation system. This comprises a relational database schema for storing the manual annotation data, an application-programming interface (API) to access it, an extensible markup language (XML) format to allow transfer of the data, and a server to allow multiuser/multimachine access to the data. We have also written a data-adaptor plugin for the Apollo Browser/Editor to enable it to utilize an Otter server. The otter database is currently used by the Vertebrate Genome Annotation (VEGA) site (http://vega.sanger.ac.uk), which provides access to manually curated human chromosomes. Support is also being developed for using the AceDB annotation editor, FMap, via a perl wrapper called Lace. The Human and Vertebrate Annotation (HAVANA) group annotators at the Sanger center are using this to annotate human chromosomes 1 and 20.

Ensembl (http://www.ensembl.org/) is a bioinformatics project to organize biological information around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of individual genomes, and of the synteny and orthology relationships between them. It is also a framework for integration of any biological data that can be mapped onto features derived from the genomic sequence. Ensembl is available as an interactive Web site, a set of flat files, and as a complete, portable open source software system for handling genomes. All data are provided without restriction, and code is freely available. Ensembl's aims are to continue to “widen” this biological integration to include other model organisms relevant to understanding human biology as they become available; to “deepen” this integration to provide an ever more seamless linkage between equivalent components in different species; and to provide further classification of functional elements in the genome that have been previously elusive.

Comparative analysis of genomic sequences is becoming a standard technique for studying gene regulation. However, only a limited number of tools are currently available for the analysis of multiple genomic sequences. An extensive data set for the testing and training of such tools is provided by the SCL gene locus. Here we have expanded the data set to eight vertebrate species by sequencing the dog SCL locus and by annotating the dog and rat SCL loci. To provide a resource for the bioinformatics community, all SCL sequences and functional annotations, comprising a collation of the extensive experimental evidence pertaining to SCL regulation, have been made available via a Web server. A Web interface to new tools specifically designed for the display and analysis of multiple sequence alignments was also implemented. The unique SCL data set and new sequence comparison tools allowed us to perform a rigorous examination of the true benefits of multiple sequence comparisons. We demonstrate that multiple sequence alignments are, overall, superior to pairwise alignments for identification of mammalian regulatory regions. In the search for individual transcription factor binding sites, multiple alignments markedly increase the signal-to-noise ratio compared to pairwise alignments.

The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort.[Supplemental material is available online at www.genome.org. Bioperl is available as open-source software free of charge and is licensed under the Perl Artistic License (http://www.perl.com/pub/a/language/misc/Artistic.html). It is available for download at http://www.bioperl.org. Support inquiries should be addressed to bioperl-l@bioperl.org.]

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.

The stem cell leukemia ( SCL ) gene encodes a tissue-specific basic helix–loop–helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes , a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5′ and 3′ flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo .

Long-range comparative sequence analysis provides a powerful strategy for identifying conserved regulatory elements. The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis, and it displays a highly conserved expression pattern. We present here a detailed sequence comparison of 193 kb of the human SCL locus to 234 kb of the mouse SCL locus. Four new genes have been identified together with an ancient mitochondrial insertion in the human locus. The SCL gene is flanked upstream by theSIL gene and downstream by the MAP17 gene in both species, but the gene order is not collinear downstream fromMAP17. To facilitate rapid identification of candidate regulatory elements, we have developed a new sequence analysis tool (SynPlot) that automates the graphical display of large-scale sequence alignments. Unlike existing programs, SynPlot can display the locus features of more than one sequence, thereby indicating the position of homology peaks relative to the structure of all sequences in the alignment. In addition, high-resolution analysis of the chromatin structure of the mouse SCL gene permitted the accurate positioning of localized zones accessible to restriction endonucleases. Zones known to be associated with functional regulatory regions were found to correspond precisely with peaks of human/mouse homology, thus demonstrating that long-range human/mouse sequence comparisons allow accurate prediction of the extent of accessible DNA associated with active regulatory regions.

Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

Polycythemia vera (PV) is an acquired clonal disorder characterized by increased production of mature red cells and growth of erythroid colonies in the absence of erythropoietin. Mutation of the erythropoietin receptor has been demonstrated to cause familial polycythemia, but no mutations have been found in PV. Moreover, both erythroid and myeloid progenitors from patients with PV have been reported to be hypersensitive to a number of different growth factors. Attention has therefore focused on post-receptor signal transduction pathways. The SHP-1 gene is an especially attractive candidate gene. Firstly, SHP-1 binds to and negatively regulates signalling from the erythropoietin receptor and is likely to regulate other cytokine receptors in a similar manner. Secondly, absence of SHP-1 protein in the motheaten mouse is accompanied by increased sensitivity of hematopoietic progenitors to a number of cytokines including erythropoietin. Thirdly, familial or sporadic polycythemia in man may result from mutations of the SHP-1 binding domain of the erythropoietin receptor. We have therefore searched for mutations of the SHP-1 gene in genomic DNA from patients with PV. In this disease the majority of peripheral blood lymphocytes are not part of the malignant clone and a variable proportion of myeloid cells may arise from normal progenitors. We have therefore chosen to study DNA from purified peripheral blood granulocytes obtained from nine women in whom the granulocytes were clonally derived. Southern analysis was used to show that the gene was not rearranged and densitometry confirmed the presence of two copies of the gene in each DNA sample. Sequencing of the entire coding region and all splice junctions revealed no mutations. Hematopoietic transcription factor binding sites in the SHP-1 promoter region were intact and the methylation status of the two SHP-1 promoters in PV patients was identical to that in three normal controls. Finally, we showed that levels of SHP-1 protein in granulocytes from patients was similar to those from normal controls. These results demonstrate that the SHP-1 gene is structurally and transcriptionally intact in patients with PV.

Abstract 726 Background: ARC1779 is an aptamer that inhibits the prothrombotic function of VWF by binding the A1 domain of VWF and thereby blocking its interaction with the platelet GPIb receptor. It has been hypothesized that effective inhibition of the formation of microthrombi by ARC1779 during the acute treatment of TTP may result in short courses of plasma exchange (PE), but may also decrease end organ complications via the rapid inhibition of the development of microthrombotic disease. Patients: Eligibility for the study included a platelet count of Methods: Patients were randomized (3:1) to either ARC1779 or placebo concurrently with therapy. ARC1779 was given as an intravenous loading dose (12.43 mg/kg) followed by a maintenance infusion (0.0006 mg/kg/min). The ARC1779 infusion continued during PE, but an additional re-loading dose (50% of the initial loading dose) was given at the completion of each PE. The infusion continued until achieving a response, defined by normalization of the platelet count (≥150 ×109) on each of 3 consecutive days, or to a maximum of 14 days. The dose was then tapered by 50% daily over two days and discontinued. Immune suppressive therapy (corticosteroids, rituximab) was permitted at investigator discretion. Treatment response, pharmacodynamic data (von Willebrand activity), and end organ injury (troponin-I, S100B) were studied prior to and throughout the treatment course. Results: All 9 patients received daily PE. 6/7 patients receiving ARC1779 achieved a response after a median of 6 days (range, 5–15) compared to the 2 placebo patients who did not achieve a response within 14 days. Serial measurement of the VWF activity in the context of ARC1779 concentration (Figure 1) and the median troponin-I and S100B concentrations for ARC1779 treated subjects are shown in Figure 2. There were no bleeding complications despite documented, prolonged inhibition of VWF activity throughout therapy with ARC1779. Conclusions: These data provide support for the concept of therapy directed at preventing the formation of microthombi in the treatment of acute TTP. The efficacy and safety of these novel therapeutic strategies will need to be assessed in the frame of large clinical trials, a challenge for clinical scientists who work on this rare disease. Disclosures: Cataland: Archemix Corp.: Consultancy. Peyvandi:Archemix Corp.: Consultancy. Mannucci:Archemix Corp.: Consultancy. Lämmle:Archemix Corp.: Consultancy. Kremer Hovinga:Archemix Corp.: Consultancy. Machin:Archemix Corp.: Consultancy. Scully:Archemix Corp.: Consultancy. Rock:Archemix Corp.: Consultancy. Gilbert:Archemix Corp.: Consultancy. Knoebl:Archemix Corp.: Consultancy. Jilma:Archemix Corp.: Consultancy.

This article is part of the supplement: Beyond the Genome: The true gene count, human evolution and disease genomics, Boston, MA, USA. 11-13 October 2010., The GENCODE consortium is a sub group of the ENCODE consortium. Its aim is to provide complete annotation of genes in the human genome including protein-coding loci, non-coding loci and pseudogenes, based on experimental evidence. The final aim is for the HAVANA team to manually annotate the complete genome. This is a time-consuming process which will be completed over the course of the ENCODE project. Currently, to provide a set of annotation covering the complete genome, rather than just the regions that have been manually annotated, a merge of manual annotation from HAVANA with automatic annotation from the Ensembl automatically annotated gene set is created. This process also adds unique full-length CDS predictions from the Ensembl protein coding set into manually annotated genes, to provide the most complete up to date annotation of the genome possible. Also included in the set are short and long ncRNA genes predicted by the Ensembl prediction pipelines and a consensus set of pseudogene predictions agreed between Havana, Yale and UCSC. The CCDS set is also fully represented within the GENCODE set. The GENCODE set is the default annotation available in Ensembl and is also available in the UCSC genome browser. All the annotation is tagged as to whether it is produced by manual annotation alone, automatic annotation alone, or by both approaches. We are currently working to provide confidence levels for annotation, based on depth and type of evidence supporting it.

Background The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results.

Background. Host defense peptides are a critical component of the innate immune system. Human alpha- and beta-defensin genes are subject to copy number variation (CNV) and historically the organization of mouse alpha-defensin genes has been poorly defined. Here we present the first full manual genomic annotation of the mouse defensin region on Chromosome 8 of the reference strain C57BL/6J, and the analysis of the orthologous regions of the human and rat genomes. Problems were identified with the reference assemblies of all three genomes. Defensins have been studied for over two decades and their naming has become a critical issue due to incorrect identification of defensin genes derived from different mouse strains and the duplicated nature of this region. Results The defensin gene cluster region on mouse Chromosome 8 A2 contains 98 gene loci: 53 are likely active defensin genes and 22 defensin pseudogenes. Several TATA box motifs were found for human and mouse defensin genes that likely impact gene expression. Three novel defensin genes belonging to the Cryptdin Related Sequences (CRS) family were identified. All additional mouse defensin loci on Chromosomes 1, 2 and 14 were annotated and unusual splice variants identified. Comparison of the mouse alpha-defensins in the three main mouse reference gene sets Ensembl, Mouse Genome Informatics (MGI), and NCBI RefSeq reveals significant inconsistencies in annotation and nomenclature. We are collaborating with the Mouse Genome Nomenclature Committee (MGNC) to establish a standardized naming scheme for alpha-defensins. Conclusions Prior to this analysis, there was no reliable reference gene set available for the mouse strain C57BL/6J defensin genes, demonstrating that manual intervention is still critical for the annotation of complex gene families and heavily duplicated regions. Accurate gene annotation is facilitated by the annotation of pseudogenes and regulatory elements. Manually curated gene models will be incorporated into the Ensembl and Consensus Coding Sequence (CCDS) reference sets. Elucidation of the genomic structure of this complex gene cluster on the mouse reference sequence, and adoption of a clear and unambiguous naming scheme, will provide a valuable tool to support studies on the evolution, regulatory mechanisms and biological functions of defensins in vivo.