FANTOM Consortium Research Articles

Identification of the macrophage-specific promoter signature in FANTOM5 mouse embryo developmental time course data.

The FANTOM5 consortium used cap analysis of gene expression (CAGE) to analyze the time course of gene expression over development from 11 days postcoitum (dpc) to adult in 16 developing organs and the whole body of the mouse. Every tissue in the body contains a large number of resident macrophages that initially infiltrate the embryo from the yolk sac. These cells contribute to organogenesis, and their functions diversify during development as they acquire tissue-specific adaptations. In each of the FANTOM5 time courses, the expression of known macrophage-specific genes, including CSF1 receptor (Csf1r), epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (Emr1), and mer receptor tyrosine kinase (Mertk), was readily detectable and increased with time. We reasoned that genes expressed by macrophages would be strongly correlated in their expression with these known markers and might vary between tissues. We used the network analysis tool, Miru, to extract the sets of coexpressed genes from the time course and identified a core set of coexpressed genes attributable to embryonic macrophages, including some, such as dehydrogenase/reductase 3 (Dhrs3), that may have unique functions in development. The FANTOM5 data also detected the appearance of tissue-specific macrophage-expressed genes, such as T cell Ig and mucin domain-containing 4 (Timd4) and V-set and Ig domain-containing 4 (Vsig4) in liver and sialic acid-binding Ig-like lectin 5 (Siglec5) in lung, and confirmed that macrophage content increases with time in each organ as the proliferative phases end, and tissue-specific gene-expression increases. The FANTOM5 data are available on a comprehensive browser (http://fantom.gsc.riken.jp/zenbu/), which provides a resource for the study of macrophage transcriptional regulation and roles in mouse development.

Relatively frequent switching of transcription start sites during cerebellar development

BackgroundAlternative transcription start site (TSS) usage plays important roles in transcriptional control of mammalian gene expression. The growing interest in alternative TSSs and their role in genome diversification spawned many single-gene studies on differential usages of tissue-specific or temporal-specific alternative TSSs. However, exploration of the switching usage of alternative TSS usage on a genomic level, especially in the central nervous system, is largely lacking.ResultsIn this study, We have prepared a unique set of time-course data for the developing cerebellum, as part of the FANTOM5 consortium (http://fantom.gsc.riken.jp/5/) that uses their innovative capturing of 5′ ends of all transcripts followed by Helicos next generation sequencing. We analyzed the usage of all transcription start sites (TSSs) at each time point during cerebellar development that provided information on multiple RNA isoforms that emerged from the same gene. We developed a mathematical method that systematically compares the expression of different TSSs of a gene to identify temporal crossover and non-crossover switching events. We identified 48,489 novel TSS switching events in 5433 genes during cerebellar development. This includes 9767 crossover TSS switching events in 1511 genes, where the dominant TSS shifts over time.ConclusionsWe observed a relatively high prevalence of TSS switching in cerebellar development where the resulting temporally-specific gene transcripts and protein products can play important regulatory and functional roles.

Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease.

The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis of published GWA studies. We propose that dysregulation of monocyte adaptation to the environment of the gastrointestinal mucosa is the key process leading to inflammatory bowel disease.

Abstract 2897: Recurrent transcriptome alterations across multiple cancer types

Abstract Genes that are frequently deregulated in cancer are clinically attractive as both potential pan-cancer diagnostic markers and therapeutic targets. Here we compared Cap Analysis of Gene Expression (CAGE) profiles from 225 cancer cell lines and 339 corresponding primary cell samples to identify transcripts that are recurrently deregulated in a broad range of cancer types. CAGE is a 5’ sequence tag technology that globally determines transcription start sites (TSS) in the genome and their expression levels. This allowed us to assess novel aspects of the cancer transcriptome. First, we identified (at promoter resolution) hundreds of protein coding and long non-coding transcripts that are commonly de-regulated in cancer cell lines. Next, we showed that promoters that overlap repetitive elements (especially SINE/Alu and LTR/ERV1 elements) are often upregulated in cancer. In particular, a specific repeat family, REP522 (largely palindromic, unclassified interspersed repeat of ∼1.8Kb in size), was strongly enriched for the most up-regulated promoters. To our knowledge this is the first report implicating REP522 activation in cancer. Then, taking advantage of the fact that CAGE data can be used to estimate the activity of enhancers from balanced bidirectional transcription we identified 90 enhancer RNA producing regions that are recurrently activated in cancer cell lines. With ENCODE ChIA-PET data, we linked 16 of the cancer-activated enhancers to promoters of known cancer related genes. Finally, to confirm that our results are relevant to clinical tumors we performed complementary analysis in RNA-seq data from 4,055 tumors and 563 normal tissues profiled by The Cancer Genome Atlas (TCGA) and we identified a core set of pan-cancer biomarkers (of both coding and non-coding transcripts) that are recurrently perturbed in both the FANTOM5 and TCGA datasets. In summary, our extensive transcriptome analysis identified a comprehensive set of candidate biomarkers with pan-cancer potential, and extended the perspective of enhancers and repetitive elements that are recurrently activated during carcinogenesis. Citation Format: Bogumil Kaczkowski, Yuji Tanaka, Hideya Kawaji, Albin Sandelin, Robin Andersson, Masayoshi Itoh, Timo Lassmann, Yoshihide Hayashizaki, Piero Carninci, Alistair R.R. Forrest, FANTOM5 Consortium. Recurrent transcriptome alterations across multiple cancer types. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2897.

CAGEd-oPOSSUM: motif enrichment analysis from CAGE-derived TSSs

With the emergence of large-scale Cap Analysis of Gene Expression (CAGE) datasets from individual labs and the FANTOM consortium, one can now analyze the cis-regulatory regions associated with gene transcription at an unprecedented level of refinement. By coupling transcription factor binding site (TFBS) enrichment analysis with CAGE-derived genomic regions, CAGEd-oPOSSUM can identify TFs that act as key regulators of genes involved in specific mammalian cell and tissue types. The webtool allows for the analysis of CAGE-derived transcription start sites (TSSs) either provided by the user or selected from ∼1300 mammalian samples from the FANTOM5 project with pre-computed TFBS predicted with JASPAR TF binding profiles. The tool helps power insights into the regulation of genes through the study of the specific usage of TSSs within specific cell types and/or under specific conditions.Availability and Implementation: The CAGEd-oPOSUM web tool is implemented in Perl, MySQL and Apache and is available at http://cagedop.cmmt.ubc.ca/CAGEd_oPOSSUM.Contacts: anthony.mathelier@ncmm.uio.no or wyeth@cmmt.ubc.caSupplementary information: Supplementary data are available at Bioinformatics online.

Application of Gene Expression Trajectories Initiated from ErbB Receptor Activation Highlights the Dynamics of Divergent Promoter Usage.

Understanding how cells use complex transcriptional programs to alter their fate in response to specific stimuli is an important question in biology. For the MCF-7 human breast cancer cell line, we applied gene expression trajectory models to identify the genes involved in driving cell fate transitions. We modified trajectory models to account for the scenario where cells were exposed to different stimuli, in this case epidermal growth factor and heregulin, to arrive at different cell fates, i.e. proliferation and differentiation respectively. Using genome-wide CAGE time series data collected from the FANTOM5 consortium, we identified the sets of promoters that were involved in the transition of MCF-7 cells to their specific fates versus those with expression changes that were generic to both stimuli. Of the 1,552 promoters identified, 1,091 had stimulus-specific expression while 461 promoters had generic expression profiles over the time course surveyed. Many of these stimulus-specific promoters mapped to key regulators of the ERK (extracellular signal-regulated kinases) signaling pathway such as FHL2 (four and a half LIM domains 2). We observed that in general, generic promoters peaked in their expression early on in the time course, while stimulus-specific promoters tended to show activation of their expression at a later stage. The genes that mapped to stimulus-specific promoters were enriched for pathways that control focal adhesion, p53 signaling and MAPK signaling while generic promoters were enriched for cell death, transcription and the cell cycle. We identified 162 genes that were controlled by an alternative promoter during the time course where a subset of 37 genes had separate promoters that were classified as stimulus-specific and generic. The results of our study highlighted the degree of complexity involved in regulating a cell fate transition where multiple promoters mapping to the same gene can demonstrate quite divergent expression profiles.

Identification and annotation of conserved promoters and macrophage-expressed genes in the pig genome.

BackgroundThe FANTOM5 consortium used Cap Analysis of Gene Expression (CAGE) tag sequencing to produce a comprehensive atlas of promoters and enhancers within the human and mouse genomes. We reasoned that the mapping of these regulatory elements to the pig genome could provide useful annotation and evidence to support assignment of orthology.ResultsFor human transcription start sites (TSS) associated with annotated human-mouse orthologs, 17% mapped to the pig genome but not to the mouse, 10% mapped only to the mouse, and 55% mapped to both pig and mouse. Around 17% did not map to either species. The mapping percentages were lower where there was not clear orthology relationship, but in every case, mapping to pig was greater than to mouse, and the degree of homology was also greater. Combined mapping of mouse and human CAGE-defined promoters identified at least one putative conserved TSS for >16,000 protein-coding genes. About 54% of the predicted locations of regulatory elements in the pig genome were supported by CAGE and/or RNA-Seq analysis from pig macrophages.ConclusionsComparative mapping of promoters and enhancers from humans and mice can provide useful preliminary annotation of other animal genomes. The data also confirm extensive gain and loss of regulatory elements between species, and the likelihood that pigs provide a better model than mice for human gene regulation and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2111-2) contains supplementary material, which is available to authorized users.

Paradigm shifts in genomics through the FANTOM projects.

Big leaps in science happen when scientists from different backgrounds interact. In the past 15 years, the FANTOM Consortium has brought together scientists from different fields to analyze and interpret genomic data produced with novel technologies, including mouse full-length cDNAs and, more recently, expression profiling at single-nucleotide resolution by cap-analysis gene expression. The FANTOM Consortium has provided the most comprehensive mouse cDNA collection for functional studies and extensive maps of the human and mouse transcriptome comprising promoters, enhancers, as well as the network of their regulatory interactions. More importantly, serendipitous observations of the FANTOM dataset led us to realize that the mammalian genome is pervasively transcribed, even from retrotransposon elements, which were previously considered junk DNA. The majority of products from the mammalian genome are long non-coding RNAs (lncRNAs), including sense-antisense, intergenic, and enhancer RNAs. While the biological function has been elucidated for some lncRNAs, more than 98 % of them remain without a known function. We argue that large-scale studies are urgently needed to address the functional role of lncRNAs.

Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells.

The analysis of CAGE (Cap Analysis of Gene Expression) time-course has been proposed by the FANTOM5 Consortium to extend the understanding of the sequence of events facilitating cell state transition at the level of promoter regulation. To identify the most prominent transcriptional regulations induced by growth factors in human breast cancer, we apply here the Complexity Invariant Dynamic Time Warping motif EnRichment (CIDER) analysis approach to the CAGE time-course datasets of MCF-7 cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). We identify a multi-level cascade of regulations rooted by the Serum Response Factor (SRF) transcription factor, connecting the MAPK-mediated transduction of the HRG stimulus to the negative regulation of the MAPK pathway by the members of the DUSP family phosphatases. The finding confirms the known primary role of FOS and FOSL1, members of AP-1 family, in shaping gene expression in response to HRG induction. Moreover, we identify a new potential regulation of DUSP5 and RARA (known to antagonize the transcriptional regulation induced by the estrogen receptors) by the activity of the AP-1 complex, specific to HRG response. The results indicate that a divergence in AP-1 regulation determines cellular changes of breast cancer cells stimulated by ErbB receptors.

EpiFactors: a comprehensive database of human epigenetic factors and complexes.

Epigenetics refers to stable and long-term alterations of cellular traits that are not caused by changes in the DNA sequence per se. Rather, covalent modifications of DNA and histones affect gene expression and genome stability via proteins that recognize and act upon such modifications. Many enzymes that catalyse epigenetic modifications or are critical for enzymatic complexes have been discovered, and this is encouraging investigators to study the role of these proteins in diverse normal and pathological processes. Rapidly growing knowledge in the area has resulted in the need for a resource that compiles, organizes and presents curated information to the researchers in an easily accessible and user-friendly form. Here we present EpiFactors, a manually curated database providing information about epigenetic regulators, their complexes, targets and products. EpiFactors contains information on 815 proteins, including 95 histones and protamines. For 789 of these genes, we include expressions values across several samples, in particular a collection of 458 human primary cell samples (for approximately 200 cell types, in many cases from three individual donors), covering most mammalian cell steady states, 255 different cancer cell lines (representing approximately 150 cancer subtypes) and 134 human postmortem tissues. Expression values were obtained by the FANTOM5 consortium using Cap Analysis of Gene Expression technique. EpiFactors also contains information on 69 protein complexes that are involved in epigenetic regulation. The resource is practical for a wide range of users, including biologists, pharmacologists and clinicians.Database URL: http://epifactors.autosome.ru

Enrichment of Genetic Variants for Rheumatoid Arthritis within T-Cell and NK-Cell Enhancer Regions.

To identify disease-causative variants, we intersected the published results of a metaanalysis of genome-wide association studies (GWAS) for rheumatoid arthritis (RA) with the set of enhancer regions for 71 primary cell types that was provided by the FANTOM consortium. We first retrieved all single nucleotide polymorphisms (SNPs) that are associated (P < 5 × 10(8)) with RA in the GWAS meta-analysis and that are located in any of these enhancer regions. After excluding the major histocompatibility complex (MHC) region, we identified 50 such RA-associated SNPs that are located in enhancer regions. Enhancer sets from different cell types were then compared with each other for their number of RA-associated SNPs by permutation analysis. This analysis showed that RA-associated SNPs are preferentially located in enhancers from several immunological cell types. In particular, we see a strong relative enrichment in enhancer regions that are active in T cells (P < 0.001) and NK cells (P < 0.001). Several loci display multiple RA-associated SNPs in tight linkage disequilibrium that are located within the same or neighboring enhancers. These haplotypes may have a greater likelihood to influence enhancer activity than any SNP on its own. Taken together, these results support the hypothesis that RA-causative variants often act through altering the activity of immune cell enhancers. The enrichment in T-cell and NK-cell enhancer regions indicates that expression changes in these cell types are particularly relevant for the pathogenesis of RA. The specific SNPs that account for this enrichment can be used as a basis for focused genotype-phenotype studies of these cell types.

Direct reprogramming based on transcriptional regulatory network analysis

Trans-differentiation of cells through direct reprogramming offers significant potential for regenerative medicine and drug screen, but currently it requires exhaustive trials and errors before the desired target cells can be created. Based on the comprehensive transcriptome analyses by the FANTOM consortium, in particular the recently published transcriptional regulatory network analysis, we have developed a systematic method for direct reprogramming and succeeded in creating monocyte-like cells with several monocyte-specific functions. Further analysis of the created cells reveals that the transcriptional regulatory networks and the epigenomes of the original terminally differentiated cells act as strong barriers that prevent easy reprogramming by extra stimuli. These biological insights should be carefully considered for efficient and complete direct reprogramming.

LOCATE: a mammalian protein subcellular localization database

LOCATE is a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of mouse and human proteins. Over the past 2 years, the data in LOCATE have grown substantially. The database now contains high-quality localization data for 20% of the mouse proteome and general localization annotation for nearly 36% of the mouse proteome. The proteome annotated in LOCATE is from the RIKEN FANTOM Consortium Isoform Protein Sequence sets which contains 58 128 mouse and 64 637 human protein isoforms. Other additions include computational subcellular localization predictions, automated computational classification of experimental localization image data, prediction of protein sorting signals and third party submission of literature data. Collectively, this database provides localization proteome for individual subcellular compartments that will underpin future systematic investigations of these regions. It is available at http://locate.imb.uq.edu.au/

Transcript Annotation in FANTOM3: Mouse Gene Catalog Based on Physical cDNAs

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

How Many New Genes Are There?

How Many New Genes Are There?

ゲノミクス・プロテオミクスを中心に‐ｏｍｉｃｓへの電気泳動法の応用 ゲノム科学新時代に向けて

The FANTOM Consortium analyzed over 2 million sequences of RNAs produced from the mouse genome, obtaining more than 100, 000 full length copies of these RNAs, and used several new technologies to generate more than 20 million tags representing their starts and ends in a technical tour-de-force that provides a amazing new view of our genome. These studies clearly indicate that while proteins comprise the essential components of our cells, the development of multicellular organisms like mammals is controlled by vast amounts of regulatory noncoding RNAs that until recently have not been suspected to exist or be relevant to biology. Moreover, since most proteins are similar among mammals it also suggests that many of the differences between species may be embedded in differences in the RNA regulatory control systems, which are evolving much faster than the protein components. If correct, these findings will radically alter our understanding of genetics and how information is stored in our genome, and how this information is transacted to control the incredibly complex process of mammalian development, with major implications for the future of biological research, medicine and biotechnology.

The New Phase of Transcriptome Analysis

The FANTOM Consortium analyzed over 2 million sequences of RNAs produced from the mouse genome, obtaining more than 100, 000 full length copies of these RNAs, and used several new technologies to generate more than 20 million tags representing their starts and ends in a technical tour-de-force that provides a amazing new view of our genome. These studies clearly indicate that while proteins comprise the essential components of our cells, the development of multicellular organisms like mammals is controlled by vast amounts of regulatory noncoding RNAs that until recently have not been suspected to exist or be relevant to biology. Moreover, since most proteins are similar among mammals it also suggests that many of the differences between species may be embedded in differences in the RNA regulatory control systems, which are evolving much faster than the protein components. If correct, these findings will radically alter our understanding of genetics and how information is stored in our genome, and how this information is transacted to control the incredibly complex process of mammalian development, with major implications for the future of biological research, medicine and biotechnology.

Special FANTOM2 Issue

It is my great pleasure to be able to publish a special issue of Genome Research focusing on the functional annotation of mouse cDNA (FANTOM). Publication of this FANTOM special issue is quite timely,as not only the mouse full-length cDNA collection but also the first draft of mouse genome sequences were just published at the end of last year. Moreover,completion of the human genome sequence was announced this spring and entered the next phase. This is a very exciting series of events. In the history of life sciences,the mouse is the first organism for which large-scale analyses of the genome and transcriptome were undertaken at the same time. This data will form the basic platform of life sciences in the 21st century. In this issue,we present papers on FANTOM activity, describing analyses based on the first data for the genome and transcriptome. The international FANTOM consortium was established to annotate functions for sequences in the full-length cDNA sequence database,which was developed by the Riken Mouse Gene Encyclopedia Project. The background of this project should first be explained. The project was started in 1995 and originally aimed at the overall collection of full-length cDNAs for all genes supported by the Science Technology Agency (currently the Ministry of Education,Culture,Sports,and Technology [MEXT]). At that time,the international human genome consortium started large-scale sequencing analysis of the entire human genome,and the EST project was almost saturated. Under circumstances in which genome science was rapidly gaining prominence in the world,we faced the following question: “What significant contribution would Japan make to this scientific field?” Our answer was mouse full-length cDNA. We started this mouse gene encyclopedia project by developing original technologies for full-length cDNA library preparation and a high-throughput system for sequence determination. Our system,the RIKEN integrated sequence analysis (RISA) system,achieved throughput of 40,000 samples per day,facilitating the establishment of the largest collection of full-length cDNAs for the mouse in the world. The full-length cDNA library was prepared by Spring 2000 from 263 tissues,finishing sequence analysis of 60,770 clones in total with full sequences selected from about 2 million end-sequenced clones in Summer 2001. After determining full-length cDNAs,functional annotation of these clones required the kind of enormous effort that can only be achieved with worldwide cooperation. We contacted researchers from various fields of biology and advertised in journals and on the Internet,opening our meetings to anyone who wished to participate,contribute,and help. Many researchers agreed with the goal of founding an international de facto standard of well-annotated full-length cDNA resources,and they voluntarily joined the FANTOM consortium. We greatly appreciate the dedication of the more than 80 researchers in FANTOM1 and 100 researchers in FANTOM2. Integrated analysis of the genome and transcriptome created a synergistic effect,providing extremely valuable insights into the nature of life. The transcriptome provides the exact location of genes,whereas genome sequences provide information such as promoters and exon–intron junctions. Analyses of the mouse genome sequence and 60,770 mouse transcripts were fortunately completed at the same time,with results published late last year. After publication,further studies by the FANTOM consortium based on the enormous amounts of data aimed for systematic understanding of the mouse transcriptome as a model for the human system,and they form the contents of this special issue. One notable feature of this special issue is a new proposal for clone transportation. With the development of genome science,handling of numerous clones as a platform for research has become indispensable. A new type of transportation system to facilitate such handling has long been warranted. The proposal described herein,in a paper entitled “DNA Book,” may well represent a timely solution, and is expected to work very effectively. The DNA book forms an easily transposable bank of full-length cDNA clones printed on paper and bound like a book,and DNA is actually incorporated onto paper that is included in this report. This issue marks the first large-scale trial of the technique,and the cooperation of subscribers worldwide with this project would be deeply appreciated. Subscribers can get involved by filling out the feedback form at the end of this issue for submission to the URL provided. We would like to report the results of subscriber feedback and assist in further improvements to this new technology. RIKEN and others who are concerned with the large-scale distribution of clones hope that the DNA book will offer a new standard of clone distribution that will soon see practical use.

Exon Structure Analysis, Ortholog Identification, and SNP Candidate Screening by Mapping Mouse RIKEN Sequences to Multiple Genome Assemblies

Mapping RIKEN full-length cDNAs (The FANTOM Consortium and the RIKEN Genome Exploration Research Group Phase I and II Team 2002) to the genome assemblies enables a variety of analyses to be performed. First, exon structure can be determined, and coding and noncoding regions can be inferred, due to different average exon length. In some cases, the alternative exon structures may be identified at this stage. Second, chromosome position can be used to identify the correct ortholog in human, for which functional data may be available. Third, intronless genes can be identified and examined carefully to determine whether they are retransposition events, pseudogenes, or genomic contamination. Finally, high-quality sequence discrepancies can be identified as potential SNPs by use of the fact that RIKEN and Mouse Genome Sequencing Consortium sequenced the C57BL/6J mouse strain, whereas the four mouse strains sequenced by Celera included 129X1/SvJ, 129S1/SvImJ, DBA/2J, and A/J. We have mapped 60,770 RIKEN clones by BLAT (Kent 2002) to the MGSC (Mouse Genome Sequencing Consortium 2002) genome assembly versions 1 and 3, Ensembl human genome assembly v.28 and the Celera mouse genome assembly releases R12 and R13, and human assembly Release R26i (http://cds.celera.com/; Table 1). The single-exon clones longer than 1 Kb are candidates for further investigation as bona fide intronless genes, retransposition events, or possible genomic DNA contamination. For this investigation, the expression profile of an intronless clone can be very informative (Su et al. 2002). This analysis also allows us to roughly compare the completeness of the assemblies. Of 60,172 RIKEN cDNAs containing >100 non-masked bases, >99% were mapped at >70% length to both latest assemblies, much more complete than the earlier assemblies. Figure 1 illustrates the comparison of the mapping to four assemblies. MGSC v.3 in green as left bars, Celera R13 in blue as right bars for each chromosome. Where RIKEN clones mapped to both assemblies, a cyan line connects the mapping positions, whereas triangles mark clones mapped exclusively to one assembly. The large-scale discrepancies are marked in red. One can observe a 10-Mb contig inversion on chromosome X (later detected and corrected by MGSC) and smaller ones on chr.5, 17, 18, 19, etc. The up-to-date scalable version of the mapping comparison is available at http://www.gnf.org/RIKEN/. The extra bars represent superimposed syntenic regions identified by mapping to human assemblies, NCBI v.28 left from MGSC v.3, Celera R26h right from the mouse Celera R13. The two-digit electric color code for human chromosomes is shown at the bottom. Several cases of different syntenic assignment deserve further investigation.

MGAlign, a Reduced Search Space Approach to the Alignment of mRNA Sequences to Genomic Sequences

Alternative splicing is a cellular process that accounts for protein diversity in higher eukaryotes. As much as 60% of the human genome (Kan et al., 2001) and 41% of the mouse genome (FANTOM Consortium, 2002) are alternatively spliced. Methods currently available specifically for aligning mRNA sequences to their cognate genomic sequence has include sim4 (Florea et al., 1998) and Spidey (Wheelan et al., 2001).