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Introduction: We performed an exploratory analysis of circulating tumor DNA (ctDNA) in patients (pts) with RAS wild-type (WT) chemotherapy-naïve stage IV metastatic colorectal cancer (mCRC) from the ongoing phase III PARADIGM study (NCT02394834). The clonal and subclonal mutational landscapes of RAS WT pts were analyzed using a mCRC-focused custom ctDNA sequencing panel. Methods: Randomized pts (1:1) received mFOLFOX6 plus either panitumumab or bevacizumab. Pre-treatment plasma samples (DNA yield >10ng/mL and >10nM) were sequenced using the custom ctDNA panel (PlasmaSELECTTM-R 91, PGDx) to detect mCRC mutations, amplifications, and rearrangements in 90, 26, and 3 genes, respectively, as well as microsatellite instability (MSI), in 250kb targeted regions using stringent quality criteria. Pre-treatment archival tissue samples from 590 pts were analyzed by the Broad Institute Solid Tumor panel, which covered 1,072 genes with 7.3Mb targeted regions. Results: Between May 29, 2015 and June 8, 2017, 823 pts were enrolled across 197 sites. Plasma samples were collected from 756 pts, with 747 (98.8%) samples meeting the quality criteria. The average total and distinct sequencing coverages were 34,200 and 5,865, respectively, with 49.1% of bases mapping to regions of interest. Mutation frequencies in ctDNA were generally consistent with those observed in RAS-WT TCGA CRC and rectal cancers (n=283), except for TET2 and DNMT3A potentially due to clonal hematopoiesis. Mutations in BRAF, APC, CTNNB1, PTEN, PIK3CA, and MSI-status observed in ctDNA were significantly different between left and right-sided tumors, consistent with known differences in tumor sidedness. In the 747 plasma samples, 4,072 mutations were observed; 1,871 had a mutant allele frequency (MAF) Conclusion: The validated ultra-deep plasma sequencing panel was concordant with tissue sequencing and detected tumor heterogeneity. The PARADIGM study will report the efficacy of anti-EGFR/VEGF therapies; post-treatment collection of ctDNA is ongoing. The relationship between therapeutic effects and clonal and subclonal mutational landscapes will be examined in the future. Funded by Takeda Pharmaceutical Company Limited, Tokyo, Japan. ClinicalTrial.gov number: NCT02394834 Citation Format: Katsuya Tsuchihara, Riu Yamashita, Takayuki Yoshino, Kohei Shitara, Jun Watanabe, Hirofumi Yasui, Hisatsugu Ohori, Manabu Shiozawa, Kentaro Yamazaki, Eiji Oki, Takeo Sato, Takeshi Naitoh, Yoshito Komatsu, Takeshi Kato, Kazunori Yamanaka, Ikuo Mori, Masamitsu Hihara, Junpei Soeda, Takeharu Yamanaka, Kiwamu Akagi, Atsushi Ochiai, Kei Muro, Victor E. Velculescu, Hiroyuki Uetake. Clonal and subclonal mutational landscapes in circulating tumor DNA in metastatic colorectal cancer: An exploratory analysis from the phase III PARADIGM study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1974.

DBTSS (http://dbtss.hgc.jp/) was originally constructed as a collection of uniquely determined transcriptional start sites (TSSs) in humans and some other species in 2002. Since then, it has been regularly updated and in recent updates epigenetic information has also been incorporated because such information is useful for characterizing the biological relevance of these TSSs/downstream genes. In the newest release, Release 9, we further integrated public and original single nucleotide variation (SNV) data into our database. For our original data, we generated SNV data from genomic analyses of various cancer types, including 97 lung adenocarcinomas and 57 lung small cell carcinomas from Japanese patients as well as 26 cell lines of lung cancer origin. In addition, we obtained publically available SNV data from other cancer types and germline variations in total of 11,322 individuals. With these updates, users can examine the association between sequence variation pattern in clinical lung cancers with its corresponding TSS-seq, RNA-seq, ChIP-seq and BS-seq data. Consequently, DBTSS is no longer a mere storage site for TSS information but has evolved into an integrative platform of a variety of genome activity data.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the “Ohanami Project”, to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis ‘Somei-yoshino’ across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained across a wide distribution; this DNA likely originated from pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the “Ohanami Project”, to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis ‘Somei-yoshino’ across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

3528 Background: Anti-EGFR therapy (tx) in RAS wild-type (wt) metastatic colorectal cancer (mCRC) induces resistance through acquired genomic alterations. We aimed to define such alterations in Nationwide Cancer Genome Screening Project tissue and blood specimens, using next generation sequencing (NGS). Methods: Tumor specimens in patients (pts) with RAS wt mCRC were obtained from SCRUM-Japan GI-SCREEN (tissue) and GOZILA (circulating tumor DNA [ctDNA] from blood). Genomic alterations were compared using the Oncomine Comprehensive Assay (SCRUM) and Guardant360 (GOZILA), before anti-EGFR tx and after progression. Results: 373 total actionable alterations were identified in 71 pts with available matched tissue and ctDNA; 255 (68%) were acquired after anti-EGFR tx progression. Frequently seen acquired oncogenic alterations included KRAS mutations (27%) and amplifications (amps) of EGFR (41%), CDK6 (24%), BRAF (20%), MYC (17%), MET (14%), PIK3CA (11%), FGFR1 (11%), and KRAS (10%). Fusions of RET, ALK, and FGFR3 were newly acquired in 1-4%. Acquired alterations co-arose in multiple pathways, including the cell cycle, PI3K-AKT, and MAPK, although 29% of pts had none. Acquired mutations were less frequently clonal versus primary mutations (p7) adjusted plasma copy numbers (ApCN). Acquired ERBB2 amps were identified in 3 pts (4%) with a median ApCN of 4, one of whom (ApCN=4.2), treated with dual HER2 blockade, progressed after 2 cycles. Conclusions: Our integrated analysis revealed that anti-EGFR tx of pts with RAS WT mCRC led to acquired genomic alterations in multiple oncogenic pathways. Although most acquired alterations were subclonal, a subset of oncogenic alterations had relatively high clonality and ApCN, suggesting potential targets for overcoming acquired resistance to anti-EGFR tx. Early progression in a pt with an ApCN of 4.2 suggests low-level/subclonal acquired alterations may not be effective treatment targets.

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit + Sca1 + Lineage − cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

Medullary thymic epithelial cells (mTECs) are essential for thymic negative selection to prevent autoimmunity. Previous studies show that mTEC development is dependent on the signal transducers TRAF6 and NIK. However, the downstream target genes of signals controlled by these molecules remain unknown. We performed a microarray analysis on mRNAs down-regulated by deficiencies in TRAF6 or functional NIK in an in vitro organ culture of fetal thymic stromata (2DG-FTOC). An in silico analysis of transcription factor binding sites in plausible promoter regions of differentially expressed genes suggests that STAT1 is involved in TRAF6- and NIK-dependent gene expression. Indeed, the signal of RANK, a TNF receptor family member that activates TRAF6 and NIK, induces the activation of STAT1 in 2DG-FTOC. Moreover, RANK signaling induces the up-regulation of interferon (IFN)-stimulated gene (ISG) expression, suggesting that the RANKL-dependent activation of STAT1 up-regulates ISG expression. The RANKL-dependent expression levels of ISGs were reduced but not completely abolished in interferon α receptor 1-deficient (Ifnar1(-/-)) 2DG-FTOC. Our data suggest that RANK signaling induces ISG expression in both type I interferon-independent and interferon-dependent mechanisms.

We performed a genome-wide analysis of transcriptional start sites (TSSs) in human genes by multifaceted use of a massively parallel sequencer. By analyzing 800 million sequences that were obtained from various types of transcriptome analyses, we characterized 140 million TSS tags in 12 human cell types. Despite the large number of TSS clusters (TSCs), the number of TSCs was observed to decrease sharply with increasing expression levels. Highly expressed TSCs exhibited several characteristic features: Nucleosome-seq analysis revealed highly ordered nucleosome structures, ChIP-seq analysis detected clear RNA polymerase II binding signals in their surrounding regions, evaluations of previously sequenced and newly shotgun-sequenced complete cDNA sequences showed that they encode preferable transcripts for protein translation, and RNA-seq analysis of polysome-incorporated RNAs yielded direct evidence that those transcripts are actually translated into proteins. We also demonstrate that integrative interpretation of transcriptome data is essential for the selection of putative alternative promoter TSCs, two of which also have protein consequences. Furthermore, discriminative chromatin features that separate TSCs at different expression levels were found for both genic TSCs and intergenic TSCs. The collected integrative information should provide a useful basis for future biological characterization of TSCs.

On the basis of integrated transcriptome analysis, we show that not all transcriptional start site clusters (TSCs) in the intergenic regions (iTSCs) have the same properties; thus, it is possible to discriminate the iTSCs that are likely to have biological relevance from the other noise-level iTSCs. We used a total of 251,933,381 short-read sequence tags generated from various types of transcriptome analyses in order to characterize 6039 iTSCs, which have significant expression levels. We analyzed and found that 23% of these iTSCs were located in the proximal regions of the RefSeq genes. These RefSeq-linked iTSCs showed similar expression patterns with the neighboring RefSeq genes, had widely fluctuating transcription start sites and lacked ordered nucleosome positioning. These iTSCs seemed not to form independent transcriptional units, simply representing the by-products of the neighboring RefSeq genes, in spite of their significant expression levels. Similar features were also observed for the TSCs located in the antisense regions of the RefSeq genes. Furthermore, for the remaining iTSCs that were not associated with any RefSeq genes, we demonstrate that integrative interpretation of the transcriptome data provides essential information to specify their biological functions in the hypoxic responses of the cells.

DataBase of Transcription Start Sites (DBTSS) is a database which contains precise positional information for transcription start sites (TSSs) of eukaryotic mRNAs. In this update, we included 330 million new tags generated by massively sequencing the 5′-end of oligo-cap selected cDNAs in humans and mice. The tags were collected from normal fetal or adult human tissues, including brain, thymus, liver, kidney and heart, from 6 human cell lines in 21 diverse growth conditions as well as from mouse NIH3T3 cell line: altogether 31 different cell types or culture conditions are represented. This unprecedented increase in depth of data now allows DBTSS to faithfully represent the dynamically changing landscape of TSSs in different cell types and conditions, during development and in the course of evolution. Differential usage of alternative 5′-ends across cell types and conditions can be viewed in a series of new interfaces. Promoter sequence information is now displayed in a comparative genomics viewer where evolutionary turnover of the TSSs can be evaluated. DBTSS can be accessed at http://dbtss.hgc.jp/.

Combining our full-length cDNA method and the massively parallel sequencing technology, we developed a simple method to collect precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high throughput manner. We applied this method to observe gene-expression changes in a colon cancer cell line cultured in normoxic and hypoxic conditions. We generated more than 100 million 36-base TSS-tag sequences and revealed comprehensive features of hypoxia responsive alterations in the transcriptional landscape of the human genome. The features include presence of inducible ‘hot regions’ in 54 genomic regions, 220 novel hypoxia inducible promoters that may drive non-protein-coding transcripts, 191 hypoxia responsive alternative promoters and detailed views of 120 novel as well as known hypoxia responsive genes. We further analyzed hypoxic response of different cells using additional 60 million TSS-tags and found that the degree of the gene-expression changes were different among cell lines, possibly reflecting cellular robustness against hypoxia. The novel dynamic figure of the human gene transcriptome will deepen our understanding of the transcriptional program of the human genome as well as bringing new insights into the biology of cancer cells in hypoxia.

Although the knowledge accumulated on the transcriptional regulations of eukaryotes is significant, the knowledge on their translational regulations remains limited. Thus, we performed a comprehensive detection of terminal oligo-pyrimidine (TOP), which is one of the well-characterized cis-regulatory motifs for translational controls located immediately downstream of the transcriptional start sites of mRNAs. Utilizing our precise 5'-end information of the full-length cDNAs, we could screen 1645 candidate TOP genes by position specific matrix search. Among them, not only 75 out of 78 ribosomal protein genes but also eight previously identified non-ribosomal-protein TOP genes were included. We further experimentally validated the translational activities of 83 TOP candidate genes. Clear translational regulations exerted on the stimulation of 12-O-tetradecanoyl-1-phorbol-13-acetate for at least 41 of them was observed, indicating that there should be a few hundreds of human genes which are subjected to regulation at translation levels via TOPs. Our result suggests that TOP genes code not only formerly characterized ribosomal proteins and translation-related proteins but also a wider variety of proteins, such as lysosome-related proteins and metabolism-related proteins, playing pivotal roles in gene expression controls in the majority of cellular mRNAs.

Although recent studies have revealed that the majority of human genes are subject to regulation of alternative promoters, the biological relevance of this phenomenon remains unclear. We have also demonstrated that roughly half of the human RefSeq genes examined contain putative alternative promoters (PAPs). Here we report large-scale comparative studies of PAPs between human and mouse counterpart genes. Detailed sequence comparison of the 17,245 putative promoter regions (PPRs) in 5463 PAP-containing human genes revealed that PPRs in only a minor fraction of genes (807 genes) showed clear evolutionary conservation as one or more pairs. Also, we found that there were substantial qualitative differences between conserved and non-conserved PPRs, with the latter class being AT-rich PPRs of relative minor usage, enriched in repetitive elements and sometimes producing transcripts that encode small or no proteins. Systematic luciferase assays of these PPRs revealed that both classes of PPRs did have promoter activity, but that their strength ranges were significantly different. Furthermore, we demonstrate that these characteristic features of the non-conserved PPRs are shared with the PPRs of previously discovered putative non-protein coding transcripts. Taken together, our data suggest that there are two distinct classes of promoters in humans, with the latter class of promoters emerging frequently during evolution.

In order to understand an overview of promoter activities intrinsic to primary DNA sequences in the human genome within a particular cell type, we carried out systematic quantitative luciferase assays of DNA fragments corresponding to putative promoters for 472 human genes which are expressed in HEK (human embryonic kidney epithelial) 293 cells. We observed the promoter activities of them were distributed in a bimodal manner; putative promoters belonging to the first group (with strong promoter activities) were designated as P1 and the latter (with weak promoter activities) as P2. The frequencies of the TATA-boxes, the CpG islands, and the overall G + C-contents were significantly different between these two populations, indicating there are two separate groups of promoters. Interestingly, similar analysis using 251 randomly isolated genomic DNA fragments showed that P2-type promoter occasionally occurs within the human genome. Furthermore, 35 DNA fragments corresponding to putative promoters of non-protein-coding transcripts (ncRNAs) shared similar features with the P2 in both promoter activities and sequence compositions. At least, a part of ncRNAs, which have been massively identified by full-length cDNA projects with no functional relevance inferred, may have originated from those sporadic promoter activities of primary DNA sequences inherent to the human genome.

We characterized the DNA methylation status at 144 tissue-biased and 37 non-tissue-biased alternative promoters of 61 human genes in five normal tissues. Analysis of the collected data revealed that (i) DNA methylation status differed greatly among alternative promoters belonging to the same gene; (ii) DNA methylation status differed between tissues for the majority of the individual promoters, and (iii) 80-90% of CpG-island-containing promoters were not methylated on either allele throughout the five tissues examined. Furthermore, although the statistical significance was not as clear as for the above features, we also found that (iv) the DNA methylation patterns of tissue-biased promoters changed more drastically than those of non-tissue-biased promoters; (v) tissue-biased promoters tended to be less methylated than their respective alternative promoters in the tissues where they were preferentially expressed, and (vi) the 'null' methylation pattern of a given promoter was enriched in the tissues where the transcription was most active. These findings together indicate that there are dynamic physiological changes of DNA methylation. DNA methylation appears to play a significant role in differential usage of alternative promoters and may be related to functional diversification between CpG-island-containing promoters and CpG-island-less promoters.

Using the 5'-end sequence data from 'oligo-capped' cDNAs, we generated a representative full-length cDNA dataset for 4870 RefSeq entries, and analyzed the 5' untranslated region (UTR) of these genes. To our surprise, about half of the 4870 genes had an upstream ATG before the ATG that starts the longest open reading frame (ORF), suggesting that about half of them have small ORFs in their 5' UTR of average length of 31 amino acids. They require attention for further analysis to identify their biological role.

In order to obtain precise information of 5′ end of mRNAs, we have developed the oligo-capping method that replaces the cap of mRNA with synthetic oligonucleotides. Using the data from oligo-capped cDNAs, we constructed database of transcriptional start sites (DBTSS; http://elmo.ims.u-tokyo.ac.jp/dbtss/ ). This database shows the information on distribution of transcriptional start sites and can be used to identify the core promoter region. Here we show the summery and the update of DBTSS and its future use on identification of regulatory motifs within the promoter.

Although the information of cDNAs is indispensable for analyzing gene function, most of the cDNA sequences stored in current databases are imperfect in the sense that they lack the precise information of 5' end termini. To overcome this difficulty, we have developed the oligo-capping method to obtain full-length cDNAs, the information of which has been partly deposited in public databases. In this study, we further constructed human cDNA libraries enriched in clones containing the cap structure to systematically explore the 5' end structure of expressed genes. Of approximately 217 402 5' end sequences obtained, 111 382 have been matched to cDNA sequences of known genes (7889 genes) and are presented in our new database, DataBase of Transcriptional Start Sites (DBTSS; http://elmo.ims.u-tokyo.ac.jp/dbtss/). Sequence comparison between our entries and those of a reference sequence database, RefSeq, revealed that 4683 (34%) of RefSeq sequences should be extended towards the 5' ends. We also mapped each sequence on the human draft genome sequence to identify its transcriptional start site, which provides us with more detailed information on distribution patterns of transcriptional start sites and adjacent regulatory regions.

Background Babesia bovis is an apicomplexan parasite that causes babesiosis in infected cattle. Genomes of pathogens contain promising information that can facilitate the development of methods for controlling infections. Although the genome of B. bovis is publically available, annotated gene models are not highly reliable prior to experimental validation. Therefore, we validated a preproposed gene model of B. bovis and extended the associated annotations on the basis of experimentally obtained full-length expressed sequence tags (ESTs). Results From in vitro cultured merozoites, 12,286 clones harboring full-length cDNAs were sequenced from both ends using the Sanger method, and 6,787 full-length cDNAs were assembled. These were then clustered, and a nonredundant referential data set of 2,115 full-length cDNA sequences was constructed. The comparison of the preproposed gene model with our data set identified 310 identical genes, 342 almost identical genes, 1,054 genes with potential structural inconsistencies, and 409 novel genes. The median length of 5' untranslated regions (UTRs) was 152 nt. Subsequently, we identified 4,086 transcription start sites (TSSs) and 2,023 transcriptionally active regions (TARs) by examining 5' ESTs. We identified ATGGGG and CCCCAT sites as consensus motifs in TARs that were distributed around -50 bp from TSSs. In addition, we found ACACA, TGTGT, and TATAT sites, which were distributed periodically around TSSs in cycles of approximately 150 bp. Moreover, related periodical distributions were not observed in mammalian promoter regions. Conclusions The observations in this study indicate the utility of integrated bioinformatics and experimental data for improving genome annotations. In particular, full-length cDNAs with one-base resolution for TSSs enabled the identification of consensus motifs in promoter sequences and demonstrated clear distributions of identified motifs. These observations allowed the illustration of a model promoter composition, which supports the differences in transcriptional regulation frameworks between apicomplexan parasites and mammals.

The application of semantic technologies to the integration of biological data and the interoperability of bioinformatics analysis and visualization tools has been the common theme of a series of annual BioHackathons hosted in Japan for the past five years. Here we provide a review of the activities and outcomes from the BioHackathons held in 2011 in Kyoto and 2012 in Toyama. In order to efficiently implement semantic technologies in the life sciences, participants formed various sub-groups and worked on the following topics: Resource Description Framework (RDF) models for specific domains, text mining of the literature, ontology development, essential metadata for biological databases, platforms to enable efficient Semantic Web technology development and interoperability, and the development of applications for Semantic Web data. In this review, we briefly introduce the themes covered by these sub-groups. The observations made, conclusions drawn, and software development projects that emerged from these activities are discussed. ispartof: Journal of Biomedical Semantics vol:5 issue:5 pages:1-13 ispartof: location:England status: published

The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

To support transcriptional regulation studies, we have constructed DBTSS (DataBase of Transcriptional Start Sites), which contains exact positions of transcriptional start sites (TSSs), determined with our own technique named TSS-seq, in the genomes of various species. In its latest version, DBTSS covers the data of the majority of human adult and embryonic tissues: it now contains 418 million TSS tag sequences from 28 tissues/cell cultures. Moreover, we integrated a series of our own transcriptomic data, such as the RNA-seq data of subcellular-fractionated RNAs as well as the ChIP-seq data of histone modifications and the binding of RNA polymerase II/several transcription factors in cultured cell lines into our original TSS information. We also included several external epigenomic data, such as the chromatin map of the ENCODE project. We further associated our TSS information with public or original single-nucleotide variation (SNV) data, in order to identify SNVs in the regulatory regions. These data can be browsed in our new viewer, which supports versatile search conditions of users. We believe that our new DBTSS will be an invaluable resource for interpreting the differential uses of TSSs and for identifying human genetic variations that are associated with disordered transcriptional regulation. DBTSS can be accessed at http://dbtss.hgc.jp.

Background Understanding the genome sequence-specific positioning of nucleosomes is essential to understand various cellular processes, such as transcriptional regulation and replication. As a typical example, the 10-bp periodicity of AA/TT and GC dinucleotides has been reported in several species, but it is still unclear whether this feature can be observed in the whole genomes of all eukaryotes. Results With Fourier analysis, we found that this is not the case: 84-bp and 167-bp periodicities are prevalent in primates. The 167-bp periodicity is intriguing because it is almost equal to the sum of the lengths of a nucleosomal unit and its linker region. After masking Alu elements, these periodicities were greatly diminished. Next, using two independent large-scale sets of nucleosome mapping data, we analyzed the distribution of nucleosomes in the vicinity of Alu elements and showed that (1) there are one or two fixed slot(s) for nucleosome positioning within the Alu element and (2) the positioning of neighboring nucleosomes seems to be in phase, more or less, with the presence of Alu elements. Furthermore, (3) these effects of Alu elements on nucleosome positioning are consistent with inactivation of promoter activity in Alu elements. Conclusions Our discoveries suggest that the principle governing nucleosome positioning differs greatly across species and that the Alu family is an important factor in primate genomes.

Ciona intestinalis is a useful model organism to analyze chordate development and genetics. However, unlike vertebrates, it shares a unique mechanism called trans-splicing with lower eukaryotes. In the computational analysis of trans-splicing in C. intestinalis we report here, we discovered that although the amount of non-trans-spliced and trans-spliced genes is usually equivalent, the expression ratio between the two groups varies significantly with tissues and developmental stages. Among the seven tissues studied, the observed ratios ranged from 2.53 in "gonad" to 19.53 in "endostyle", and during development they increased from 1.68 at the "egg" stage to 7.55 at the "juvenile" stage. We further hypothesize that this enrichment in trans-spliced mRNAs in early developmental stages might be related to the abundance of trans-spliced mRNAs in "gonad". Our analysis indicates that in C. intestinalis, although there may not exist strong fundamental requirements for genes to be trans-spliced, the populations of non-trans-spliced and trans-spliced genes are likely to be spatially and temporally regulated differently.

DBTSS is a database of transcriptional start sites, based on our unique collection of precise, experimentally determined 5′-end sequences of full-length cDNAs. Since its first release in 2002, several major updates have been made. In this update, we expanded the human transcriptional start site dataset by 19 million uniquely mapped, and RefSeq-associated, 5′-end sequences, which were generated by a newly introduced Solexa sequencer. Moreover, in order to provide means for interpreting those massive TSS data, we implemented two new analytical tools: one for connecting expression information with predicted transcription factor binding sites; the other for examining evolutionary conservation or species-specificity of promoters and transcripts, which can be browsed by our own comparative genome viewer. With the expanded dataset and the enhanced functionalities, DBTSS provides a unique platform that enables in-depth transcriptome analyses. DBTSS is accessible at http://dbtss.hgc.jp/.

Epigenetic regulation including DNA methylation plays an important role in several differentiation processes. We profiled global DNA methylation in the neural differentiation of P19 embryonic carcinoma cells using a microarray-based method called MIAMI. We found a genome-wide demethylation of genes. This suggests demethylation rather than methylation is important in neural differentiation.

We present the second version of Melina, a web-based tool for promoter analysis. Melina II shows potential DNA motifs in promoter regions with a combination of several available programs, Consensus, MEME, Gibbs sampler, MDscan and Weeder, as well as several parameter settings. It allows running a maximum of four programs simultaneously, and comparing their results with graphical representations. In addition, users can build a weight matrix from a predicted motif and apply it to upstream sequences of several typical genomes (human, mouse, S. cerevisiae, E. coli, B. subtilis or A. thaliana) or to public motif databases (JASPAR or DBTBS) in order to find similar motifs. Melina II is a client/server system developed by using Adobe (Macromedia) Flash and is accessible over the web at http://melina.hgc.jp.

Given the advent of massively parallel DNA sequencing, human microbiome is analyzed comprehensively by metagenomic approaches. However, the inter- and intra-individual variability and stability of the human microbiome remain poorly characterized, particularly at the intra-day level. This issue is of crucial importance for studies examining the effects of microbiome on human health. Here, we focused on bacteriome of oral plaques, for which repeated, time-controlled sampling is feasible. Eighty-one supragingival plaque subjects were collected from healthy individuals, examining multiple sites within the mouth at three time points (forenoon, evening, and night) over the course of 3 days. Bacterial composition was estimated by 16S rRNA sequencing and species-level profiling, resulting in identification of a total of 162 known bacterial species. We found that species compositions and their relative abundances were similar within individuals, and not between sampling time or tooth type. This suggests that species-level oral bacterial composition differs significantly between individuals, although the number of subjects is limited and the intra-individual variation also occurs. The majority of detected bacterial species (98.2%; 159/162), however, did not fluctuate over the course of the day, implying a largely stable oral microbiome on an intra-day time scale. In fact, the stability of this data set enabled us to estimate potential interactions between rare bacteria, with 40 co-occurrences supported by the existing literature. In summary, the present study provides a valuable basis for studies of the human microbiome, with significant implications in terms of biological and clinical outcomes.

By analyzing 1,780,295 5′-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

Gene expression profiling of cancer tissues is expected to contribute to our understanding of cancer biology as well as developments of new methods of diagnosis and therapy. Our collaborative efforts in Japan have been mainly focused on solid tumors such as breast, colorectal and hepatocellular cancers. The expression data are obtained by a high-throughput RT–PCR technique, and patients are recruited mainly from a single hospital. In the cancer gene expression database (CGED), the expression and clinical data are presented in a way useful for scientists interested in specific genes or biological functions. The data can be retrieved either by gene identifiers or by functional categories defined by Gene Ontology terms or the Swiss-Prot annotation. Expression patterns of multiple genes, selected by names or similarity search of the patterns, can be compared. Visual presentation of the data with sorting function enables users to easily recognize of relationships between gene expression and clinical parameters. Data for other cancers such as lung and thyroid cancers will be added in the near future. The URL of CGED is http://cged.hgc.jp.

It has been envisaged that CpG islands are often observed near the transcriptional start sites (TSS) of housekeeping genes. However, neither the precise positions of CpG islands relative to TSS of genes nor the correlation between the presence of the CpG islands and the expression specificity of these genes is well-understood. Using thousands of sequences with known TSS in human and mouse, we found that there is a clear peak in the distribution of CpG islands around TSS in the genes of these two species. Thus, we classified human (mouse) genes into 6600 (2948) CpG+ genes and 2619 (1830) CpG- ones, based on the presence of a CpG island within the -100: +100 region. We estimated the degree of each gene being a housekeeper by the number of cDNA libraries where its ESTs were detected. Then, the tendency that a gene lacking CpG islands around its TSS is expressed with a higher degree of tissue specificity turned out to be evolutionarily conserved. We also confirmed this tendency by analyzing the gene ontology annotation of classified genes. Since no such clear correlation was found in the control data (mRNAs, pre-mRNAs, and chromosome banding pattern), we concluded that the effect of a CpG island near the TSS should be more important than the global GC content of the region where the gene resides.

We report the generation and initial characterization of a large-scale collection of sequences of putative promoter regions (PPRs) of human and mouse genes. Based on our unique collection of 400,225 and 580,209 human and mouse full-length cDNAs, we determined exact transcriptional start sites (TSSs). Using positional information of the TSSs, we could retrieve adjacent sequences as PPRs for 8,793 and 6,875 human and mouse genes, respectively. The positions of the PPRs were 4 kb upstream to previously reported 5'-ends of cDNAs on average, demonstrating that full-length cDNA information is indispensable for this purpose. Among those PPRs supported by experimentally validated TSSs, 3,324 could be paired as mutually homologous genes between human and mouse and were used for the comprehensive comparative studies. The sequence identities in the proximal regions of the TSSs were 45% on average, and 22,794 putative transcription factor binding sites that are conserved between human and mouse were identified. The data resource created in the present work and the results of the sequences' initial characterization should lay the firm foundation for deciphering the transcriptional modulations of human genes. All the data were deposited and made available through a database for comparative studies, DBTSS.

Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a “block” structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5′ ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

The C 2 H 2 zinc finger motif found in many transcription factors is thought to be for nucleic acid binding and/or dimerization. Nearly 1% of eukaryote genes are estimated to encode this motif. We investigated the gene family encoding this motif in the Mus musculus mRNA by molecular indexing, a technique used to select a subpopulation of cDNA by ligation of adapters to cDNA fragments digested by a class IIS restriction enzyme(s). In place of oligo-dT primers in the original method, a polymerase chain reaction primer designed based on the conserved sequence of the C 2 H 2 zinc finger protein stranded cDNA was made from various mouse tissue mRNAs, digested with Fok I and Bsm AI, and joined with adapters. Amplification of the cDNA with an adapter primer and zinc finger-specific primer yielded products enriched in zinc finger protein genes. Fragments were separated by subsequent denaturing polyacrylamide gel electrophoresis, and characterized by DNA sequencing. Consequently, 259 C 2 H 2 zinc finger motif sequences were obtained, among which 166 were novel. Combined with the reported sequences, these mouse motif sequences were compared with those of other species such as Saccharomyces cerevisiae and Caenorhabditis elegans . Some of the amino acids in the motif sequence showed strong bias among species. Most of the novel sequences were supposed to be DNA-binding according to the surface potential of predicted tertiary structures.

Background CpG islands are observed in mammals and other vertebrates, generally escape DNA methylation, and tend to occur in the promoters of widely expressed genes. Another class of promoter has lower G+C and CpG contents, and is thought to be involved in the spatiotemporal regulation of gene expression. Non-vertebrate deuterostomes are reported to have a single class of promoter with high-frequency CpG dinucleotides, suggesting that this is the original type of promoter. However, the limited annotation of these genes has impeded the large-scale analysis of their promoters. Results To determine the origins of the two classes of vertebrate promoters, we chose Ciona intestinalis, an invertebrate that is evolutionarily close to the vertebrates, and identified its transcription start sites genome-wide using a next-generation sequencer. We indeed observed a high CpG content around the transcription start sites, but their levels in the promoters and background sequences differed much less than in mammals. The CpG-rich stretches were also fairly restricted, so they appeared more similar to mammalian CpG-poor promoters. Conclusions From these data, we infer that CpG islands are not sufficiently ancient to be found in invertebrates. They probably appeared early in vertebrate evolution via some active mechanism and have since been maintained as part of vertebrate promoters.

We developed a computer program that can predict the intrinsic promoter activities of primary human DNA sequences. We observed promoter activity using a quantitative luciferase assay and generated a prediction model using multiple linear regression. Our program achieved a prediction accuracy correlation coefficient of 0.87 between the predicted and observed promoter activities. We evaluated the prediction accuracy of the program using massive sequencing analysis of transcriptional start sites in vivo. We found that it is still difficult to predict transcript levels in a strictly quantitative manner in vivo; however, it was possible to select active promoters in a given cell from the other silent promoters. Using this program, we analyzed the transcriptional landscape of the entire human genome. We demonstrate that many human genomic regions have potential promoter activity, and the expression of some previously uncharacterized putatively non-protein-coding transcripts can be explained by our prediction model. Furthermore, we found that nucleosomes occasionally formed open chromatin structures with RNA polymerase II recruitment where the program predicted significant promoter activities, although no transcripts were observed.

Background: Individual human carcinomas have distinct biological and clinical properties: geneexpression profiling is expected to unveil the underlying molecular features. Particular interest has been focused on potential diagnostic and therapeutic applications. Solid tumors, such as colorectal carcinoma, present additional obstacles for experimental and data analysis. Results: We analyzed the expression levels of 1,536 genes in 100 colorectal cancer and 11 normal tissues using adaptor-tagged competitive PCR, a high-throughput reverse transcription-PCR technique. A parametric clustering method using the Gaussian mixture model and the Bayes inference revealed three groups of expressed genes. Two contained large numbers of genes. One of these groups correlated well with both the differences between tumor and normal tissues and the presence or absence of distant metastasis, whereas the other correlated only with the tumor/normal difference. The third group comprised a small number of genes. Approximately half showed an identical expression pattern, and cancer tissues were classified into two groups by their expression levels. The high-expression group had strong correlation with distant metastasis, and a poorer survival rate than the low-expression group, indicating possible clinical applications of these genes. In addition to c-yes, a homolog of a viral oncogene, prognostic indicators included genes specific to glial cells, which gives a new link between malignancy and ectopic gene expression. Conclusions: The malignancy of human colorectal carcinoma is correlated with a unique expression pattern of a specific group of genes, allowing the classification of tumor tissues into two clinically distinct groups.

SummaryThe transcription factor Foxp3 is essential for the development of regulatory T (Treg) cells, yet its expression is insufficient for establishing the Treg cell lineage. Here we showed that Treg cell development was achieved by the combination of two independent processes, i.e., the expression of Foxp3 and the establishment of Treg cell-specific CpG hypomethylation pattern. Both events were induced by T cell receptor stimulation. The Treg cell-type CpG hypomethylation began in the thymus and continued to proceed in the periphery and could be fully established without Foxp3. The hypomethylation was required for Foxp3+ T cells to acquire Treg cell-type gene expression, lineage stability, and full suppressive activity. Thus, those T cells in which the two events have concurrently occurred are developmentally set into the Treg cell lineage. This model explains how Treg cell fate and plasticity is controlled and can be exploited to generate functionally stable Treg cells.

The high similarity of tunicates and vertebrates during their development coupled with the transparency of tunicate larvae, their well-studied cell lineages and the availability of simple and efficient transgenesis methods makes of this subphylum an ideal system for the investigation of vertebrate physiological and developmental processes. Recently, the sequencing of two different Ciona genomes has lead to the identification of numerous genes. In order to better understand the regulation of these genes, a database was created containing information on regulation of tunicate genes collected from literature. It includes for instance information regarding the minimal promoter length, the transcription factors involved and their binding sites, as well as the localization of the gene expression. Additionally, binding sites for characterized transcription factors were predicted based on published in vitro recognition sites. Comparison of the promoters of homologous genes in different species is also provided to allow identification of conserved cis elements. At the time of writing, information about 184 promoters, containing 73 identified binding sites and >2000 newly predicted binding sites is available. This database is accessible at http://dbtgr.hgc.jp.

Background Interspecies sequence comparison is a powerful tool to extract functional or evolutionary information from the genomes of organisms. A number of studies have compared protein sequences or promoter sequences between mammals, which provided many insights into genomics. However, the correlation between protein conservation and promoter conservation remains controversial. Results We examined promoter conservation as well as protein conservation for 6,901 human and mouse orthologous genes, and observed a very weak correlation between them. We further investigated their relationship by decomposing it based on functional categories, and identified categories with significant tendencies. Remarkably, the 'ribosome' category showed significantly low promoter conservation, despite its high protein conservation, and the 'extracellular matrix' category showed significantly high promoter conservation, in spite of its low protein conservation. Conclusion Our results show the relation of gene function to protein conservation and promoter conservation, and revealed that there seem to be nonparallel components between protein and promoter sequence evolution.

Background The interaction between biological researchers and the bioinformatics tools they use is still hampered by incomplete interoperability between such tools. To ensure interoperability initiatives are effectively deployed, end-user applications need to be aware of, and support, best practices and standards. Here, we report on an initiative in which software developers and genome biologists came together to explore and raise awareness of these issues: BioHackathon 2009. Results Developers in attendance came from diverse backgrounds, with experts in Web services, workflow tools, text mining and visualization. Genome biologists provided expertise and exemplar data from the domains of sequence and pathway analysis and glyco-informatics. One goal of the meeting was to evaluate the ability to address real world use cases in these domains using the tools that the developers represented. This resulted in i) a workflow to annotate 100,000 sequences from an invertebrate species; ii) an integrated system for analysis of the transcription factor binding sites (TFBSs) enriched based on differential gene expression data obtained from a microarray experiment; iii) a workflow to enumerate putative physical protein interactions among enzymes in a metabolic pathway using protein structure data; iv) a workflow to analyze glyco-gene-related diseases by searching for human homologs of glyco-genes in other species, such as fruit flies, and retrieving their phenotype-annotated SNPs. Conclusions Beyond deriving prototype solutions for each use-case, a second major purpose of the BioHackathon was to highlight areas of insufficiency. We discuss the issues raised by our exploration of the problem/solution space, concluding that there are still problems with the way Web services are modeled and annotated, including: i) the absence of several useful data or analysis functions in the Web service "space"; ii) the lack of documentation of methods; iii) lack of compliance with the SOAP/WSDL specification among and between various programming-language libraries; and iv) incompatibility between various bioinformatics data formats. Although it was still difficult to solve real world problems posed to the developers by the biological researchers in attendance because of these problems, we note the promise of addressing these issues within a semantic framework.