Nobuaki Kono

Pathway Projector: Web-Based Zoomable Pathway Browser Using KEGG Atlas and Google Maps API

Background Biochemical pathways provide an essential context for understanding comprehensive experimental data and the systematic workings of a cell. Therefore, the availability of online pathway browsers will facilitate post-genomic research, just as genome browsers have contributed to genomics. Many pathway maps have been provided online as part of public pathway databases. Most of these maps, however, function as the gateway interface to a specific database, and the comprehensiveness of their represented entities, data mapping capabilities, and user interfaces are not always sufficient for generic usage. Methodology/Principal Findings We have identified five central requirements for a pathway browser: (1) availability of large integrated maps showing genes, enzymes, and metabolites; (2) comprehensive search features and data access; (3) data mapping for transcriptomic, proteomic, and metabolomic experiments, as well as the ability to edit and annotate pathway maps; (4) easy exchange of pathway data; and (5) intuitive user experience without the requirement for installation and regular maintenance. According to these requirements, we have evaluated existing pathway databases and tools and implemented a web-based pathway browser named Pathway Projector as a solution. Conclusions/Significance Pathway Projector provides integrated pathway maps that are based upon the KEGG Atlas, with the addition of nodes for genes and enzymes, and is implemented as a scalable, zoomable map utilizing the Google Maps API. Users can search pathway-related data using keywords, molecular weights, nucleotide sequences, and amino acid sequences, or as possible routes between compounds. In addition, experimental data from transcriptomic, proteomic, and metabolomic analyses can be readily mapped. Pathway Projector is freely available for academic users at http://www.g-language.org/PathwayProjector/.

Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes

BackgroundDuring the replication process of bacteria with circular chromosomes, an odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. However, many bacteria harbor a conserved dimer resolution machinery consisting of one or two tyrosine recombinases, XerC and XerD, and their 28-bp target site, dif.ResultsTo study the evolution of the dif/ XerCD system and its relationship with replication termination, we report the comprehensive prediction of dif sequences in silico using a phylogenetic prediction approach based on iterated hidden Markov modeling. Using this method, dif sites were identified in 641 organisms among 16 phyla, with a 97.64% identification rate for single-chromosome strains. The dif sequence positions were shown to be strongly correlated with the GC skew shift-point that is induced by replicational mutation/selection pressures, but the difference in the positions of the predicted dif sites and the GC skew shift-points did not correlate with the degree of replicational mutation/selection pressures.ConclusionsThe sequence of dif sites is widely conserved among many bacterial phyla, and they can be computationally identified using our method. The lack of correlation between dif position and the degree of GC skew suggests that replication termination does not occur strictly at dif sites.

Genome Projector: zoomable genome map with multiple views.

BackgroundMolecular biology data exist on diverse scales, from the level of molecules to -omics. At the same time, the data at each scale can be categorised into multiple layers, such as the genome, transcriptome, proteome, metabolome, and biochemical pathways. Due to the highly multi-layer and multi-dimensional nature of biological information, software interfaces for database browsing should provide an intuitive interface that allows for rapid migration across different views and scales. The Zoomable User Interface (ZUI) and tabbed browsing have proven successful for this purpose in other areas, especially to navigate the vast information in the World Wide Web.ResultsThis paper presents Genome Projector, a Web-based gateway for genomics information with a zoomable user interface using Google Maps API, equipped with four seamlessly accessible and searchable views: a circular genome map, a traditional genome map, a biochemical pathways map, and a DNA walk map. The Web application for 320 bacterial genomes is available at http://www.g-language.org/GenomeProjector/. All data and software including the source code, documentations, and development API are freely available under the GNU General Public License. Zoomable maps can be easily created from any image file using the development API, and an online data mapping service for Genome Projector is also available at our Web site.ConclusionGenome Projector is an intuitive Web application for browsing genomics information, implemented with a zoomable user interface and tabbed browsing utilising Google Maps API and Asynchronous JavaScript and XML (AJAX) technology.

Quantitative assay for TALEN activity at endogenous genomic loci

Summary Artificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshift-mediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZ&agr; sequence for the conventional blue–white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.

Metabolic Flux Analysis and Visualization

One of the ultimate goals of systems biology research is to obtain a comprehensive understanding of the control mechanisms of complex cellular metabolisms. Metabolic Flux Analysis (MFA) is a important method for the quantitative estimation of intracellular metabolic flows through metabolic pathways and the elucidation of cellular physiology. The primary challenge in the use of MFA is that many biological networks are underdetermined systems; it is therefore difficult to narrow down the solution space from the stoichiometric constraints alone. In this tutorial, we present an overview of Flux Balance Analysis (FBA) and (13)C-Metabolic Flux Analysis ((13)C-MFA), both of which are frequently used to solve such underdetermined systems, and we demonstrate FBA and (13)C-MFA using the genome-scale model and the central carbon metabolism model, respectively. Furthermore, because such comprehensive study of intracellular fluxes is inherently complex, we subsequently introduce various pathway mapping and visualization tools to facilitate understanding of these data in the context of the pathways. Specific visualization of MFA results using the BioCyc Omics Viewer and Pathway Projector are shown as illustrative examples.

Restauro-G: a rapid genome re-annotation system for comparative genomics.

Annotations of complete genome sequences submitted directly from sequencing projects are diverse in terms of annotation strategies and update frequencies. These inconsistencies make comparative studies difficult. To allow rapid data preparation of a large number of complete genomes, automation and speed are important for genome re-annotation. Here we introduce an open-source rapid genome re-annotation software system, Restauro-G, specialized for bacterial genomes. Restauro-G re-annotates a genome by similarity searches utilizing the BLAST-Like Alignment Tool, referring to protein databases such as UniProt KB, NCBI nr, NCBI COGs, Pfam, and PSORTb. Re-annotation by Restauro-G achieved over 98% accuracy for most bacterial chromosomes in comparison with the original manually curated annotation of EMBL releases. Restauro-G was developed in the generic bioinformatics workbench G-language Genome Analysis Environment and is distributed at http://restauro-g.iab.keio.ac.jp/ under the GNU General Public License.

Evaluation of the impact of RNA preservation methods of spiders for de novo transcriptome assembly

With advances in high‐throughput sequencing technologies, de novo transcriptome sequencing and assembly has become a cost‐effective method to obtain comprehensive genetic information of a species of interest, especially in nonmodel species with large genomes such as spiders. However, high‐quality RNA is essential for successful sequencing, and sample preservation conditions require careful consideration for the effective storage of field‐collected samples. To this end, we report a streamlined feasibility study of various storage conditions and their effects on de novo transcriptome assembly results. The storage parameters considered include temperatures ranging from room temperature to −80°C; preservatives, including ethanol, RNAlater, TRIzol and RNAlater‐ICE; and sample submersion states. As a result, intact RNA was extracted and assembly was successful when samples were preserved at low temperatures regardless of the type of preservative used. The assemblies as well as the gene expression profiles were shown to be robust to RNA degradation, when 30 million 150‐bp paired‐end reads are obtained. The parameters for sample storage, RNA extraction, library preparation, sequencing and in silico assembly considered in this work provide a guideline for the study of field‐collected samples of spiders.

Undesigned selection for replication termination of bacterial chromosomes.

The oriC DNA replication origin in bacterial chromosomes, the location of which appears to be physically identified, is genetically regulated by relevant molecular machinery. In contrast, the location of the terminus remains obscure for many bacterial replicons, except for terC, the proposed and well-studied chromosome termination site in certain bacteria. The terC locus, which is composed of specific sequences for its binding protein, is located at a site opposite from oriC, exhibiting a symmetric structure around the oriC-terC axis. Here, we investigated Bacillus subtilis 168 strains whose axes were hindered and found that the native terC function was robust. However, eradication of terminus region specific binding protein resulted in the natural terC sites not being used for termination; instead, new termini were selected at a location exactly opposite to oriC. We concluded that replication generally terminates at the loci where the two approaching replisomes meet. This site was automatically selected, and two replisomes moving at the same rate supported symmetrical chromosome structures relative to oriC. The rule, which was even validated by artificial chromosomes irrespective of oriC, should be general for replicons administered by two replisomes.

The complete mitochondrial genome of Eumeta variegata (Lepidoptera: Psychidae)

Abstract The complete mitochondrial genome of Eumeta variegate, largest bagworm moth in Japan, has been sequenced using a nanopore sequencer as a single long read. The genome has a total length of 16,601 bp, consisting of 13 protein-coding genes, 20 tRNA, 2 rRNA genes, and an AT-rich control region. The nucleotide composition was extremely AT-rich, with 42.4% A, 40.4% T, 6.67% G, and 10.6% C. This is the second report of a complete mitochondrial genome of Psychidae, and the sequence information together with a phylogenetic analysis would provide a reference data in the future studies of Lepidoptera and Psychidae.

Far rapid synthesis of giant DNA in the Bacillus subtilis genome by a conjugation transfer system

Bacillus subtilis offers a platform for giant DNA synthesis, which is mediated by the connection of overlapping DNA segments called domino DNA, in the cloning locus of the host. The domino method was successfully used to produce DNA fragments as large as 3500 kbp. However, domino DNA is limited to <100 kbp because of size restrictions regarding the transformation (TF) of B. subtilis competent cells. A novel conjugal transfer (CT) method was designed to eliminate the TF size limit. The CT method enables rapid and efficient domino reactions in addition to the transfer of giant DNA molecules of up to 875 kbp to another B. subtilis genome within 4 hours. The combined use of the TF and CT should enable significantly rapid giant DNA production.