Bacterial genome mapper: A comparative bacterial genome mapping tool

Recently, next generation sequencing (NGS) technologies have led to a revolutionary increase in sequencing speed and costefficacy. Consequently, a vast number of contigs from many recently sequenced bacterial genomes remain to be accurately mapped and annotated, requiring the development of more convenient bioinformatics programs. In this paper, we present a newly developed web-based bioinformatics program, Bacterial Genome Mapper, which is suitable for mapping and annotating contigs that have been assembled from bacterial genome sequence raw data. By constructing a multiple alignment map between target contig sequences and two reference bacterial genome sequences, this program also provides very useful comparative genomics analysis of draft bacterial genomes. AVAILABILITY: The database is available for free at http://mbgm.kribb.re.kr.     

全基因组测序在病原菌分型与溯源中的应用研究进展

细菌分子分型已成为监测细菌感染性疾病的暴发流行与明确病原菌传播途径的重要工具.随着全基因组测序技术的日益兴起,公共数据库中已产生大量的细菌基因组数据,迫切需要研究人员充分认识和理解该技术,并掌握多种生物信息学工具挖掘并解读测序数据.本文系统概述了全基因组测序技术与生物信息学工具在病原菌分型与溯源中的应用,并对全基因组测...     

Comparison of direct genome restriction enzyme analysis and pulsed-field gel electrophoresis for typing of Vibrio vulnificus and their correspondence with multilocus sequence typing data

We compared the potential of direct genome restriction enzyme analysis (DGREA) and pulsed-field gel electrophoresis (PFGE) for discriminating Vibrio vulnificus isolates from clinical (23) and environmental (17) sources. The genotypes generated by both methodologies were compared to previous multilocus sequence typing (MLST) data. DGREA established clearer relationships among V. vulnificus strains and was more consistent with MLST than with PFGE. DGREA is a very promising tool for epidemiological and ecological studies of V. vulnificus.     

A whole genome screen for HIV restriction factors

Background

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8⁺ T-cells in various clinical status of HTLV-1 infection.

Results

By using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8⁺ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8⁺ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8⁺ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8⁺ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8⁺ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8⁺ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8⁺ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8⁺ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8⁺ T-cells.

Conclusions

These findings indicated that Tax-specific CD8⁺ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8⁺ T-cells in early stages.     

Automatic typing of bacterial isolates

An oligonucleotide probe was selected from the 16S rRNA gene of Clavibacter michiganensis subsp. sepedonicus for specific in situ hybridization. The rhodamine-labelled oligonucleotide probe was used in conjunction with an indirect immunofluorescence procedure based on a specific monoclonal antibody detected with a fluorescein-labelled conjugate. Simultaneous labelling of bacterial cells with the oligonucleotide and antibody probes allows accurate microscopic identification of single cells when isolation and other methods of confirming bacterial identity are not possible.     

Multi-locus sequence typing: a tool for global epidemiology

The characterization of pathogenic isolates plays a pivotal role in the epidemiology of infectious diseases, generating the information necessary for identifying, tracking, and intervening against disease outbreaks. In 1998 multi-locus sequence typing (MLST) was proposed as a nucleotide sequence-based approach that could be applied to many bacterial pathogens. It combined developments in high-throughput sequencing and bioinformatics with established population genetics techniques to provide a portable, reproducible, and scalable typing system that reflected the population and evolutionary biology of bacterial pathogens. MLST schemes have been developed for a variety of procaryotic and eucaryotic pathogens and the data generated have contributed to both epidemiological surveillance and fundamental studies of pathogen biology.     

DNA-guided genome editing tool

正Technologies for targeted modification of eukaryotic genomes are of great value for elucidating and manipulating gene functions in fundamental and applied biological research.In the past two decades,genome editing tools have evolved quickly from random DNA mutagenesis-inducing chemicals or ionizing radiation to programmable sequencespecific nucleases(SSNs)with the CRISPR/Cas technology as the most significant breakthrough(Gaj et al.,2013).     

基于基因组序列的脑膜炎奈瑟菌分型方法

[目的]建立并评估1种适宜的脑膜炎奈瑟菌(Neisseria meningitidis,Nm)基因组分子分型方法.[方法]本研究以125株代表性Nm菌株的基因组序列为对象,建立了基于核心基因SNP的基因组分型方法,并与pubMLST网站公布的MLST和cgMLST分型方法进行比较.[结果]基于核心基因SNP的基因组分型...     

Web-based visualization tools for bacterial genome alignments

With the increase in the flow of sequence data, both in contigs and whole genomes, visual aids for comparison and analysis studies are becoming imperative. We describe three web-based tools for visualizing alignments of bacterial genomes. The first, called Enteric, produces a graphical, hypertext view of pairwise alignments between a reference genome and sequences from each of several related organisms, covering 20 kb around a user-specified position. Insertions, deletions and rearrangements relative to the reference genome are color-coded, which reveals many intriguing differences among genomes. The second, Menteric, computes and displays nucleotide-level multiple alignments of the same sequences, together with annotations of ORFs and regulatory sites, in a 1 kb region surrounding a given address. The third, a Java-based viewer called Maj, combines some features of the previous tools, and adds a zoom-in mechanism. We compare the Escherichia coli K-12 genome with the partially sequenced genomes of Klebsiella pneumoniae, Yersinia pestis, Vibrio cholerae, and the Salmonella enterica serovars Typhimurium, Typhi and Paratyphi A. Examination of the pairwise and multiple alignments in a region allows one to draw inferences about regulatory patterns and functional assignments. For example, these tools revealed that rffH, a gene involved in enterobacterial common antigen (ECA) biosynthesis, is partly deleted in one of the genomes. We used PCR to show that this deletion occurs sporadically in some strains of some serovars of S.enterica subspecies I but not in any strains tested from six other subspecies. The resulting cell surface diversity may be associated with selection by the host immune response.     

The quest for the minimal bacterial genome

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New ultrarare restriction site-carrying transposons for bacterial genomics

Electrophoretic separation of macrorestriction fragments containing a particular genomic interval has until recently depended on fortuitously placed native rare restriction sites. We present new IS10-based transposons carrying the yeast intron-encoded I-SceI restriction site which is absent from most prokaryotic and eukaryotic genomes. Construction of the plasmid vectors containing them is described. Analysis by conventional or Pulsed Field gel electrophoresis of the DNA fragments generated by the I-SceI digestion reveals the physical distance between genomic insertions of these transposons: use of the same approach to subdivide the chromosome of Escherichia coli K-12 into equivalently sized contiguous/nonoverlapping I-SceI fragments is demonstrated. Because coordinates for the loci delimited by their insertions can be readily determined in different isolates by either physical or genetic manipulations, these transposons allow sufficient flexibility for species-wide bacterial genomics.     

AgdbNet – antigen sequence database software for bacterial typing

Background  

Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences.     

Dynamic bacterial genome organization

Recently completed projects of sequencing chromosomal fragments and entire chromosomes, as well as physical mapping of genomes, have opened novel inroads to the understanding of the biology of bacterial genomes. From these studies one may draw some conclusions. (i) The organization of orthologous genes on the bacterial chromosome is not conserved during evolution. (ii) The bacterial genome is more complex and also more flexible than hitherto thought. Genetic elements are sometimes part of the chromosome, while at other times they are independent elements or parts of alternative replicons (e.g. large plasmids). Such replicons, carrying essential genes, now seem to deserve the designation 'secondary chromosomes'. A study of the regulation of replication and segregation of these essential genetic elements will be of great interest.     

A graphic tool for circular genome maps.

A program has been developed for drawing map of circular DNA such as organelle or plasmid genome. Total size of the genome, gene names and positions, and other details, if required, should be prepared in a simple format text file then the program process it to a PostScript(R) (PS) file with which you can print a image of the map on suitable device(s). The final touch on the map can be given through editing the PS file.     

Lysogenicity as a tool for bacteriophage typing ofSalmonella Paratyphi B

Summary and Conclusions The serological identity of the phage produced by a spontaneously lysogenic strain ofS. paratyphi B depends on the bacterial type of that particular strain. The frequency of the phenomenon of spontaneous lysogenicity is such, in the case ofS. paratyphi B, that it can be utilised as a tool for typing.It is clear, however, that the possibilities of the method are limited. Firstly, types exist that do not as a rule produce bacteriophages. Secondly, alysogenic strains may exist of types that as a rule are lysogenic.Therefore the method can be utilised only together with, and as a check upon, a system of phage reactions.In Holland the types Kampen and Leeuwarden are frequent types, that can readily be recognised by the identity of the phages they produce. This is sufficient to justify the use of the method.The relation shown between true lysogenicity and bacterial types may be of theoretical interest. Perhaps it will be possible in this way to relate every existing phage to a bacterial type. The theoretical aspects will, however, be discussed elsewhere.     

Structural proteomics: a tool for genome annotation

In any newly sequenced genome, 30% to 50% of genes encode proteins with unknown molecular or cellular function. Fortunately, structural genomics is emerging as a powerful approach of functional annotation. Because of recent developments in high-throughput technologies, ongoing structural genomics projects are generating new structures at an unprecedented rate. In the past year, structural studies have identified many new structural motifs involved in enzymatic catalysis or in binding ligands or other macromolecules (DNA, RNA, protein). The efficiency by which function is deduced from structure can be further improved by the integration of structure with bioinformatics and other experimental approaches, such as screening for enzymatic activity or ligand binding.     

Genomic methylation: a tool for typing Helicobacter pylori isolates

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.     

AuberGene--a sensitive genome alignment tool

MOTIVATION: The accumulation of genome sequences will only accelerate in the coming years. We aim to use this abundance of data to improve the quality of genomic alignments and devise a method which is capable of detecting regions evolving under weak or no evolutionary constraints. RESULTS: We describe a genome alignment program AuberGene, which explores the idea of transitivity of local alignments. Assessment of the program was done based on a 2 Mbp genomic region containing the CFTR gene of 13 species. In this region, we can identify 53% of human sequence sharing common ancestry with mouse, as compared with 44% found using the usual pairwise alignment. Between human and tetraodon 93 orthologous exons are found, as compared with 77 detected by the pairwise human-tetraodon comparison. AuberGene allows the user to (1) identify distant, previously undetected, conserved orthogonal regions such as ORFs or regulatory regions; (2) identify neutrally evolving regions in related species which are often overlooked by other alignment programs; (3) recognize false orthologous genomic regions. The increased sensitivity of the method is not obtained at the cost of reduced specificity. Our results suggest that, over the CFTR region, human shares 10% more sequence with mouse than previously thought ( approximately 50%, instead of 40% found with the pairwise alignment).