Arabidopsis genome analysis as exemplified by analysis of chromosome 4

During the last decade the small cruciferous plant Arabidopsis thaliana has become a model organism for flowering plants. Sequencing and analysis of the Arabidopsis genome is nearing completion. Beside an overview on methods and strategies for Arabidopsis genome analysis, a summary of the results from the first analysis is presented.This includes an overview on chromosomal organisation and topological features as well as a first comparison with other genomes.     

The (in)complete organelle genome: exploring the use and nonuse of available technologies for characterizing mitochondrial and plastid chromosomes

Not long ago, scientists paid dearly in time, money and skill for every nucleotide that they sequenced. Today, DNA sequencing technologies epitomize the slogan ‘faster, easier, cheaper and more’, and in many ways, sequencing an entire genome has become routine, even for the smallest laboratory groups. This is especially true for mitochondrial and plastid genomes. Given their relatively small sizes and high copy numbers per cell, organelle DNAs are currently among the most highly sequenced kind of chromosome. But accurately characterizing an organelle genome and the information it encodes can require much more than DNA sequencing and bioinformatics analyses. Organelle genomes can be surprisingly complex and can exhibit convoluted and unconventional modes of gene expression. Unravelling this complexity can demand a wide assortment of experiments, from pulsed‐field gel electrophoresis to Southern and Northern blots to RNA analyses. Here, we show that it is exactly these types of ‘complementary’ analyses that are often lacking from contemporary organelle genome papers, particularly short ‘genome announcement’ articles. Consequently, crucial and interesting features of organelle chromosomes are going undescribed, which could ultimately lead to a poor understanding and even a misrepresentation of these genomes and the genes they express. High‐throughput sequencing and bioinformatics have made it easy to sequence and assemble entire chromosomes, but they should not be used as a substitute for or at the expense of other types of genomic characterization methods.     

Artemis: sequence visualization and annotation

SUMMARY: Artemis is a DNA sequence visualization and annotation tool that allows the results of any analysis or sets of analyses to be viewed in the context of the sequence and its six-frame translation. Artemis is especially useful in analysing the compact genomes of bacteria, archaea and lower eukaryotes, and will cope with sequences of any size from small genes to whole genomes. It is implemented in Java, and can be run on any suitable platform. Sequences and annotation can be read and written directly in EMBL, GenBank and GFF format. AVAILABITLTY: Artemis is available under the GNU General Public License from http://www.sanger.ac.uk/Software/Artemis     

原核生物蛋白质基因组学研究进展

随着基因组测序技术的不断发展,大量微生物基因组序列可以在短时间内得以准确鉴定。为了进一步探究基因组的结构与功能,基于序列特征与同源特征的基因组注释算法广泛应用于新测序物种。然而受基因组测序质量以及算法本身准确性偏低等问题的影响,现有的基因组注释存在着相当比例的假基因以及注释错误,尤其是蛋白质N端的注释错误。为了弥补基因组注释的不足,以基因芯片或RNA-seq为核心的转录组测序技术和以串联质谱为核心的蛋白质组测序技术可以高通量地对基因的转录和翻译产物进行精确测定,进而实现预测基因结构的实验验证。然而,原核生物细胞中存在的大量非编码RNA给转录组测序技术引入了污染数据,限制了其对基因组注释的应用。相对而言,以串联质谱技术为核心的蛋白质组学测序可以在短时间内鉴定到生物体内大量的蛋白质,实现注释基因的验证甚至校准。已成为基因组注释和重注释的重要依据,并因而衍生了"蛋白质基因组学"的新研究方向。文中首先介绍传统的基于序列预测和同源比对的基因组注释算法,指出其中存在的不足。在此基础上,结合转录组学与蛋白质组学的技术特点,分析蛋白质组学对于原核生物基因组注释的优势,总结现阶段大规模蛋白质基因组学研究的进展情况。最后从信息学角度指出当前蛋白质组数据进行基因组重注释存在的问题与相应的解决方案,进而探讨未来蛋白质基因组学的发展方向。     

DNAPlotter: circular and linear interactive genome visualization

Summary: DNAPlotter is an interactive Java application for generatingcircular and linear representations of genomes. Making use ofthe Artemis libraries to provide a user-friendly method of loadingin sequence files (EMBL, GenBank, GFF) as well as data fromrelational databases, it filters features of interest to displayon separate user-definable tracks. It can be used to producepublication quality images for papers or web pages. Availability: DNAPlotter is freely available (under a GPL licence)for download (for MacOSX, UNIX and Windows) at the WellcomeTrust Sanger Institute web sites: http://www.sanger.ac.uk/Software/Artemis/circular/ Contact: artemis{at}sanger.ac.uk Associate Editor: John Quackenbush     

Automated interpretation of metabolic capacity from genome and metagenome sequences

The KEGG pathway maps are widely used as a reference data set for inferring high-level functions of the organism or the ecosystem from its genome or metagenome sequence data. The KEGG modules, which are tighter functional units often corresponding to subpathways in the KEGG pathway maps, are designed for better automation of genome interpretation. Each KEGG module is represented by a simple Boolean expression of KEGG Orthology (KO) identifiers (K numbers), enabling automatic evaluation of the completeness of genes in the genome. Here we focus on metabolic functions and introduce reaction modules for improving annotation and signature modules for inferring metabolic capacity. We also describe how genome annotation is performed in KEGG using the manually created KO database and the computationally generated SSDB database. The resulting KEGG GENES database with KO (K number) annotation is a reference sequence database to be compared for automated annotation and interpretation of newly determined genomes.     

三种蚜虫的线粒体基因组数据

完整的线粒体基因组已被广泛应用于分子进化、基因组学、系统发育等方面的研究。蚜虫是一类重要的农林业害虫, 但目前公开报道的蚜虫完整线粒体基因组非常有限, 因此获得更多的基因组数据对相关研究具有重要价值。本文报道了榕毛管蚜(Greenidea ficicola)、橘二叉蚜(Aphis aurantia)和油杉纩蚜(Mindarus keteleerifoliae) 3种蚜虫的完整线粒体基因组的序列、详细注释信息、基因结构图、密码子使用情况等。该数据集可为昆虫系统发育关系、种群分化格局、害虫防治等方面的工作提供帮助。     

啮总目昆虫的线粒体基因组多样性及系统发育研究进展

啮总目包括啮虫目（皮虱和书虱）和虱目（羽虱和吸虱）,是农业和医学等领域具有重要经济意义和研究价值的类群,目前已鉴定和描述的物种超过10 000个。啮总目昆虫线粒体基因组的变异性在昆虫各类群中最为剧烈,这些变异包括基因组的结构、基因排序、基因含量和链上分布等诸多方面。本文全面分析和总结了啮总目昆虫裂化线粒体基因组的进化属性,并结合两侧对称动物线粒体基因组的裂化特征重构了线粒体基因组环裂化的过程。引入“线粒体基因组核型”的概念来描述动物线粒体基因组丰富的变异程度。动物线粒体的染色体有减小的趋势,而线粒体基因组的裂化正是体现这种趋势的一种重要策略。同时,总结和探讨了目前具有争议的啮总目主要类群间的系统发育关系。本综述为啮总目昆虫线粒体基因组学、啮总目系统发生关系以及两侧对称动物线粒体基因组进化模式的研究提供一个新的视角。     

Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus

We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time.The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.     

Constructing plant radiation hybrid panels

Radiation hybrid (RH) mapping, a somatic cell genetic technique, has been developed in animal systems as a general approach for the construction of long-range physical maps of chromosomes. This statistical method relies on X-ray induced breakage of chromosomes to determine the physical distance between markers, as well as their order on the chromosome. The method can be applied to single chromosomes or across the whole genome. The generation of plant (barley) radiation hybrids and their culture in vitro is described here. PCR-based marker systems are used to verify hybrid status and to demonstrate genome coverage. RH panels of the type generated can be used for physical mapping, map-based cloning, or sequence contig assembly. RH resources will greatly aid the physical characterisation of crop plants with large genomes.     

蛋白质基因组学：运用蛋白质组技术注释基因组

随着高通量DNA测序技术的飞速发展,越来越多的物种完成了基因组测序．定位编码基因、确定编码基因结构是基因组注释的基本任务,然而以往的基因组注释方法主要依赖于DNA及RNA序列信息．为了更加精确地解读完成测序的基因组,我们需要整合多种类型的组学数据进行基因组注释．近年来,基于串联质谱技术的蛋白质组学已经发展成熟,实现了对蛋白质组的高覆盖,使得利用串联质谱数据进行基因组注释成为可能．串联质谱数据一方面可以对已注释的基因进行表达验证,另一方面还可以校正原注释基因,进而发现新基因,实现对基因组序列的重新注释．这正是当前进展较快的蛋白质基因组学的研究内容．利用该方法系统地注释已完成测序的基因组已成为解读基因组的一个重要补充．本文综述了蛋白质基因组学的主要研究内容和研究方法,并展望了该研究方向未来的发展．     

Sequencing and assembly of low copy and genic regions of isolated Triticum aestivum chromosome arm 7DS

The genome of bread wheat (Triticum aestivum) is predicted to be greater than 16 Gbp in size and consist predominantly of repetitive elements, making the sequencing and assembly of this genome a major challenge. We have reduced genome sequence complexity by isolating chromosome arm 7DS and applied second‐generation technology and appropriate algorithmic analysis to sequence and assemble low copy and genic regions of this chromosome arm. The assembly represents approximately 40% of the chromosome arm and all known 7DS genes. Comparison of the 7DS assembly with the sequenced genomes of rice (Oryza sativa) and Brachypodium distachyon identified large regions of conservation. The syntenic relationship between wheat, B. distachyon and O. sativa, along with available genetic mapping data, has been used to produce an annotated draft 7DS syntenic build, which is publicly available at http://www.wheatgenome.info . Our results suggest that the sequencing of isolated chromosome arms can provide valuable information of the gene content of wheat and is a step towards whole‐genome sequencing and variation discovery in this important crop.     

Identification of efflux proteins using efficient radial basis function networks with position‐specific scoring matrices and biochemical properties

Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25–30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C‐terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78 and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using fivefold cross‐validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7 and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/～sachen/EFFLUXpredict/Efflux‐RBF.php . We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences.Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Assembly and RNA‐free annotation of highly heterozygous genomes: The case of the thick‐billed murre (Uria lomvia)

Thanks to a dramatic reduction in sequencing costs followed by a rapid development of bioinformatics tools, genome assembly and annotation have become accessible to many researchers in recent years. Among tetrapods, birds have genomes that display many features that facilitate their assembly and annotation, such as small genome size, low number of repeats and highly conserved genomic structure. However, we found that high genomic heterozygosity could have a great impact on the quality of the genome assembly of the thick‐billed murre (Uria lomvia), an arctic colonial seabird. In this study, we tested the performance of three genome assemblers, ray /sscape , soapdenovo 2 and platanus , in assembling the highly heterozygous genome of the thick‐billed murre. Our results show that platanus , an assembler specifically designed for heterozygous genomes, outperforms the other two approaches and produces a highly contiguous (N50 = 15.8 Mb) and complete genome assembly (93% presence of genes from the Benchmarking Universal Single Copy Ortholog [BUSCO] gene set). Additionally, we annotated the thick‐billed murre genome using a homology‐based approach that takes advantage of the genomic resources available for birds and other taxa. Our study will be useful for those researchers who are approaching assembly and annotation of highly heterozygous genomes, or genomes of species of conservation concern, and/or who have limited financial resources.     

Current Status of Echinoderm Genome Analysis - What do we Know?

Echinoderms have long served as model organisms for a variety of biological research, especially in the field of developmental biology. Although the genome of the purple sea urchin Strongylocentrotus purpuratus has been sequenced, it is the only echinoderm whose whole genome sequence has been reported. Nevertheless, data is rapidly accumulating on the chromosomes and genomic sequences of all five classes of echinoderms, including the mitochondrial genomes and Hox genes. This blossoming new data will be essential for estimating the phylogenetic relationships among echinoderms, and also to examine the underlying mechanisms by which the diverse morphologies of echinoderms have arisen.     

Complete nucleotide sequence of the chloroplast genome from a leptosporangiate fern, Adiantum capillus-veneris L.

We determined the complete nucleotide sequence of the chloroplast genome of the leptosporangiate fern, Adiantum capillus-veneris L. (Pteridaceae). The circular genome is 150,568 bp, with a large single-copy region (LSC) of 82,282 bp, a small-single copy region (SSC) of 21,392 bp and inverted repeats (IR) of 23,447 bp each. We compared the sequence to other published chloroplast genomes to infer the location of putative genes. When the IR is considered only once, we assigned 118 genes, of which 85 encode proteins, 29 encode tRNAs and 4 encode rRNAs. Four protein-coding genes, all four rRNA genes and six tRNA genes occur in the IR. Most (57) putative protein-coding genes appear to start with an ATG codon, but we also detected five other possible start codons, some of which suggest tRNA editing. We also found 26 apparent stop codons in 18 putative genes, also suggestive of RNA editing. We found all but one of the tRNA genes necessary to encode the complete repertoire required for translation. The missing trnK gene appears to have been disrupted by a large inversion, relative to other published chloroplast genomes. We detected several structural rearrangements that may provide useful information for phylogenetic studies.     

Sequence analysis of the long arm of rice chromosome 11 for rice–wheat synteny

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10× redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.