The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003

The SWISS-PROT protein knowledgebase (http://www.expasy.org/sprot/ and http://www.ebi.ac.uk/swissprot/) connects amino acid sequences with the current knowledge in the Life Sciences. Each protein entry provides an interdisciplinary overview of relevant information by bringing together experimental results, computed features and sometimes even contradictory conclusions. Detailed expertise that goes beyond the scope of SWISS-PROT is made available via direct links to specialised databases. SWISS-PROT provides annotated entries for all species, but concentrates on the annotation of entries from human (the HPI project) and other model organisms to ensure the presence of high quality annotation for representative members of all protein families. Part of the annotation can be transferred to other family members, as is already done for microbes by the High-quality Automated and Manual Annotation of microbial Proteomes (HAMAP) project. Protein families and groups of proteins are regularly reviewed to keep up with current scientific findings. Complementarily, TrEMBL strives to comprise all protein sequences that are not yet represented in SWISS-PROT, by incorporating a perpetually increasing level of mostly automated annotation. Researchers are welcome to contribute their knowledge to the scientific community by submitting relevant findings to SWISS-PROT at swiss-prot@expasy.org.     

Applications of InterPro in protein annotation and genome analysis

The applications of InterPro span a range of biologically important areas that includes automatic annotation of protein sequences and genome analysis. In automatic annotation of protein sequences InterPro has been utilised to provide reliable characterisation of sequences, identifying them as candidates for functional annotation. Rules based on the InterPro characterisation are stored and operated through a database called RuleBase. RuleBase is used as the main tool in the sequence database group at the EBI to apply automatic annotation to unknown sequences. The annotated sequences are stored and distributed in the TrEMBL protein sequence database. InterPro also provides a means to carry out statistical and comparative analyses of whole genomes. In the Proteome Analysis Database, InterPro analyses have been combined with other analyses based on CluSTr, the Gene Ontology (GO) and structural information on the proteins.     

GI-POP: A combinational annotation and genomic island prediction pipeline for ongoing microbial genome projects

Sequencing of microbial genomes is important because of microbial-carrying antibiotic and pathogenetic activities. However, even with the help of new assembling software, finishing a whole genome is a time-consuming task. In most bacteria, pathogenetic or antibiotic genes are carried in genomic islands. Therefore, a quick genomic island (GI) prediction method is useful for ongoing sequencing genomes. In this work, we built a Web server called GI-POP (http://gipop.life.nthu.edu.tw) which integrates a sequence assembling tool, a functional annotation pipeline, and a high-performance GI predicting module, in a support vector machine (SVM)-based method called genomic island genomic profile scanning (GI-GPS). The draft genomes of the ongoing genome projects in contigs or scaffolds can be submitted to our Web server, and it provides the functional annotation and highly probable GI-predicting results. GI-POP is a comprehensive annotation Web server designed for ongoing genome project analysis. Researchers can perform annotation and obtain pre-analytic information include possible GIs, coding/non-coding sequences and functional analysis from their draft genomes. This pre-analytic system can provide useful information for finishing a genome sequencing project.     

Automated genome sequence analysis and annotation.

MOTIVATION: Large-scale genome projects generate a rapidly increasing number of sequences, most of them biochemically uncharacterized. Research in bioinformatics contributes to the development of methods for the computational characterization of these sequences. However, the installation and application of these methods require experience and are time consuming. RESULTS: We present here an automatic system for preliminary functional annotation of protein sequences that has been applied to the analysis of sets of sequences from complete genomes, both to refine overall performance and to make new discoveries comparable to those made by human experts. The GeneQuiz system includes a Web-based browser that allows examination of the evidence leading to an automatic annotation and offers additional information, views of the results, and links to biological databases that complement the automatic analysis. System structure and operating principles concerning the use of multiple sequence databases, underlying sequence analysis tools, lexical analyses of database annotations and decision criteria for functional assignments are detailed. The system makes automatic quality assessments of results based on prior experience with the underlying sequence analysis tools; overall error rates in functional assignment are estimated at 2.5-5% for cases annotated with highest reliability ('clear' cases). Sources of over-interpretation of results are discussed with proposals for improvement. A conservative definition for reporting 'new findings' that takes account of database maturity is presented along with examples of possible kinds of discoveries (new function, family and superfamily) made by the system. System performance in relation to sequence database coverage, database dynamics and database search methods is analysed, demonstrating the inherent advantages of an integrated automatic approach using multiple databases and search methods applied in an objective and repeatable manner. AVAILABILITY: The GeneQuiz system is publicly available for analysis of protein sequences through a Web server at http://www.sander.ebi.ac. uk/gqsrv/submit     

Solving the Problem: Genome Annotation Standards before the Data Deluge

The promise of genome sequencing was that the vast undiscovered country would be mapped out by comparison of the multitude of sequences available and would aid researchers in deciphering the role of each gene in every organism. Researchers recognize that there is a need for high quality data. However, different annotation procedures, numerous databases, and a diminishing percentage of experimentally determined gene functions have resulted in a spectrum of annotation quality. NCBI in collaboration with sequencing centers, archival databases, and researchers, has developed the first international annotation standards, a fundamental step in ensuring that high quality complete prokaryotic genomes are available as gold standard references. Highlights include the development of annotation assessment tools, community acceptance of protein naming standards, comparison of annotation resources to provide consistent annotation, and improved tracking of the evidence used to generate a particular annotation. The development of a set of minimal standards, including the requirement for annotated complete prokaryotic genomes to contain a full set of ribosomal RNAs, transfer RNAs, and proteins encoding core conserved functions, is an historic milestone. The use of these standards in existing genomes and future submissions will increase the quality of databases, enabling researchers to make accurate biological discoveries.     

DNA Data Bank of Japan dealing with large-scale data submission.

The DNA Data Bank of Japan (DDBJ) (http//:www.ddbj.nig.ac.jp) has developed a software system for mass submissions to cope with a recent expansion of EST and genome data submissions. The system is composed of four parts, the WWW data submission, large-scale submission, submission management and storing. Using this system one can submit data on a large number of sequences or a very long sequence while checking the consistency between the annotation and sequence without much effort. DDBJ has received large scale data of Homo sapiens, Arabidopsis and Pyrococcus from Japanese researchers who made full use of the new submission system.     

probeBase: an online resource for rRNA-targeted oligonucleotide probes

Ribosomal RNA-(rRNA)-targeted oligonucleotide probes are widely used for culture-independent identification of microorganisms in environmental and clinical samples. ProbeBase is a comprehensive database containing more than 700 published rRNA-targeted oligonucleotide probe sequences (status August 2002) with supporting bibliographic and biological annotation that can be accessed through the internet at http://www.probebase.net. Each oligonucleotide probe entry contains information on target organisms, target molecule (small- or large-subunit rRNA) and position, G+C content, predicted melting temperature, molecular weight, necessity of competitor probes, and the reference that originally described the oligonucleotide probe, including a link to the respective abstract at PubMed. In addition, probes successfully used for fluorescence in situ hybridization (FISH) are highlighted and the recommended hybridization conditions are listed. ProbeBase also offers difference alignments for 16S rRNA-targeted probes by using the probe match tool of the ARB software and the latest small-subunit rRNA ARB database (release June 2002). The option to directly submit probe sequences to the probe match tool of the Ribosomal Database Project II (RDP-II) further allows one to extract supplementary information on probe specificities. The two main features of probeBase, 'search probeBase' and 'find probe set', help researchers to find suitable, published oligonucleotide probes for microorganisms of interest or for rRNA gene sequences submitted by the user. Furthermore, the 'search target site' option provides guidance for the development of new FISH probes.     

The DOE-JGI Standard Operating Procedure for the Annotations of Microbial Genomes

The DOE-JGI Microbial Annotation Pipeline (DOE-JGI MAP) supports gene prediction and/or functional annotation of microbial genomes towards comparative analysis with the Integrated Microbial Genome (IMG) system. DOE-JGI MAP annotation is applied on nucleotide sequence datasets included in the IMG-ER (Expert Review) version of IMG via the IMG ER submission site. Users can submit the sequence datasets consisting of one or more contigs in a multi-fasta file. DOE-JGI MAP annotation includes prediction of protein coding and RNA genes, as well as repeats and assignment of product names to these genes.     

PhyloGena--a user-friendly system for automated phylogenetic annotation of unknown sequences

MOTIVATION: Phylogenomic approaches towards functional and evolutionary annotation of unknown sequences have been suggested to be superior to those based only on pairwise local alignments. User-friendly software tools making the advantages of phylogenetic annotation available for the ever widening range of bioinformatically uninitiated biologists involved in genome/EST annotation projects are, however, not available. We were particularly confronted with this issue in the annotation of sequences from different groups of complex algae originating from secondary endosymbioses, where the identification of the phylogenetic origin of genes is often more problematic than in taxa well represented in the databases (e.g. animals, plants or fungi). RESULTS: We present a flexible pipeline with a user-friendly, interactive graphical user interface running on desktop computers that automatically performs a basic local alignment search tool (BLAST) search of query sequences, selects a representative subset of them, then creates a multiple alignment from the selected sequences, and finally computes a phylogenetic tree. The pipeline, named PhyloGena, uses public domain software for all standard bioinformatics tasks (similarity search, multiple alignment, and phylogenetic reconstruction). As the major technological innovation, selection of a meaningful subset of BLAST hits was implemented using logic programming, mimicing the selection procedure (BLAST tables, multiple alignments and phylogenetic trees) are displayed graphically, allowing the user to interact with the pipeline and deduce the function and phylogenetic origin of the query. PhyloGena thus makes phylogenomic annotation available also for those biologists without access to large computing facilities and with little informatics background. Although phylogenetic annotation is particularly useful when working with composite genomes (e.g. from complex algae), PhyloGena can be helpful in expressed sequence tag and genome annotation also in other organisms. AVAILABILITY: PhyloGena (executables for LINUX and Windows 2000/XP as well as source code) is available by anonymous ftp from http://www.awi.de/en/phylogena.     

基于Web服务的流感病毒基因组自动化翻译注释系统

随着流感病毒基因组测序数据的急剧增加,深入挖掘流感病毒基因组大数据蕴含的生物学信息成为研究热点。基于中国流感病毒流行特征数据,建设一个集自动化、一体化和信息化的序列库系统,对于实现流感病毒基因组批量快速翻译、注释、存储、查询、分析具有重要的应用价值。本课题组通过集成一系列软件和工具包,并结合自主研发的其他功能,在底层维护的2个关键的参考数据集基础上另外追加了翻译注释信息最佳匹配的精细化筛选规则,构建具有流感病毒基因组信息存储、自动化翻译、蛋白序列精准注释、同源序列比对和进化树分析等功能的自动化系统。结果显示,通过Web端输入fasta格式的流感病毒基因序列,本系统可针对参考序列片段数据集(blastdb.fasta)进行Blast同源性检索,可以鉴定流感病毒的型别(A、B或C)、亚型和基因片段(1~8片段);在此基础上,通过查询数据库底层用于翻译、注释的基因片段参考数据集,可以获得一组肽段数据集,然后通过循环调用ProSplign软件对其进行预测。结合精细化的筛选准入规则,选出与输入序列匹配最好的翻译后产物,作为该输入序列的预测蛋白,输出为gbk,asn和fasta等通用格式的文件,给出序列长度、是否全长、病毒型别、亚型、片段等信息。基于以上工作,另外自主研发了系统其他的附加功能如进化树分析展示、基因组数据存储等功能,构建成基于Web服务的流感病毒基因组自动化翻译注释系统。本研究提示,系统高度集成系列软件以及自有的注释翻译数据库文件,实现从序列存储、翻译、注释到序列分析和展示的功能,可全面满足我国高通量基因检测数据共享化、本土化、一体化、自动化的需求。     

Construction of Customized Sub-Databases from NCBI-nr Database for Rapid Annotation of Huge Metagenomic Datasets Using a Combined BLAST and MEGAN Approach

We developed a fast method to construct local sub-databases from the NCBI-nr database for the quick similarity search and annotation of huge metagenomic datasets based on BLAST-MEGAN approach. A three-step sub-database annotation pipeline (SAP) was further proposed to conduct the annotation in a much more time-efficient way which required far less computational capacity than the direct NCBI-nr database BLAST-MEGAN approach. The 1^st BLAST of SAP was conducted using the original metagenomic dataset against the constructed sub-database for a quick screening of candidate target sequences. Then, the candidate target sequences identified in the 1^st BLAST were subjected to the 2^nd BLAST against the whole NCBI-nr database. The BLAST results were finally annotated using MEGAN to filter out those mistakenly selected sequences in the 1^st BLAST to guarantee the accuracy of the results. Based on the tests conducted in this study, SAP achieved a speedup of ∼150–385 times at the BLAST e-value of 1e–5, compared to the direct BLAST against NCBI-nr database. The annotation results of SAP are exactly in agreement with those of the direct NCBI-nr database BLAST-MEGAN approach, which is very time-consuming and computationally intensive. Selecting rigorous thresholds (e.g. e-value of 1e–10) would further accelerate SAP process. The SAP pipeline may also be coupled with novel similarity search tools (e.g. RAPsearch) other than BLAST to achieve even faster annotation of huge metagenomic datasets. Above all, this sub-database construction method and SAP pipeline provides a new time-efficient and convenient annotation similarity search strategy for laboratories without access to high performance computing facilities. SAP also offers a solution to high performance computing facilities for the processing of more similarity search tasks.     

IDconverter and IDClight: Conversion and annotation of gene and protein IDs

Background  

Researchers involved in the annotation of large numbers of gene, clone or protein identifiers are usually required to perform a one-by-one conversion for each identifier. When the field of research is one such as microarray experiments, this number may be around 30,000.     

CHIKVPRO - a protein sequence annotation database for chikungunya virus

In the recent past, there has been a resurgence of interest in Chikungunya virus (CHIKV) attributed to massive outbreaks of Chikungunya fever in the South-East Asia Region. This has reflected in substantial increase in submission of CHIKV genome sequences to NCBI (National Center for Biotechnology Information) database. Hereby we submit a database "CHIKVPRO" containing structural and functional annotation of Chikungunya virus proteins (25 strains) submitted in the NCBI repository. The CHIKV genome encodes for 9 proteins:4 non-structural and 5 structural. The CHIKVPRO database aims to provide the virology community with a single accession authoritative resource for CHIKV proteome- with reference to physiochemical and molecular properties, proteolytic cleavage sites, hydrophobicity, transmembrane prediction, and classification into functional families using SVMProt and other Expasy tools. AVAILABILITY: The database is freely available at http://www.chikvpro.info/     

Combined evidence annotation of transposable elements in genome sequences

Transposable elements (TEs) are mobile, repetitive sequences that make up significant fractions of metazoan genomes. Despite their near ubiquity and importance in genome and chromosome biology, most efforts to annotate TEs in genome sequences rely on the results of a single computational program, RepeatMasker. In contrast, recent advances in gene annotation indicate that high-quality gene models can be produced from combining multiple independent sources of computational evidence. To elevate the quality of TE annotations to a level comparable to that of gene models, we have developed a combined evidence-model TE annotation pipeline, analogous to systems used for gene annotation, by integrating results from multiple homology-based and de novo TE identification methods. As proof of principle, we have annotated "TE models" in Drosophila melanogaster Release 4 genomic sequences using the combined computational evidence derived from RepeatMasker, BLASTER, TBLASTX, all-by-all BLASTN, RECON, TE-HMM and the previous Release 3.1 annotation. Our system is designed for use with the Apollo genome annotation tool, allowing automatic results to be curated manually to produce reliable annotations. The euchromatic TE fraction of D. melanogaster is now estimated at 5.3% (cf. 3.86% in Release 3.1), and we found a substantially higher number of TEs (n = 6,013) than previously identified (n = 1,572). Most of the new TEs derive from small fragments of a few hundred nucleotides long and highly abundant families not previously annotated (e.g., INE-1). We also estimated that 518 TE copies (8.6%) are inserted into at least one other TE, forming a nest of elements. The pipeline allows rapid and thorough annotation of even the most complex TE models, including highly deleted and/or nested elements such as those often found in heterochromatic sequences. Our pipeline can be easily adapted to other genome sequences, such as those of the D. melanogaster heterochromatin or other species in the genus Drosophila.     

A mutation spectra database for bacterial and mammalian genes.

Each mutation spectrum in this database is a dataset of changes in DNA base sequence in mutations induced in a gene by a particular mutagen (including spontaneous processes) under defined conditions. There are 240 datasets with 24 500 mutants in nine bacterial genes, two phage genes, five mammalian genes and one yeast gene. The database is available on the Web at http://info.med.yale.edu/mutbase/ . The data tables can be viewed on the Web and downloaded in text form for local use. The data are also available in dBASE III, a format which can be utilized by essentially any desktop computer database program or spreadsheet, and makes feasible analyses of a large number of mutants. Researchers are invited to submit additional data. A data entry program, MUTSIN, diagrams each mutation on the computer screen as the data are entered and alerts the user to any discrepancies between the entry and the gene sequence.     

PipTools: a computational toolkit to annotate and analyze pairwise comparisons of genomic sequences

Sequence conservation between species is useful both for locating coding regions of genes and for identifying functional noncoding segments. Hence interspecies alignment of genomic sequences is an important computational technique. However, its utility is limited without extensive annotation. We describe a suite of software tools, PipTools, and related programs that facilitate the annotation of genes and putative regulatory elements in pairwise alignments. The alignment server PipMaker uses the output of these tools to display detailed information needed to interpret alignments. These programs are provided in a portable format for use on common desktop computers and both the toolkit and the PipMaker server can be found at our Web site (http://bio.cse.psu.edu/). We illustrate the utility of the toolkit using annotation of a pairwise comparison of the mouse MHC class II and class III regions with orthologous human sequences and subsequently identify conserved, noncoding sequences that are DNase I hypersensitive sites in chromatin of mouse cells.