RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) contains nearly 200 000 bacterial and archaeal genomes and 150 million proteins with up-to-date annotation. Changes in the Prokaryotic Genome Annotation Pipeline (PGAP) since 2018 have resulted in a substantial reduction in spurious annotation. The hierarchical collection of protein family models (PFMs) used by PGAP as evidence for structural and functional annotation was expanded to over 35 000 protein profile hidden Markov models (HMMs), 12 300 BlastRules and 36 000 curated CDD architectures. As a result, >122 million or 79% of RefSeq proteins are now named based on a match to a curated PFM. Gene symbols, Enzyme Commission numbers or supporting publication attributes are available on over 40% of the PFMs and are inherited by the proteins and features they name, facilitating multi-genome analyses and connections to the literature. In adherence with the principles of FAIR (findable, accessible, interoperable, reusable), the PFMs are available in the Protein Family Models Entrez database to any user. Finally, the reference and representative genome set, a taxonomically diverse subset of RefSeq prokaryotic genomes, is now recalculated regularly and available for download and homology searches with BLAST. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/.

RefSeq: an update on prokaryotic genome annotation and curation.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) provides annotation for over 95 000 prokaryotic genomes that meet standards for sequence quality, completeness, and freedom from contamination. Genomes are annotated by a single Prokaryotic Genome Annotation Pipeline (PGAP) to provide users with a resource that is as consistent and accurate as possible. Notable recent changes include the development of a hierarchical evidence scheme, a new focus on curating annotation evidence sources, the addition and curation of protein profile hidden Markov models (HMMs), release of an updated pipeline (PGAP-4), and comprehensive re-annotation of RefSeq prokaryotic genomes. Antimicrobial resistance proteins have been reannotated comprehensively, improved structural annotation of insertion sequence transposases and selenoproteins is provided, curated complex domain architectures have given upgraded names to millions of multidomain proteins, and we introduce a new kind of annotation rule-BlastRules. Continual curation of supporting evidence, and propagation of improved names onto RefSeq proteins ensures that the functional annotation of genomes is kept current. An increasing share of our annotation now derives from HMMs and other sets of annotation rules that are portable by nature, and available for download and for reuse by other investigators. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/.

NCBI prokaryotic genome annotation pipeline.

Recent technological advances have opened unprecedented opportunities for large-scale sequencing and analysis of populations of pathogenic species in disease outbreaks, as well as for large-scale diversity studies aimed at expanding our knowledge across the whole domain of prokaryotes. To meet the challenge of timely interpretation of structure, function and meaning of this vast genetic information, a comprehensive approach to automatic genome annotation is critically needed. In collaboration with Georgia Tech, NCBI has developed a new approach to genome annotation that combines alignment based methods with methods of predicting protein-coding and RNA genes and other functional elements directly from sequence. A new gene finding tool, GeneMarkS+, uses the combined evidence of protein and RNA placement by homology as an initial map of annotation to generate and modify ab initio gene predictions across the whole genome. Thus, the new NCBI's Prokaryotic Genome Annotation Pipeline (PGAP) relies more on sequence similarity when confident comparative data are available, while it relies more on statistical predictions in the absence of external evidence. The pipeline provides a framework for generation and analysis of annotation on the full breadth of prokaryotic taxonomy. For additional information on PGAP see https://www.ncbi.nlm.nih.gov/genome/annotation_prok/ and the NCBI Handbook, https://www.ncbi.nlm.nih.gov/books/NBK174280/.

Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation.

The RefSeq project at the National Center for Biotechnology Information (NCBI) maintains and curates a publicly available database of annotated genomic, transcript, and protein sequence records (http://www.ncbi.nlm.nih.gov/refseq/). The RefSeq project leverages the data submitted to the International Nucleotide Sequence Database Collaboration (INSDC) against a combination of computation, manual curation, and collaboration to produce a standard set of stable, non-redundant reference sequences. The RefSeq project augments these reference sequences with current knowledge including publications, functional features and informative nomenclature. The database currently represents sequences from more than 55,000 organisms (>4800 viruses, >40,000 prokaryotes and >10,000 eukaryotes; RefSeq release 71), ranging from a single record to complete genomes. This paper summarizes the current status of the viral, prokaryotic, and eukaryotic branches of the RefSeq project, reports on improvements to data access and details efforts to further expand the taxonomic representation of the collection. We also highlight diverse functional curation initiatives that support multiple uses of RefSeq data including taxonomic validation, genome annotation, comparative genomics, and clinical testing. We summarize our approach to utilizing available RNA-Seq and other data types in our manual curation process for vertebrate, plant, and other species, and describe a new direction for prokaryotic genomes and protein name management.

Improvements to pairwise sequence comparison (PASC): a genome-based web tool for virus classification.

The number of viral genome sequences in the public databases is increasing dramatically, and these sequences are playing an important role in virus classification. Pairwise sequence comparison is a sequence-based virus classification method. A program using this method calculates the pairwise identities of virus sequences within a virus family and displays their distribution, and visual analysis helps to determine demarcations at different taxonomic levels such as strain, species, genus and subfamily. Subsequent comparison of new sequences against existing ones allows viruses from which the new sequences were derived to be classified. Although this method cannot be used as the only criterion for virus classification in some cases, it is a quantitative method and has many advantages over conventional virus classification methods. It has been applied to several virus families, and there is an increasing interest in using this method for other virus families/groups. The Pairwise Sequence Comparison (PASC) classification tool was created at the National Center for Biotechnology Information. The tool's database stores pairwise identities for complete genomes/segments of 56 virus families/groups. Data in the system are updated every day to reflect changes in virus taxonomy and additions of new virus sequences to the public database. The web interface of the tool ( http://www.ncbi.nlm.nih.gov/sutils/pasc/ ) makes it easy to navigate and perform analyses. Multiple new viral genome sequences can be tested simultaneously with this system to suggest the taxonomic position of virus isolates in a specific family. PASC eliminates potential discrepancies in the results caused by different algorithms and/or different data used by researchers.

Virus Variation Resource--recent updates and future directions.

Virus Variation (http://www.ncbi.nlm.nih.gov/genomes/VirusVariation/) is a comprehensive, web-based resource designed to support the retrieval and display of large virus sequence datasets. The resource includes a value added database, a specialized search interface and a suite of sequence data displays. Virus-specific sequence annotation and database loading pipelines produce consistent protein and gene annotation and capture sequence descriptors from sequence records then map these metadata to a controlled vocabulary. The database supports a metadata driven, web-based search interface where sequences can be selected using a variety of biological and clinical criteria. Retrieved sequences can then be downloaded in a variety of formats or analyzed using a suite of tools and displays. Over the past 2 years, the pre-existing influenza and Dengue virus resources have been combined into a single construct and West Nile virus added to the resultant resource. A number of improvements were incorporated into the sequence annotation and database loading pipelines, and the virus-specific search interfaces were updated to support more advanced functions. Several new features have also been added to the sequence download options, and a new multiple sequence alignment viewer has been incorporated into the resource tool set. Together these enhancements should support enhanced usability and the inclusion of new viruses in the future.

PAirwise Sequence Comparison (PASC) and its application in the classification of filoviruses.

PAirwise Sequence Comparison (PASC) is a tool that uses genome sequence similarity to help with virus classification. The PASC tool at NCBI uses two methods: local alignment based on BLAST and global alignment based on Needleman-Wunsch algorithm. It works for complete genomes of viruses of several families/groups, and for the family of Filoviridae, it currently includes 52 complete genomes available in GenBank. It has been shown that BLAST-based alignment approach works better for filoviruses, and therefore is recommended for establishing taxon demarcations criteria. When more genome sequences with high divergence become available, these demarcation will most likely become more precise. The tool can compare new genome sequences of filoviruses with the ones already in the database, and propose their taxonomic classification.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI Website. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central (PMC), Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Primer-BLAST, COBALT, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, Genome and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, BioProject, BioSample, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Probe, Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), Biosystems, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central (PMC), Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Primer-BLAST, COBALT, Electronic PCR, OrfFinder, Splign, ProSplign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Entrez Probe, GENSAT, Online Mendelian Inheritance in Man (OMIM), Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), IBIS, Biosystems, Peptidome, OMSSA, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, Reference Sequence, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Entrez Probe, GENSAT, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool, Biosystems, Peptidome, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. All these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups (COGs), Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Entrez Probe, GENSAT, Online Mendelian Inheritance in Man (OMIM), Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART) and the PubChem suite of small molecule databases. Augmenting many of the web applications is custom implementation of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data available through NCBI's web site. NCBI resources include Entrez, the Entrez Programming Utilities, My NCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link, Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genome, Genome Project and related tools, the Trace, Assembly, and Short Read Archives, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Influenza Viral Resources, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Entrez Probe, GENSAT, Database of Genotype and Phenotype, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting the web applications are custom implementations of the BLAST program optimized to search specialized data sets. These resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, the Entrez Programming Utilities, My NCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link(BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genome, Genome Project and related tools, the Trace and Assembly Archives, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups (COGs), Viral Genotyping Tools, Influenza Viral Resources, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Entrez Probe, GENSAT, Online Mendelian Inheritance in Man (OMIM), Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART) and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. These resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Retroviral Genotyping Tools, HIV-1, Human Protein Interaction Database, SAGEmap, Gene Expression Omnibus, Entrez Probe, GENSAT, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized datasets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.

Plant genome resources at the national center for biotechnology information.

The National Center for Biotechnology Information (NCBI) integrates data from more than 20 biological databases through a flexible search and retrieval system called Entrez. A core Entrez database, Entrez Nucleotide, includes GenBank and is tightly linked to the NCBI Taxonomy database, the Entrez Protein database, and the scientific literature in PubMed. A suite of more specialized databases for genomes, genes, gene families, gene expression, gene variation, and protein domains dovetails with the core databases to make Entrez a powerful system for genomic research. Linked to the full range of Entrez databases is the NCBI Map Viewer, which displays aligned genetic, physical, and sequence maps for eukaryotic genomes including those of many plants. A specialized plant query page allow maps from all plant genomes covered by the Map Viewer to be searched in tandem to produce a display of aligned maps from several species. PlantBLAST searches against the sequences shown in the Map Viewer allow BLAST alignments to be viewed within a genomic context. In addition, precomputed sequence similarities, such as those for proteins offered by BLAST Link, enable fluid navigation from unannotated to annotated sequences, quickening the pace of discovery. NCBI Web pages for plants, such as Plant Genome Central, complete the system by providing centralized access to NCBI's genomic resources as well as links to organism-specific Web pages beyond NCBI.