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GenBank® is a comprehensive database that contains publicly available nucleotide sequences for more than 300 000 organisms named at the genus level or lower, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or standalone Sequin programs, and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the European Molecular Biology Laboratory Nucleotide Sequence Database in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through the NCBI Entrez retrieval system, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bi-monthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI homepage: www.ncbi.nlm.nih.gov.
GenBank (1) is a comprehensive public database of nucleotide sequences and supporting bibliographical and biological annotation. GenBank is built and distributed by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine (NLM), located on the campus of the US National Institutes of Health (NIH) in Bethesda, MD, USA.
NCBI builds GenBank primarily from the submission of sequence data from authors and from the bulk submission of expressed sequence tag (EST), genome survey sequence (GSS) and other high-throughput data from sequencing centers. The US Office of Patents and Trademarks also contributes sequences from issued patents. GenBank participates with the European Molecular Biology Laboratory Nucleotide Sequence Database (EMBL) (2) and the DNA Databank of Japan (DDBJ) (3) as a partner in the International Nucleotide Sequence Database Collaboration (INSDC), which exchanges data daily to ensure that a uniform and comprehensive collection of sequence information is available worldwide. NCBI makes the GenBank data available at no cost over the Internet, through FTP and a wide range of web-based retrieval and analysis services (4).
From its inception, GenBank has grown exponentially, and continues to do so with the number of sequence records doubling approximately every 35 months. The traditional GenBank divisions contain 106 billion nucleotide bases from 108 million individual sequences, with 11 million new sequences added in the past year. Contributions from whole genome shotgun (WGS) projects supplement the data in the traditional divisions to bring the total to 255 billion bases. Complete genomes (www.ncbi.nlm.nih.gov/Genomes/) continue to represent a rapidly growing segment of the database. GenBank now contains more than 1000 complete genomes from bacteria and archaea, and 30% of these were deposited during the past year. The number of eukaryote genomes with significant coverage and assembly continues to increase as well, with over 380 WGS assemblies now available.
Database sequences are classified and can be queried using a comprehensive sequence-based taxonomy (www.ncbi.nlm.nih.gov/sites/entrez?db=taxonomy) developed by NCBI in collaboration with EMBL and DDBJ and with the valuable assistance of external advisers and curators. More than 300 000 species named at the genus level or lower are represented in GenBank, and new taxa are being added at the rate of over 2500/month. About 12% of the non-WGS sequences in GenBank are of human origin and 8% are ESTs. The top species in GenBank in terms of number of non-WGS bases are listed in Table 1.
Each GenBank entry includes a concise description of the sequence, the scientific name and taxonomy of the source organism, bibliographical references and a table of features (www.ncbi.nlm.nih.gov/collab/FT/) listing areas of biological significance, such as coding regions and their protein translations, transcription units, repeat regions and sites of mutations or modifications. The files in the GenBank distribution have traditionally been partitioned into ‘divisions’ that roughly correspond to taxonomic groups such as bacteria (BCT), viruses (VRL), primates (PRI) and rodents (ROD). Additional divisions support specific sequencing strategies, such as those for EST, GSS, high-throughput genomic (HTG), high-throughput cDNA (HTC) and environmental sample (ENV) sequences, making 20 divisions. For convenience in file transfer, the GenBank data are partitioned into multiple files, currently more than 1800 (release 173), for the bi-monthly GenBank releases on the NCBI FTP site.
ESTs continue to be a major source of new sequence records and gene sequences, comprising over 34 billion nucleotide bases representing more than 1800 different organisms in GenBank release 173. The number of ESTs has increased by 14% in the past year to 62.8 million sequences. The top organisms represented in the EST division are listed in Table 1. As part of its daily processing of GenBank EST data, NCBI identifies through BLAST searches all similar sequences for each new EST sequence and incorporates that information into the companion database, dbEST (www.ncbi.nlm.nih.gov/dbEST/) (5). The data in dbEST are processed further to produce the UniGene database (www.ncbi.nlm.nih.gov/unigene) of more than 3.8 million gene-oriented sequence clusters representing over 110 organisms (4).
The STS division of GenBank (www.ncbi.nlm.nih.gov/dbSTS/) contains 1.3 million sequences, including anonymous STSs based on genomic sequence as well as gene-based STSs derived from the 3′-ends of genes and ESTs. These STS records usually include mapping information.
The GSS division of GenBank (www.ncbi.nlm.nih.gov/dbGSS/) has grown over the past year by 6% to a total of 25.8 million records from over 870 organisms and contributes over 16.7 billion nucleotide bases. GSS sequences are produced by as many as 80 different experimental techniques and are similar to EST sequences, except that they are derived from genomic DNA. The human data have been used (www.ncbi.nlm.nih.gov/projects/genome/clone/) along with the STS records in tiling the BACs for the Human Genome Project (6).
The ENV division of GenBank accommodates non-WGS sequences obtained via environmental sampling methods in which the source organism is unknown. Many ENV sequences arise from metagenome samples derived from microbiota in various animal tissues, such as within the gut or skin, or from particular environments, such as freshwater sediment, hot springs or areas of mine drainage. Records in the ENV division contain ‘ENV’ in the keyword field and use an ‘/environmental_sample’ qualifier in the source feature. As of GenBank release 173, the ENV division of GenBank contained 1.3 million sequences and 1.1 billion base pairs.
The HTG division of GenBank (www.ncbi.nlm.nih.gov/HTGS/) contains unfinished large-scale genomic records, which are in transition to a finished state (7). These records are designated as Phase 0–3 depending on the quality of the data, with Phase 3 being the finished state. Upon reaching Phase 3, HTG records are moved into the appropriate organism division of GenBank. As of GenBank release 173, the HTG division contained 142 000 sequences and 23.9 billion base pairs.
The HTC division of GenBank contains HTC sequences that are of draft quality but may contain 5′-UTRs, 3′-UTRs, partial coding regions and introns. HTC sequences which are finished and of high quality are moved to the appropriate organism division of GenBank. GenBank release 173 contained more than 550 000 HTC sequences totaling 636 million bases. A project generating HTC data is described in Ref. (8).
More than 148 billion bases of WGS sequence appear in GenBank as sets of WGS sequence overlap contigs, each of which is issued an accession number consisting of a four-letter project ID, followed by a two-digit version number and a six-digit contig ID. Hence, the WGS accession number ‘AAAA01072744’ is assigned to CON number ‘072744’ of the first version of project ‘AAAA’. WGS sequencing projects have contributed over 48 million contigs to GenBank, and these primary sequences have been used to construct 6.4 million large-scale assemblies of scaffolds and chromosomes. WGS project contigs for nearly 1800 organisms and environmental samples are available. For a complete list of WGS projects with links to the data, see www.ncbi.nlm.nih.gov/projects/WGS/WGSprojectlist.cgi.
Although WGS project sequences may be annotated, many low-coverage genome projects do not contain annotation. Because these sequence projects are ongoing and incomplete, these annotations may not be tracked from one assembly version to the next and should be considered preliminary. Submitters of genomic sequences, including WGS sequences, are urged to use evidence tags of the form ‘/experimental = text’ and ‘/inference = TYPE:text’, where TYPE is one of a number of standard inference types and text consists of structured text.
In recent years, a growing number of sequencing traces have been deposited in the NCBI Trace Archive (TA), which now contains over 2 billion records (4). Given the advent of next-generation sequencing technologies, including those from Roche-454 Life Sciences, Illumina Solexa and Applied Biosystems SOLiD, NCBI created a Sequence Read Archive (SRA) in 2007. For more information about the TA and SRA, please see the accompanying articles in this issue (4,9). Neither the TA nor SRA is a part of GenBank, but beginning with release 166, GenBank added a new TSA division for Transcriptome Shotgun Assembly sequences, which are shotgun assemblies of sequences deposited in TA, SRA and the EST division of GenBank. TSA records (e.g. EZ000001) have ‘TSA’ as their keyword and a primary block that provides the base ranges and identifiers of the sequences used in the TSA assembly.
Third party annotation (TPA) records are sequence annotations published by someone other than the original submitter of the primary sequence record in DDBJ/EMBL/GenBank (www.ncbi.nlm.nih.gov/Genbank/TPA.html). TPA records fall into one of three categories: (i) experimental, in which case there is direct experimental evidence for the existence of the annotated molecule; (ii) inferential, in which case the experimental evidence is indirect; and (iii) reassembly, where the focus is on providing a better assembly of the raw reads. TPA sequences may be created by assembling a number of primary sequences. The format of a TPA record (e.g. BK000016) is similar to that of a conventional GenBank record but includes the label ‘TPA_exp:’, ‘TPA_inf:’ or ‘TPA_reasm:’ at the beginning of each Definition Line and the keywords ‘Third Party Annotation; TPA’ and either ‘TPA:experimental’, ‘TPA:inferential’ or ‘TPA:reassembly’. TPA experimental and inferential records also contain a primary block similar to that in a TSA record. Currently GenBank contains over 6500 TPA records, with the top organisms being Drosophila melanogaster (2200 sequences), Homo sapiens (990), Oryza sativa (640) and Mus musculus (350). TPA sequences are not released to the public until their accession numbers or sequence data and annotation appear in a peer-reviewed biological journal. TPA submissions to GenBank may be made using either BankIt or Sequin.
Small genomes, such as those from bacteria, can generally be conveniently represented, transferred between computers and analyzed as single sequences. For very long sequences, such as a eukaryotic chromosome, where the sequence is not complete but consists of several contig (CON) records with uncharacterized gaps between them, the entire chromosome is represented in GenBank as a CON record. Rather than listing the sequence itself, CON records contain assembly instructions involving the several component sequences. An example of such a CON record is DP000010 for rice chromosome 11.
The data in GenBank and the collaborating databases, EMBL and DDBJ, are submitted primarily by individual authors to one of the three databases, or by sequencing centers as batches of EST, STS, GSS, HTC, WGS or HTG sequences. Data are exchanged daily with DDBJ and EMBL so that the daily updates from NCBI servers incorporate the most recently available sequence data from all sources.
Virtually all records enter GenBank as direct electronic submissions (www.ncbi.nlm.nih.gov/Genbank/), with the majority of authors using the BankIt or Sequin programs. Many journals require authors with sequence data to submit the data to a public database as a condition of publication. GenBank staff can usually assign an accession number to a sequence submission within two working days of receipt, and do so at a rate of almost 1600/day. The accession number serves as confirmation that the sequence has been submitted and provides a means for readers of articles in which the sequence is cited to retrieve the data. Direct submissions receive a quality assurance review that includes checks for vector contamination, proper translation of coding regions, correct taxonomy and correct bibliographic citations. A draft of the GenBank record is passed back to the author for review before it enters the database.
Authors may ask that their sequences be kept confidential until the time of publication. Since GenBank policy requires that the deposited sequence data be made public when the sequence or accession number is published, authors are instructed to inform GenBank staff of the publication date of the article in which the sequence is cited in order to ensure a timely release of the data. Although only the submitter is permitted to modify sequence data or annotations, all users are encouraged to report lags in releasing data or possible errors or omissions to GenBank at vog.hin.mln.ibcn@etadpu.
NCBI works closely with sequencing centers to ensure timely incorporation of bulk data into GenBank for public release. GenBank offers special batch procedures for large-scale sequencing groups to facilitate data submission, including the program tbl2asn, described at www.ncbi.nlm.nih.gov/Sequin/table.html.
About a third of author submissions are received through an NCBI web-based data submission tool named BankIt (www.ncbi.nlm.nih.gov/BankIt). Using BankIt, authors enter sequence information directly into a form and add biological annotation such as coding regions or mRNA features. Free-form text boxes, list boxes and pull-down menus allow the submitter to describe the sequence further without having to learn formatting rules or restricted vocabularies. Before creating a draft record in the GenBank flat file format for the submitter to review, BankIt validates the submissions by flagging many common errors and checking for vector contamination using a variant of BLAST called Vecscreen. BankIt is the tool of choice for simple submissions, especially when only one or a small number of records is being submitted (7). Submitters can also use BankIt to update their existing GenBank records. In 2009, NCBI released a new version of BankIt (www.ncbi.nlm.nih.gov/WebSub/?tool=genbank) that offers several improvements: a depositor’s contact information is stored and easily reused in future submissions; sets of sequences can be uploaded as one submission; feature table data can be uploaded from a file; and a submitter can leave a partially finished submission and return later to complete it.
NCBI also offers a standalone multiplatform submission program called Sequin (www.ncbi.nlm.nih.gov/projects/Sequin/) that can be used interactively with other NCBI sequence retrieval and analysis tools. Sequin handles simple sequences, such as a single cDNA, as well as segmented entries, phylogenetic studies, population studies, mutation studies, environmental samples and alignments for which BankIt and other web-based submission tools are not well suited. Sequin has convenient editing and complex annotation capabilities and contains a number of built-in validation functions for quality assurance. In addition, Sequin is able to accommodate large sequences, such as the 5.6 Mb Escherichia coli genome, and read in a full complement of annotations from simple tables. The most recent version, Sequin 9.50, was released in July 2009 and is available for Macintosh, PC and UNIX computers via anonymous FTP at ftp.ncbi.nih.gov/sequin. Once a submission is completed, submitters can e-mail the Sequin file to vog.hin.mln.ibcn@bus-bg. Submitters of large, heavily annotated genomes may find it convenient to use ‘tbl2asn’ (described above) to convert a table of annotations generated from an annotation pipeline into an ASN.1 (Abstract Syntax Notation One) record suitable for submission to GenBank.
The Consortium for the Barcode of Life (CBOL) is an international initiative to develop DNA barcoding as a tool for characterizing species of organisms using a short DNA sequence, usually a 648-bp fragment of the gene for cytochrome oxidase subunit I. NCBI, in collaboration with CBOL (www.barcoding.si.edu/) has created an online tool (BarSTool) for the bulk submission of barcode sequences to GenBank (www.ncbi.nlm.nih.gov/WebSub/?tool=barcode) that allows users to upload files containing a batch of sequences with associated source information. The Nucleotide query ‘barcode[keyword]’ retrieves almost 21 000 barcode sequences in GenBank, over 5000 of which were added in the last year.
Each GenBank record, consisting of both a sequence and its annotations, is assigned a unique identifier called an accession number that is shared across the three collaborating databases (GenBank, DDBJ and EMBL). The accession number appears on the ACCESSION line of a GenBank record and remains constant over the lifetime of the record, even when there is a change to the sequence or annotation. Changes to the sequence data itself are tracked by an integer extension of the accession number, and this Accession.version identifier appears on the VERSION line of the GenBank flat file. The initial version of a sequence has the extension ‘.1’. In addition, each version of the DNA sequence is also assigned a unique NCBI identifier called a ‘GI’ number that also appears on the VERSION line following the Accession.version:
VERSION AF000001.1 GI: 987654321
When a change is made to a sequence in a GenBank record, a new GI number is issued to the updated sequence and the version extension of the Accession.version identifier is incremented. The accession number for the record as a whole remains unchanged, and will always retrieve the most recent version of the record; the older versions remain available under the old Accession.version identifiers and their original GI numbers.
A similar system tracks changes in the corresponding protein translations. These identifiers appear as qualifiers for CDS features in the FEATURES portion of a GenBank entry, e.g. /protein_id=‘AAA00001.1’. Protein sequence translations also receive their own unique GI number, which appears as a second qualifier on the CDS feature:/db_xref=‘GI: 1233445’.
The sequence records in GenBank are accessible through the NCBI Entrez retrieval system (4). Records from the EST and GSS divisions of GenBank are stored in the Entrez EST and GSS databases, while all other GenBank records are stored in Entrez Nucleotide. Other Entrez databases contain protein sequences derived from GenBank and other sources; genome maps; population, phylogenetic and environmental sequence sets; gene expression data; the NCBI taxonomy; and protein domain information and protein structures from the Molecular Modeling Database (MMDB) (10). Each database is linked to the scientific literature via PubMed and PubMed Central.
The ability to identify all GenBank records submitted by a specific group or those with a particular focus, such as metagenomic surveys, is essential for the analysis of large volumes of sequence data. The use of organism or submitter names as a means to define such a set of sequences is unreliable. The Projects database (www.ncbi.nlm.nih.gov/genomeprj), developed at NCBI and subsequently adopted across the INSDC, allows sequencing centers to register projects under a unique project identifier, enabling reliable linkage between sequencing projects and the data they produce. The Projects database is currently expanding to include a wider variety of projects, such as metagenome and environmental sampling projects, comparative genomics projects and transcriptome projects as well as projects focused on a particular locus, such as 16S ribosomal RNA, or a notable medical event, such as the 2009 H1N1 flu outbreak.
A ‘DBLINK’ line appearing in GenBank flat files identifies the sequencing projects with which a GenBank sequence record is associated and, as of GenBank release 172, replaces the earlier ‘PROJECT’ line. As an example, the DBLINK line below associates a GenBank sequence record with Project record 18787.
Project record 18787 provides details of the progress made in the effort to sequence the green anole, Anolis carolinensis (www.broad.mit.edu/models/anole/). Within the Entrez system, such a sequence record is linked directly to the appropriate Genome Project record; these links are bidirectional, so that the Project records also link back to associated sequence records.
Sequence-similarity searches are the most fundamental and frequent type of analysis performed on the GenBank data. NCBI offers the BLAST family of programs (blast.ncbi.nlm.nih.gov) to detect similarities between a query sequence and database sequences (11,12). BLAST searches may be performed on the NCBI web site (13) or by using a set of standalone programs distributed by FTP (4).
NCBI distributes GenBank releases in the traditional flat file format as well as in the ASN.1 format used for internal maintenance. The full bi-monthly GenBank release along with the daily updates, which incorporate sequence data from EMBL and DDBJ, is available by anonymous FTP from NCBI at ftp.ncbi.nih.gov/genbank. The full release in flat file format is available as a set of compressed files with a non-cumulative set of updates at ftp.ncbi.nih.gov/daily-nc/. Uncompressed, a complete copy of release 173 occupies 437 GB. A script is provided in ftp.ncbi.nih.gov/tools/ to convert a set of daily updates into a cumulative update.
GenBank, National Center for Biotechnology Information, Building 45, Room 6AN12D-37, 45 Center Drive, Bethesda, MD 20892, USA. Tel: +1 301 496 2475; Fax: +1 301 480 9241.
NCBI Home Page: www.ncbi.nlm.nih.gov.
Submission of sequence data to GenBank: vog.hin.mln.ibcn@bus-bg.
Revisions to, or notification of release of, ‘confidential’ GenBank entries: vog.hin.mln.ibcn@etadpu.
General information about NCBI resources: vog.hin.mln.ibcn@ofni.
If you use the GenBank database in your published research, we ask that this article be cited.
Funding for open access charge: The Intramural Research Program of the National Institutes of Health; National Library of Medicine.
Conflict of interest statement. None declared.