The data in GenBank and the collaborating databases, EMBL-Bank and DDBJ, are submitted either 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-Bank so that the daily updates from NCBI servers incorporate the most recently available sequence data from all sources.
Direct electronic submission
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 sequence 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 ~3500/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 update@ncbi.nlm.nih.gov.
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/genbank/tbl2asn2.html.
Submission using BankIt
About a third of author submissions are received through an NCBI web-based data submission tool named BankIt. Using BankIt, authors enter sequence information and biological annotations, such as coding regions or mRNA features, directly into a series of tabbed forms that allow the submitter to describe the sequence further without having to learn formatting rules or controlled vocabularies. Additionally, BankIt allows submitters to upload source and annotation using tab-delimited tables. 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.
Submission using Sequin and tbl2asn
NCBI also offers a standalone multi-platform 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), phylogenetic studies, population studies, mutation studies, environmental samples with or without alignments and sequences with complex annotation. Sequin has a number of wizards that guide the submitter in preparing their submission with proper annotation for a number of data types, like viral genomic sequences and ribosomal RNA from cultured and uncultured microbes. 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 11.7, was released in September 2011 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 gb-sub@ncbi.nlm.nih.gov. Submitters of large, heavily annotated genomes may find it convenient to use
tbl2asn 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.
Submission of barcode sequences
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. For animal species, a 648-bp fragment of the gene for cytochrome oxidase subunit I is used as the barcode. The plant and fungal communities are using other loci. NCBI, in collaboration with CBOL (
www.barcoding.si.edu/), provides 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 the over 500
000 barcode sequences in GenBank, over 300
000 of which were added in the last year.
Notes on particular divisions
Transcriptome shotgun assembly sequences The TSA division contains transcriptome shotgun assembly (TSA) sequences that are assembled from sequences deposited in the NCBI Trace Archive, the Sequence Read Archive (SRA) and the EST division of GenBank. While neither the Trace Archive nor SRA is a part of GenBank, they are part of the INSDC and provide access to the data underlying these assemblies (
4,
5). TSA records (for example, EZ000001) have ‘TSA’ as their keyword and can be retrieved with the query ‘tsa[properties]’.
Environmental sample sequences The environmental sample sequences (ENV) division of GenBank accommodates 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.
WGS sequences WGS sequences appear in GenBank as groups of sequence-overlap contigs collected under a master WGS record. Each master record represents a WGS project and has an accession number in the Nucleotide database consisting of a four-letter prefix followed by eight zeroes and a version suffix as found in standard GenBank records. The number of zeroes increases to nine for WGS projects with one million or more contigs. Master records contain no sequence data; rather, they are linked to their set of individual contigs that can be viewed using the new WGS browser (see above). Contig records have accessions consisting of the same four-letter prefix as their master accession, followed by a two-digit version number and a six-digit contig ID. For example, the WGS accession number ‘AAAA02002744’ is assigned to contig number ‘002744’ of the second version of project ‘AAAA’, whose accession number is ‘AAAA00000000.2’. Currently, there are over 3400 WGS sequencing projects, many of whose data have been used to build more than 9 million scaffolds and chromosomes for genome assemblies. For a complete list of WGS projects with links to the data, see
www.ncbi.nlm.nih.gov/Traces/wgs/.
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.
Expressed sequence tags Expressed sequence tags (ESTs) continue to be a major source of data for gene expression and annotation studies, and at 39 billion base pairs, it remains the largest non-WGS division in GenBank. EST data are available for download from
ftp.ncbi.nih.gov/repository/dbEST/ (
6) as well as from the GenBank FTP site. The data in dbEST are clustered using the BLAST programs to produce the UniGene database (
www.ncbi.nlm.nih.gov/unigene) of more than 5.3 million gene-oriented sequence clusters representing almost 140 organisms (
4).
High-throughput genomic and high-throughput cDNA sequences The high-throughput genomic (HTG) division of GenBank (
www.ncbi.nlm.nih.gov/genbank/htgs/) contains unfinished large-scale genomic records, which are in transition to a finished state (
7). These records are designated as belonging to Phases 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.
The HTC division of GenBank contains high-throughput cDNA (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. A project generating HTC data is described in Ref. (
8).
Special record types
Third party annotation 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). Each of the current 160
000 TPA records falls into one of three categories:
experimental, in which case there is direct experimental evidence for the existence of the annotated molecule;
inferential, in which case the experimental evidence is indirect; and
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 as well as corresponding keywords. TPA experimental and inferential records also contain a Primary block that provides the base ranges and identifier for the sequences used to build the TPA. 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.
Contig (CON) records for assemblies of smaller records Within GenBank, CON records are used to represent very long sequences, such as a eukaryotic chromosome, where the sequence is not complete but consists of several contig records with uncharacterized gaps between them. Rather than listing the sequence itself, CON records contain assembly instructions involving the several component sequences. An example of such a CON record is CM000663 for human chromosome 1.
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