ABSTRACT
The GenBank sequence database incorporates DNA sequences from all available
public sources, primarily through the direct submission of sequence data from
authors and from large-scale sequencing projects. Data exchange with the EMBL Data Library and
the DNA Data Bank of Japan helps ensure comprehensive coverage. GenBank
continues to focus on quality control and annotation while expanding data
coverage and retrieval services. An integrated retrieval system, known as
Entrez
, incorporates data from the major DNA and protein sequence databases, along
with genome maps and protein structure information. MEDLINE abstracts from
published articles describing the sequences are also included as an additional
source of biological annotation. Sequence similarity searching is offered
through the BLAST family of programs. All of NCBI's services are offered
through the World Wide Web. In addition, there are specialized server/client
versions as well as FTP and e-mail server access.
GenBank
®
(
1
) is a public database of all known nucleotide and protein sequences with
supporting bibliographic and biological annotation, 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). NCBI was created by Congress in 1988 to develop
information systems, such as GenBank, to support the biomedical research
community. NCBI was also mandated to conduct basic and applied research and, as
part of the NIH Intramural Program, NCBI scientists work in areas of gene and
genome analysis, computational structural biology, and mathematical methods for
sequence analysis.
NCBI builds GenBank primarily from the direct submission of sequence data from
authors and secondarily from scanning the journal literature. A major source of
data are bulk submissions of EST and other high-throughput data from sequencing centers. The data are supplemented by
sequences from other public databases. Through an international collaboration
with the EMBL Data Library in the UK and the DNA Databank of Japan (DDBJ), data
are exchanged daily to ensure that all three sites maintain comprehensive sets
of sequence information. The data are made available at no cost through the
Internet, either by downloading database files or by text and sequence
similarity search services.
GenBank has experienced another year of unprecedented growth. Over the past 12
months 420 000 new sequences have been added. As of Release 96 in August, 1996, GenBank contained 602 072 354 nucleotide bases from 920 588 different sequences. Notably, 1996 is the year in
which the complete genome of a eukaryotic organism, the yeast
Saccharomyces cerevisiae
was completed and added to GenBank (
2
). The complete genomic sequences of an archeon,
Methanococcus jannaschii
(
3
) also entered the database this year (see Genomes division below).
Historically, the database had been doubling in size about every 18 months, but
that rate has rapidly accelerated due to the enormous growth in data from
expressed sequence tags (ESTs). Over 65% of the sequences in the current
release are ESTs and most of the growth in terms of sequence records over the
past 2 years has come from the collaborative project between the Merck & Co. and Washington University (
4
,
5
). This growth is expected to continue because Washington University and the
Howard Hughes Medical Institute are pursuing a mouse EST project of the same
scale as human EST sequencing. Furthermore, the Human Genome Project has
entered its pilot scale-up sequencing phase and 100 million nucleotides of human genomic DNA
sequence data are expected during the next 2 years. Sequence records from
several of the six US centers funded to do this work are already beginning to
appear in GenBank.
Of the nearly one million sequences in GenBank, human entries predominate,
constituting 59% of the total, but >16 000 species are represented and ~10 new organisms enter GenBank each day. Database sequences are processed,
and can be queried, using a consistent and comprehensive sequence-based taxonomy developed by NCBI in collaboration with EMBL and DDBJ and
with the valuable assistance of external advisors and curators. Further details
along with a taxonomy browser and information on taxonomic resources may be
found via NCBI's home page.
After
Homo sapiens
, the top species in GenBank, in terms of the number of bases, include
Caenorhabditis elegans, Mus musculus,
Saccharomyces cerevisiae
, and
Arabidopsis thaliana
. For
Caenorhabditis
and
Saccharomyces
this is due to the impact of genomic sequencing projects; for
Mus
and
Arabidopsis,
sequence abundance currently reflects large-scale EST sequencing efforts.
Each GenBank entry includes a concise description of the sequence, the
scientific name and taxonomy of the source organism, and a table of features
that identifies coding regions and other sites of biological significance, such
as transcription units, intron/exon boundaries, sites of mutations or
modifications, and other sequence features. Protein translations for coding
regions are included in the feature table. Bibliographic references are
included along with links to the MEDLINE
®
unique identifiers for all published sequences.
The files in the GenBank distribution have traditionally been divided into
`divisions' which roughly correspond to taxonomic divisions, e.g. bacteria,
viruses, primates, rodents, etc. In the past year a division has been added for
Genome Survey Sequences, e.g. `single pass' reads from cosmid/BAC/YAC ends,
exon-trapped genomic sequences, and Alu PCR sequences. A new division, for High-Throughput Genomic (HTG) sequences, was added with Release 97 in
October, 1996. The HTG division is designed for the efficient processing and
distribution of long sequences expected to be primarily human and including
computer-generated annotation.
In order to produce the GenBank database, NCBI maintains internally an
Integrated Database, ID, to track and index records from the multiple sources
of sequence data. These sources include submissions from EMBL, DDBJ, Genome
Sequence Database (GSDB), and patents, plus amino acid sequences from PIR,
SWISS-PROT, Protein Research Foundation (PRF) and the Protein Data Bank (PDB).
ID represents the most current view that each data source has of its sequence
data, and allows NCBI to assign stable identifiers. Such identifiers for
nucleotide sequences are found in the `NID' field of a GenBank record, directly
following the ACCESSION field. Identifiers for encoded protein sequences are
found in the FEATURES table under CDS and are labeled `PID'. Through this
approach, sequence information from a wide variety of sources can have a
uniform identification system. These identifiers are stable and therefore help
identify sequences which have changed.
A separate section of the database has been created for the placement of
chromosome- or genome-length sequences. This section, the Genomes division, arose from the
need to store sequences >350 000 bases, the upper limit for single database
entries. The entries in this section are organized with respect to a `reference
sequence'. A reference sequence can be the complete sequence of a genome as
contained in a single GenBank record, or a specific sequence that has been
accepted by researchers in that particular field (e.g. HIV research) as a
reference sequence. In some cases, the selection of a reference sequence is
arbitrary. With respect to the reference, all other sequences in GenBank from
that organism or organelle are aligned and the alignments are presented under
the reference sequence in the Genomes division. This concept of a reference
sequence is critical for human genome data, because the complete human sequence
will actually be a composite of the sequences of several individuals (
6
).
In some cases, for example yeast and some bacterial genomes, the complete
sequences of the chromosomes are known but exist as many separate records in
GenBank. In these cases a virtual reference sequence is created that contains
instructions on how to assemble the GenBank records to make the complete
chromosome. The NCBI software tools can dynamically project the features
annotated on the GenBank records to the coordinate system of the reference
sequence, including entire chromosomes, for any region of interest. This
provides the scientist with both a large-scale view of genomic sequence data but also a view of smaller regions
around genes in the traditional GenBank record format. The breakdown of a
genome sequence into separate records makes for a convenient unit of analysis
and annotation updates because only a relatively small GenBank record need be
changed and distributed. Nevertheless, any new information will appear
automatically in the large-scale view as well.
The most complicated case is for chromosomes which are not completely sequenced
such as eukaryotic chromosomes other than yeast. As one example, NCBI has
obtained, in collaboration with a number of established mapping centers,
genetic, physical and cytogenetic maps of human chromosomes and produced cross-referenced sets of aligned maps. Using STS markers on these maps, and the
UniGene set of non-redundant human genes (
7
), aligned groups of sequences can be placed onto the framework of the whole
chromosome. These sequences appear as `islands' on the sequence map (Figs
1
and
2
) and will eventually coalesce into contiguous sequence as the Human Genome
Project is completed. This approach provides an important organizing principle
for making optimal use of traditional annotated human sequence records in
GenBank, as well as the specialized properties of EST, STS and HTG sequences.
ESTs or `expressed sequence tags' are still the most rapidly expanding source of
new sequence records and genes. Last year there were 328 905 sequences in the
EST Division of GenBank (dbEST). Over the past year there has been a doubling
in the number of ESTs for a current total of 658 698 sequences in dbEST
representing 63 different organisms. The top five organisms include: 476 148
human ESTs (72%); 74 312 mouse ESTs (11%); 30 196 nematode ESTs (4.6%); 28 951
Arabidopsis
ESTs (4.4%); and 11 316 rice ESTs (1.7%). ESTs continue to provide the major
source of new gene discoveries and NCBI has serviced more than half a million
queries (BLAST searches, e-mail retrievals, WWW accesses and anonymous ftp downloads) for dbEST data
to date.
Because of the nature of EST survey sequencing, there is much redundancy in
dbEST. For example, there are >1300 sequences representing serum albumin mRNA.
In order to organize the data in a more useful fashion, NCBI has created the
UniGene collection of unique human genes (
7
). Briefly, UniGene starts with human entries in the primate (PRI) division of
GenBank, combines these with human ESTs and creates clusters of sequences that
share virtually identical 3' untranslated regions (3'UTRs). In this manner, the nearly 500 000 human ESTs in dbEST have
been reduced 10-fold to ~50 000 sequence clusters each of which may be considered as
representing a single human gene. Access to the UniGene collection is provided
through NCBI's home page on the WWW. The UniGene collection has been
effectively utilized as a source of mapping candidates for the construction of
a human gene map (
7
). In this case, the 3'UTRs of genes and ESTs are converted to STSs which are then placed on
physical maps and integrated with preexisting genetic maps of the genome.
The ultimate purpose for creating high resolution physical maps of the human
genome is to create a scaffold for organizing large-scale sequencing (
8
). Physical maps based on `sequence tagged site' (STS) landmarks are used to
develop so-called `sequence-ready' clones consisting of overlapping cosmids or BACs. As the high-throughput sequence data derived from these clones are
submitted to GenBank, STSs become crucial reference points for organizing,
presenting and searching the data. NCBI uses a process of `electronic PCR' in
which all human sequences are compared with the contents of the STS division of
GenBank (dbSTS) to see if the former sequences contain primer binding sites in
the correct orientation and at an appropriate spacing that the expected product
would be amplified in a PCR reaction. This tool permits us to assign an initial
location on the map for sequence data and to relate previous GenBank entries to
the new reference sequence. The electronic PCR tool developed for this task is
also being made publicly available on the WWW to enable any researcher with a
new human sequence to relate that sequence to existing maps and HTG sequence
data.
The STS division of GenBank currently contains 37 261 STS sequences and includes
anonymous STSs based on genomic sequence as well gene-based STSs derived from the 3' ends of genes and ESTs. These STS records usually include primer
sequences and PCR reaction conditions.
The data in GenBank come from three sources: (i) authors who submit data
directly to the collaborating databases; (ii) bulk submissions from sequencing
centers; and (iii) annotators at NCBI who extract the information from relevant
journals. Data are exchanged daily with collaborating databases so that the
daily updates from NCBI servers incorporate the most recently available
sequence data from all sources.
The majority of entries continue to enter the database through direct author
submission. Many journals have the policy of requiring authors with sequence
data to submit data directly to the database as a condition of publication.
Even for those journals without a mandatory submission policy, author
submission has the positive benefits of acquiring annotation information
directly from the authors and reducing the time-lag between publication and the appearance of the sequence in the
database.
Over the past year several large-scale sequencing projects have begun scaling up to the goal of producing
hundreds of megabases of human genomic DNA sequence over the next 1-3 years. NCBI is working closely with sequencing centers to ensure timely
incorporation of these data for public release. In parallel, NCBI has developed
methods to display these data integrated with genetic and physical map data and
to search the sequences more effectively (e.g. options in BLAST to mask Alu and
other types of repetitive elements). GenBank offers special batch procedures
for large-scale sequencing groups to facilitate data submission.
Over 80% of individual submissions are now received through a Web-based data submission tool, BankIt. With BankIt, authors enter sequence
information directly into a form, edit as necessary, and add biological
annotation (e.g. coding regions, mRNA features). Free-form text boxes provide the option of using your own words to describe the
sequence, without having to learn formatting rules or use restricted
vocabularies. BankIt creates a draft record in GenBank flat file format for the
user to review and revise.
GenBank has developed a platform-independent submission program called Sequin, which runs stand-alone or over the network. The advantages of Sequin over the
previous submission program, Authorin, include the capacity to handle long
sequences and segmented entries, easier editing and updating, and complex
annotation capabilities. In addition, Sequin contains a number of built-in validation functions for enhanced quality assurance. Versions for
Macintoshes, PCs and Unix are available at no charge. It can be obtained by
anonymous FTP to `ncbi.nlm.nih.gov' in the `pub/sequin' directory. Once a
submission is completed, users can e-mail it to the address: gb-sub@ncbi.nlm.nih.gov
GenBank staff can usually assign an author an accession number within 1 working
day of receipt. The accession number serves as confirmation that the sequence
has been submitted and allows readers of the article to retrieve the relevant
data. All direct submissions receive a systematic quality assurance review
including screening against GenBank to identify full or partial matches,
checking for vector sequence and verifying proper translation of coding
regions. A draft of the GenBank record is passed back to the author for review
before entering the database. Authors have the right to request that their
sequences be kept confidential until the time of publication. In these cases,
authors are reminded to inform the database of the publication date in order to
have a timely release of the data. Although only the submitting scientist is
permitted to modify sequence data or annotations, all users are encouraged to
inform the database of possible errors or omissions using the e-mail address: update@ncbi.nlm.nih.gov
NCBI has begun distributing a database of macromolecular three-dimensional structure information, specifically aimed at molecular
modeling research. MMDB is based upon the data in the Brookhaven PDB. By
reorganizing and validating the PDB data, MMDB provides explicit descriptions
of a biopolymer's spatial structure, its chemical organization, and the linkage
between the two. With MMDB, there is a clear cross-referencing between the three-dimensional structure and the chemistry of a macromolecule. Explicit
linkages have been made to the sequence entries within the ID database so that,
with the appropriate graphics software, users are able to view the three-dimensional structure of proteins identified via text or similarity
searching of the sequence database. During the past year, NCBI added a new
three-dimensional structure viewer called Cn3D (`See in 3D') to the
Entrez
retrieval system.
Entrez
is an integrated database retrieval system which accesses DNA and protein
sequence data, related MEDLINE references, genome data from the GenBank genomes
division, the NCBI taxonomy and three-dimensional structures from MMDB (
9
). The DNA and protein sequence data are integrated from a variety of sources
via the ID database previously described. The MEDLINE references are
approximately one million citations indexed under the NLM's Medical Subject
Heading (MeSH), `genetics'.
The linkages among data sources are shown in Figure
3
. The DNA sequence, protein sequence, MEDLINE, genome and three-dimensional structure data are linked to provide easy traversal among the
data sets.
Entrez
provides an entry point into sequence or bibliographic records by simple
Boolean queries. From a record, hypertext links may be used to navigate through
the information space using a point-and-click interface. Some of the links are simple cross-references, for example, between a sequence and the abstract
of the paper in which the sequence was reported, or between a protein sequence
and its corresponding DNA sequence. Among these cross-references are external links to the full-text versions of articles when these are available from publishers'
WWW sites. Other links are based on computed similarities among the sequences
or among the textual documents. The pre-computed `neighbors' allow very rapid access for browsing groups of
related records.
One of the most frequent uses of GenBank is sequence similarity searching. NCBI
offers the BLAST family of search programs to perform fast searching with
rigorous statistical methods for judging the significance of matches. WWW
access to BLAST currently offers two interfaces, a `Basic' version with default
search parameters and an `Advanced' option which allows customization of the
parameters. A new graphical version called PowerBLAST, designed for rapid
analysis and annotation of large contigs of genomic sequence data (J. Zhang,
personal communication), is available as a server/client application on NCBI's
ftp site and it is being modified for use via the BLAST WWW pages. PowerBLAST
is particularly useful for assessing multiple EST matches with a query
sequence.
The primary databases for sequence similarity searching are non-redundant (`nr') versions of nucleotide and protein sequences. ESTs are
available for separate searching, as is a set of those sequences added during
the previous 30 days (designated `month' on the BLAST web pages). Frequent
users may find the server/client version of BLAST more convenient; clients are
available for several platforms. BLAST client software also incorporates
advanced features such as one-to-many alignments of the query sequence with all the matching
sequences (as opposed to the standard results that show the query sequence
aligned individually against each matching sequence). Another feature of the
client software is the ability to generate organism-specific output, for example, searches restricted to human sequences.
Information on BLAST client software can be obtained by e-mail to the address: blast-help@ncbi.nlm.nih.gov
Users on the Internet can use the file transfer protocol (FTP) program to
download the entire GenBank release or the daily updates (which also
incorporate sequence data from other public databases). Files of the full
release and daily updates of the GenBank database are available for anonymous
FTP from: ncbi.nlm.nih.gov. The full release in flat-file format is available as compressed files in the directory, `genbank'.
A cumulative update file is contained in the subdirectory, `daily', and a non-cumulative set of updates is in the subdirectory, `daily-nc'. ASN.1 formatted data is in the directory, `ncbi-asn1'. Software developers creating their own interfaces or
analysis tools for GenBank data are offered the NCBI toolkit to assist in
developing specialized applications. Software can be found in the directory:
toolbox/ncbi_tools
Users with access to electronic mail can search GenBank and several other
databases by accession number or Boolean combinations of text words. The QUERY
server (query{at}ncbi.nlm.nih.gov) performs text-based searches of the integrated
Entrez
databases. It allows access not only to sets of sequence or MEDLINE records, but
also to the neighbored data. Various output formats, such as FASTA for sequence
data, are available. The RETRIEVE e-mail server is still operating (retrieve{at}ncbi.nlm.nih.gov), but will
eventually be superseded by QUERY, which also supports the RETRIEVE query
syntax. BLAST sequence similarity searches can be performed by e-mail through the address: blast@ncbi.nlm.nih.gov. Documentation can be
obtained for each of the servers by sending the word `help' in the body of an e-mail message to the addresses above.
The flat file version of GenBank is available on CD-ROM through a subscription service with the Government Printing Office
(Tel: +1 202 512 1800; Fax: +1 202 512 2233). Order forms are also included in
each issue of
NCBI News
, a free subscription to which may be obtained by contacting NCBI. A new release
of the database appears every 2 months. Each release is a full release
incorporating all previous GenBank data supplemented by new data from direct
submissions, NCBI journal scanning, patents and the other sequence databases.
Conceptual translations of coding regions appear in feature tables. The release
contains the standard index files and is organized into divisions. No retrieval
software is provided. The distribution currently requires six CD-ROMs. (The CDROM version of
Entrez
has been discontinued because of the size of the distribution files.)
The GenBank Fellowship Program is an NCBI initiative to improve the quality of
the database and also to serve as a bioinformatics training program. GenBank
fellows are selected for strong backgrounds in biology and for motivation to
apply computational tools to the organization of electronic data in molecular
and structural biology, genetics and phylogeny. GenBank Fellows, under the
supervision of a mentor from NCBI's Computational Biology Branch, pursue
various applied research projects to improve the quality and annotation of
GenBank entries, to reduce sequence redundancy, and to establish and maintain
links to other databases. Currently five GenBank fellows are in the program and
applications are reviewed on a continuing cycle.
GenBank, National Center for Biotechnology Information, Building 38A, Room 8S-803, 8600 Rockville Pike, Bethesda, MD 20894, USA. Tel: +1 301 496 2475;
Fax: +1 301 480 9241.
http://www.ncbi.nlm.nih.gov/ (NCBI Home Page)
gb-sub@ncbi.nlm.nih.gov (submission of sequence data to GenBank)
update@ncbi.nlm.nih.gov (revisions to GenBank entries and notification of
release of `hold until published' entries).
info@ncbi.nlm.nih.gov (general information about NCBI and services).
If you use GenBank as a tool in your published research, we ask that this paper
be cited.
REFERENCES
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