Nucleic Acids Research Advance Access originally published online on October 18, 2007
Nucleic Acids Research 2008 36(Database issue):D53-D56; doi:10.1093/nar/gkm811
Nucleic Acids Research, 2008, Vol. 36, Database issue D53-D56
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
UgMicroSatdb: database for mining microsatellites from unigenes
Veenu Aishwarya and
P. C. Sharma*
University School of Biotechnology, Guru Gobind Singh Indraprastha University, Kashmere Gate, Delhi 110 006, India
*To whom correspondence should be addressed. Tel: +91 11 23900220; Fax: +91 11 23865941; Email: deansbt{at}yahoo.co.in
Received August 14, 2007. Revised September 17, 2007. Accepted September 18, 2007.
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ABSTRACT
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Microsatellites, also known as simple sequence repeats (SSRs)
or short tandem repeats (STRs), have extensively been exploited
as molecular markers for diverse applications. Recently, their
role in gene regulation and genome evolution has also been discussed
widely. We have developed UgMicroSat
db (Unigene MicroSatellite
data
base), a web-based relational database of microsatellites
present in unigene sequences covering 80 genomes. UgMicroSat
db allows microsatellite search using multiple parameters like
microsatellite type (simple perfect, compound perfect and imperfect),
repeat unit length (mono- to hexa-nucleotide), repeat number,
microsatellite length and repeat sequence class. Microsatellites
can also be retrieved by specifying EST, cDNA, CDS identity
or by using Gene Index, GenBank, UniGene IDs. The database also
provides information about trinucleotide repeats encoding various
amino acids. Such codon repeats can be searched by specifying
characteristics of coded amino acids like charge (basic, acidic
or neutral), polarity (polar or non-polar), and their hydrophobic
or hydrophilic nature. The nucleotide sequences of the target
UniGenes are also provided to facilitate primer designing for
PCR amplification of the desired microsatellite. UgMicroSat
db is available at
http://ipu.ac.in/usbt/UgMicroSatdb.htm.
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INTRODUCTION
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Microsatellites represent arrays of 1–6 bp tandem repeats
in DNA. These sequences have proved very useful as molecular
markers in diverse areas of genetic research including genome
characterization and mapping (
1). Recently, microsatellites
have also been implicated to play some role in gene regulation
and genome evolution (
2–4). Association of trinucleotide
repeats with many human diseases has been reported over the
years (
5). Some important examples include Huntington's disease
and spinocerebellar ataxia (SCA) caused by expansion of CAG
repeats (
6,
7), and oculopharyngeal muscular dystrophy caused
by GCG expansion (
8). Some other reports suggest association
of trinucleotide repeats with various forms of cancer (
9,
10).
(A)
n repeats too have been assigned both cancer causing (
11–13)
and tumor-suppressive functions (
14). Interestingly, trinucleotide
repeats are now also being considered as potential therapeutic
agents that act by triggering RNAi pathway as they are highly
specific in silencing the mutant transcripts containing complementary
repeats (
15). In Drosophila, expansion of CAG repeats in homeobox
gene
DLX6 leads to cell death (
16).
The importance of microsatellites has been appreciated in plant systems also. Microsatellites derived from EST sequences (EST–SSRs) have found immense utility in various research projects in recent years (17). EST–SSR markers have been preferred over genomic-SSR markers for plant improvement programmes owing to their higher interspecific transferability rate. Moreover, EST–SSRs are proposed to be the better candidates for gene tagging. EST based microsatellite markers have been developed for apricot and grape (18), barley (19) and wheat (20), to name a few. However, unlike animal systems, significance of microsatellites in transcriptional activities has not been well documented in plant systems.
Considering vast utility of microsatellites in the fields of medicine and agriculture, many research groups have attempted to characterize their abundance, distribution and genomic localization using in silico methods (21–23). Such tools enable research scientists to exploit microsatellites for different applications with greater efficiency and specificity. EST–SSR databases have been developed and released in public domain by different groups. Examples of such databases include PlantSSR database (http://www.genome.clemson.edu/projects/ssr/) developed by Clemson University Genomics Institute (CUGI) and COS–EST–SSRs for cereals and legumes (http://intranet.icrisat.org/gt1/ssr/ESTSSRClustersubmit.asp). CMD (Cotton Microsatellite Database; 24) and SilkSatDb (25) provide microsatellite data from genomic sequences as well as EST sequences. Satellog (26) catalogs a number of disorders associated with mutations in microsatellite sequences near or within genes in humans. InSatDb (27) provides a compilation of microsatellites in five insect species. Such databases offer useful resources for various research activities aiming towards development of microsatellite markers and also for investigations focusing on deciphering the functional roles of microsatellites.
The databases and resources described above and elsewhere (28) remain specific and limited in their content and purpose. In particular, microsatellites within the transcriptionally active regions of the genome have not received the desired attention over a wide range of taxa. Such information is, however, necessary for undertaking various transcriptome based experimental studies. Therefore, there is a need to develop a platform for mining genic microsatellites to ensure, firstly their better utilization as molecular markers, secondly to understand fundamental questions concerning their abundance, distribution and evolution and thirdly to attribute putative function, if any, to these repeats. Considering this requirement, we have developed UgMicroSatdb (Unigene MicroSatellite database), a relational database that provides information on microsatellites present within the unigenes across 80 eukaryotic genomes. A classified range of search options facilitate a user friendly and specific extraction interface. The database is so designed that unigene sequences harboring microsatellite(s) of interest can be extracted and further used, for example, in cross amplification experiments. We hope that information retrieved from the database may be helpful in opening new frontiers of basic and applied research on microsatellites.
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CONSTRUCTION OF DATABASE
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UgMicroSat
db provides a catalogue of microsatellites occurring
in unigene sequences of eukaryotic organisms belonging to a
wide range of taxonomic groups. UniGene sequences were downloaded
from The National Center for Biotechnology Information (NCBI)
and scanned using a simple sequence repeat mining tool called
MISA (
19) that extracted microsatellite motifs and wrote them
in tab delimited text files. This raw microsatellite information
was processed using a set of C++ codes and Perl scripts. VMI_PRCS,
a C++ code, processed statistics like size and position of microsatellites
within the unigene sequences. VUG_PRCS, another C++ code, processed
unigene IDs and sequences in the desired format. The data was
reassembled using a Perl script VDATA_ASMBL and a data file
was created. The data file was further formatted and then imported
as a table in a MS-ACCESS database. Similar approach was adopted
for all the individual sets of unigene sequences of different
species. The overall scheme of database construction is explained
in
Figure 1. The quick retrieval of information from UgMicroSat
db has been ensured by creating small, specific sub-databases for
different groups of organisms. Furthermore, within each sub-database,
individual organism has been represented by a separate table.
A parent database that indexes all the sub-databases and the
tables therein maintains fast, efficient and precise communication
with these sub-databases. The graphical user interface was constructed
using Active Server Pages (ASP). The overall architecture of
the database has been outlined in
Figure 2.
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ACCESSING DATABASE
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UgMicroSat
db can be accessed to extract simple (perfect) repeats
and compound (perfect and imperfect) repeats. Microsatellites
can be mined using various search options viz. repeat unit length
(mononucleotide to hexanucleotide), repeat sequence (motif search),
microsatellite length, host cDNA, CDS, EST name, GenBank ID,
gene index number or UniGene ID and microsatellite class search
(
29). For trinucleotide repeats, the database also gives data
for codon repeats i.e. repeats that code for amino acids. The
option ‘amino acid codon search’ allows search for
repeats that code for all the 20 amino acids. Further, search
can also be made for codon repeats on the basis of characteristics
of amino acids like charge (basic, acidic and neutral), polarity
(polar and non-polar) and hydrophobicity or hydrophilicity.
The database also allows batch download, such that a user can
download all the microsatellites mined in response to a particular
query in a text file. The database is linked with NCBI for the
retrieval of detailed information on unigene sequences based
on their genbank IDs. Finally, the database allows the user
to design primers for PCR amplification of the specific microsatellite
locus by providing the selected unigene sequences harboring
the particular microsatellite, and is also linked to Primer3,
a primer designing tool (
30). A quick help mode is provided
with examples on how to fill in the search options for easy
reference and navigation for the user. The search options are
extensively explained with the help of some case studies on
the database website. The database can serve as an immense source
of information in understanding the microsatellite dynamics
in the transcriptionally active regions. For example, information
pertaining to a trinucleotide repeat CTC, whose length is between
10 and 20 base pair, present in platelet-derived growth factor
alpha polypeptide (PDGFA) of
Homo sapiens can easily be searched
as shown in
Figure 3. The output also gives the GenBank IDs
and gene index number along with the unigene IDs. Further, the
details of the unigene and the localization of the microsatellite
(start and end positions) are provided and the microsatellites
are marked in lower capitals.
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UTILITY OF THE DATABASE
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UgMicroSat
db is likely to be accessed by biologists engaged
in research with diverse objectives in both plant and animal
systems primarily to develop molecular markers and also to understand
the functional significance of microsatellites in regulating
gene expression and genome evolution. UgMicroSat
db offers an
important platform for a detailed comparative analysis of microsatellite
repeats in genic regions for a wide range of species. The comprehensive
options to search for simple and compound microsatellites and
to identify the codon repeats in the genic regions allow users
to explore new avenues of investigations on these repeats. The
availability of unigene sequences for different aspects like
designing primers for PCR amplification of desired motifs will
facilitate studies on mutability, microsatellite abundance and
variability across genomes, etc. Association of these microsatellites
with a particular disease or phenotype may be explored by identifying
their expansion and contraction possibilities in a given population.
Microsatellite data can also be used to investigate various
anomalies and disorders using candidate gene approach. Further,
such information can be used to design synthetic oligonucleotides
representing complementary repeats to be used in RNA interference
based silencing to target mutant genes. Such approaches hold
much therapeutic value. The database can largely be used to
develop EST–SSR markers for various research programmes,
particularly on genome mapping and gene tagging. Apart from
hosting an extensive form of microsatellite data within the
genes, UgMicroSat
db is unique in a way as compared to the previously
developed databases as it hosts microsatellite data covering
a large number of organisms including both lower and higher
forms of plants and animals. Relative mining of imperfect repeats
of such a diverse range of organisms may provide tools to study
the dynamics of microsatellites and also their association with
similar or different types of repeats. To conclude, UgMicroSat
db will serve as an important starting point whereby extracted
information serves as an important input in designing experiments
in new directions elucidating novel roles and functions of microsatellites
in the unexplored transcriptomes.
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FUTURE PERSPECTIVES
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At present, UgMicroSat
db hosts data on microsatellites occurring
in unigene sequences of 80 genomes. UgMicroSat
db team aims to
update the database commensurating with updation of the NCBI
unigene database. The flexible design of the database makes
it feasible to increase the size of database to virtually any
size without compromising with its fast data retrieval rate.
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AVAILABILITY
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UgMicroSat
db can be freely accessed from
http://ipu.ac.in/usbt/UgMicroSatdb.htm
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ACKNOWLEDGEMENTS
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Funding to pay the Open Access publication charges for this
article was waived by the Oxford University Press.
Conflict of interest statement. None declared.
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ABSTRACT
INTRODUCTION
