microsatellites are simple sequence repeats ( ssrs ) that exhibit complex patterns in their frequency of occurrence , genomic distribution , mutability , function and evolution . apart from being the source of informative genetic markers , microsatellites per se
have attracted a lot of attention with respect to their origin , distribution , expansion , mutation and disintegration ( 17 ) .
questions are also asked about the functional role of microsatellites in particular and biological significance of the microsatellites in general ( 4,812 ) .
genetic studies and whole genome sequence analysis have established non - random distribution , variability and high mutability as characteristics of microsatellites .
evidences are accruing , which support the role of microsatellites in gene regulation , transcription and protein function ( 13 ) .
existence of qualitative and quantitative differences between microsatellites of different genomes and their role in adaptive evolution have also been theorized ( 2,8 ) .
however , such studies require information on type ( mono to hexa ) , motif ( gc% ) , abundance ( motif preferences ) , frequency , distribution ( linkage group - wise and chromosomal position ) , location ( exon , intron , regulatory element and transposon ) , nature ( perfect , imperfect and compound ) and copy number ( existence of paralogs ) of microsatellites not only on a whole genome basis but also as a comparative analysis of multiple genomes that are related by phylogeny ( for instance , fully sequenced primate genomes or fungal genomes or insect genomes ) to draw functional conclusions .
whole genome sequences of insects have lived up to the reputation of diversity and have thrown immense variability in size and organization of their genomes . among others , there are five fully sequenced insect genomes : drosophila melanogaster ( as a model organism it provides maximum annotated data ) , anopheles gambiae ( another dipteran but economically highly important as malarial vector ) , tribolium castaneum ( relatively early insect order of coleoptera ) , apis mellifera ( hymenoptera , relatively a recent insect order ) and bombyx mori ( economically important as silk - producing member of lepidoptera , members of which are crop pests ; also significant as a model for insect development ) .
researchers attempting to understand the biology and evolution of microsatellites are often faced with the following questions : ( i ) do microsatellites occur everywhere in the genome ? ( ii )
( v ) does the gc content of the motif affect the length , repeat units or mutation rate of microsatellites ?
( viii ) do microsatellites occur as families of common flanking sequences in the genomes ( paralogs ) ?
insatdb , unlike many other microsatellite databases that cater to only the needs of microsatellites as markers , allows users to address the above - mentioned questions by accessing qualitative and quantitative genome level microsatellites profile of a single insect or to carry out comparative genomic analysis using all the five genomes .
t.castaneum sequences were downloaded from genbank ( ) and bombyx mori sequences were downloaded from .
repeats were extracted employing tandem repeat finder version 4 ( 14 ) . to ensure that the extracted repeat sequences were real microsatellites , those with less than five repeat units and shorter than 15 bp in length were excluded .
tandem repeat finder does not employ minimal alignment score for detecting microsatellites ; rather a probabilistic model of random repeat sequences specified by per cent identity and frequency of insertions and deletions .
this includes calculation of average per cent identity between the copies ( pm ) and average percentage of insertions and deletions ( pi ) .
the algorithm has a pair of matching probability and indel probability values ( pm = 0.80 , pi = 0.10 ) as default to cover most divergent copies at every locus .
we used two sets of alignment parameters ( match , mismatch , gap ) , ( + 2 , 3 , 5 ) and ( + 2 , 5 , 7 ) to score the matches . all the microsatellites with a minimum alignment score of 30 are reported in the database , which means that both perfect and imperfect microsatellites are listed .
smit , r. hubley and p. green , unpublished data ; ) to obtain indices marking the occurrence of simple repeats , tandem repeats , segmental duplications , interspersed repeats including sines , dna transposons , retrotransposons , lines , etc .
further , sequences were analysed for the delineation of exons and introns using genscan ( 15 ) . flanking sequences of microsatellites
were aligned to catalogue paralogous microsatellites that exhibit identical origin and hence considered belonging to the same microsatellite family .
occurrence of two or more microsatellites contiguously with intervening non - repeat sequence of 70 bp were separately categorized as compound repeats .
it stores microsatellites from all the five insect genomes separately as well as carries complete annotations of these microsatellites .
the database also provides basic information on each of the five insects and important links to obtain further knowledge , and contains a tutorial page and a glossary page .
end users with adequate computational capabilities can batch download full complement of microsatellites ( insect - wise ) , microsatellite sequences , compound microsatellites and full list of microsatellite loci existing as families .
these data are made available as csv files , which are compatible with spreadsheet programmes such as ms excel .
alternatively , details of the microsatellites with highly specific characteristics may be queried using a multi - option query sheet ( figure 2 ) .
the options include insect ( one at a time ) ; location ( intron , exon , i.e. boundary , upstream , intergenic , repeat elements  single or in combination ) ; repeat type ( motif size , mono- to hexa - nucleotide ) or actual repeat motif ( by essentially entering up to five repeat motifs ) ; gc% ( fixed value or range ) ; repeat size in either base pairs or number of units ( fixed value or range ) ; repeat kind ( perfect or imperfect ) . once insect and location options
the output is primarily a list of microsatellites annotated for all options of the query sheet and the output table is generated as a hierarchical pre - sorted list .
each microsatellite is given a unique i d that also carries genomic sequence i d and corresponding indices .
if the number of microsatellites selected based on the options of the query sheet exceeds 500 , the output is split into sets of 500 microsatellites . in addition , a csv file containing total output is also made available for downloading . if the query options do not select any microsatellite , a message indicating zero output is displayed and a back button is provided to refine the options .
ssr motif and 100 bp each of the left and right flanking sequences are given for each microsatellite entry , which allows users to carry out sequence analysis of microsatellite vis -  - vis locus .
in addition , users can select individual microsatellites to convert them into locus - specific markers .
this is facilitated by automatic uploading of repeat and flanking sequences of the selected microsatellite into primer3 query form ( 16 ) .
screen shots of ( a ) insatdb homepage , ( b ) multi - option query sheet , ( c ) output table and ( d ) analysis page .
our analysis showed that microsatellite content of five fully sequenced insect genomes is independent of both genome size and gc content ( table 1 ) .
the database consists of a dedicated section ( analysis ) that describes the types of analysis that can be carried out using the data obtained from insatdb .
some of the quick observations and inferences from a comparative genomic analysis are given in this section .
microsatellite content of insect genomes preponderance of di- and tri - nucleotide repeats is observed in drosophila and anopheles , whereas tri- and tetra - nucleotide repeats are abundant in bombyx and tribolium . on the whole ,
shorter microsatellites are abundant in the five insect genomes ; as the length of the microsatellite increases their number decreases logarithmically typified by bombyx and drosophila microsatellites ( > 90% of the microsatellites < 50 bp ) ; on the other hand , anopheles and tribolium have longer microsatellites in a relatively high frequency . shorter microsatellites
not only predominate microsatellite population in the five insect genomes , but also seem to possess higher number of imperfect repeat units . on the other hand , microsatellites spanning > 100 bp consisted of perfect , rather than imperfect repeats .
interruptions , if at all , occur mainly in the middle region of the repeat sequence and the ends seem to be selected against decomposition . on the whole ,
there is no linear correlation between gc content and the average number of repeat units .
average length of the microsatellite across gc range is 37  9 bp and between 0 and 5% gc content , microsatellites tend to be longer than 60 bp .
compound microsatellites account for nearly 3.2% in the insect genomes analysed ; owing to high density of microsatellites , apis has higher number of compound loci ( 6.12% ) .
anopheles and apis genomes have as many as 50 and 60% of the total microsatellites in coding region , respectively .
bombyx genome has only 10% of the microsatellites in regions spanning exons , introns and their boundary .
more than 70% of the microsatellites present in exons are trinucleotide repeats except in apis , where 50% tri- and 25% dinucleotide repeats are present in exonic regions .
microsatellites in insects are at rich ( on an average 23.4% gc ) ; however , they exist within regions that are not always at rich .
incorporation of microsatellite data from additional insects , query facility for better comparative genomic analysis such as gene - based microsatellite extraction and conservation analysis are planned .
additionally , based on users ' feedback , supplementary features will be added to make insatdb a single window system for insect genome analyses using microsatellite tools .
 insatdb presents an interactive interface to query information regarding microsatellite characteristics per se of five fully sequenced insect genomes ( fruit - fly , honeybee , malarial mosquito , red - flour beetle and silkworm ) . 
 insatdb allows users to obtain microsatellites annotated with size ( in base pairs and repeat units ) ; genomic location ( exon , intron , up - stream or transposon ) ; nature ( perfect or imperfect ) ; and sequence composition ( repeat motif and gc% ) . 
 one can access microsatellite cluster ( compound repeats ) information and a list of microsatellites with conserved flanking sequences ( microsatellite family or paralogs ) . 
 insatdb is complete with the insects information , web links to find details , methodology and a tutorial . 
 a separate  analysis section illustrates the comparative genomic analysis that can be carried out using the output . 
 insatdb is available at . 