BACKGROUND
eukaryotic gene prediction has been a challenging problem, explored over the last two decades and driven by the availability of large volumes of genomic data.
the development of gene prediction methods have traditionally included  ab-initio approaches such as genscan  <cit>  that do not use any experimental evidence,  alignment-based methods such as genewise  <cit>  that attempts to align an homologous protein sequence to a genomic sequence and more recently,  hybrid approaches that incorporate cdna-defined splice junctions into ab-initio and protein alignment information  <cit> .
such hybrid approaches for automatic annotation of genome sequences have been implemented within the ensembl annotation project  <cit> .
ensembl represents a bioinformatics project aimed at annotating sequenced genomes and integrating biological data that can be mapped or assigned to features described in the genomic data.
at present, twenty fully or near-fully sequenced vertebrate genomes have been included in ensembl .
teleosts, comprising about half the number of all extant vertebrate species, are represented by only five species, namely japanese fugu , green spotted pufferfish , zebrafish , japanese medaka  and three-spined stickleback , within the ensembl data.
the zebrafish is a representative of the most abundant and widespread primary freshwater fish family, cyprinidae  <cit>  with ample genomic resources including a nearly fully sequenced genome and over a million expressed sequence tags .
however, genomic data for the rest of the cyprinids are quite scarce , partly due to polyploidy that represents a characteristic feature of several members of the cyprinidae family  <cit> .
in the absence of genome projects from closely related species, the automatic annotation of genomes relies heavily on available cdna and protein sequences of other vertebrates for sequence comparisons.
for example, mammalian and teleost genome comparisons have been used successfully to identify conserved protein-coding genes and regulatory elements despite the  <dig> million years that elapsed since their last common ancestor  <cit> .
in contrast, a recent study by thomas and colleagues  <cit>  concluded that fish-mammal comparisons were unable to detect most non-coding regions that were conserved between amniotes.
theoretically, the annotation of the zebrafish genome could benefit from sequence data for a closely related species excluding the annotated genomes of japanese fugu and the green spotted pufferfish that share a common ancestor with zebrafish more than  <dig> million years ago  <cit> .
the unigene collection  <cit>  represents a database of species-specific mrna and ests that are grouped into clusters or genes based on stringent sequence identity.
currently two cyprinid species are present in the unigene collection , namely the zebrafish and fathead minnow .
zebrafish belongs to the subfamily rasborinae, whereas fathead minnow is a member of leuciscinae  <cit> .
nearly  <dig>  ests are present in dbest  <cit>  for a third cyprinid species, common carp   <cit> , however they were not sampled in the recent unigene collection .
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common carp is the most important fish species of freshwater aquaculture, probably with the earliest domestication records among fishes  <cit> .
it has been used in fish biology and aquaculture research quite extensively .
common carp is a close relative of the zebrafish, they both belong to the same family.
the ancestors of common carp and zebrafish have split about  <dig> million years ago   <cit> , whereas the corresponding divergence data for fathead minnow is not available.
the wealth of est data for these three cyprinid species and the recent speciation event provides a valuable resource to aid the ongoing zebrafish genome annotation project.
in order to facilitate the comparative genomic analysis of gonad development in cyprinid teleosts, primarily the zebrafish  <cit>  and common carp, we set out to complement the non-gonadal common carp transcriptome data by sequencing clones from testis-derived cdna libraries.
we then performed a cross-species analysis of cyprinids by comparing common carp ests sequences to those originating from zebrafish and fathead minnow, as well as to the partially sequenced zebrafish genome.
we mapped common carp ests to un-annotated regions of the zebrafish genome.
our results identified novel testis-expressed transcripts in cyprinids and new splice variants in the common carp transcriptome.
we were able to show that the two species share a significant level of similarity in the 5'utr regions.
collectively, these results indicate that such a comparative approach, based on the usage of closely related species, could add value to the current ongoing improvements to the zebrafish genome assembly and annotation by the genomic community.
RESULTS
testis-derived common carp cdnas add nearly  <dig>  unique sequences to the public est collection
at the start of our work genbank  <cit>  and carpbase  <cit>  together contained  <dig>  common carp ests, all of which originated from non-gonadal cdna libraries.
we enriched the existing transcriptome dataset for common carp, by generating an additional  <dig>  ests by random sequencing of clones from five different cdna libraries derived from differentiating common carp testis .
we also added an additional  <dig> common carp mrnas extracted from genbank in order to assist the assembly of ests.
following cleaning and quality control, over  <dig>  ests { <dig>  from genbank plus carpbase and  <dig>  from our own data } were retained and clustered .
the clustered dataset of  <dig>  unique sequences  contained  <dig>   "testis-only" sequences, including clusters with exclusively testis-derived ests and singletons isolated from one of the testis cdna libraries .
in order to initiate functional annotation of the partial transcriptome of common carp, we identified open reading frames  in our clustered est set using estscan  <cit> .
an orf prediction was obtained for 81% of the clusters and  <dig> % of the singletons, yielding a total of  <dig>  sequences .
the orf-containing common carp transcripts were classified into functional categories using protein domain databases .
mapping of common carp ests to the zebrafish genome
in the zebrafish ensembl annotation  genes were annotated using mrna and proteins from the target species as well as a range of other vertebrates, the closest to zebrafish being japanese fugu and green spotted pufferfish.
we mapped our common carp est data to the zebrafish genome assembly  according to a multi-step protocol .
a total of  <dig>  common carp clusters  and  <dig>  singletons  showed sequence similarity to the zebrafish genome with a blast e-value cutoff of 1e- <dig>  after stringent filtering – selecting a unique zebrafish genomic location for each mapped common carp cluster  and sequence identity of 80% over 70% of the est length – we assigned  <dig> clusters  and  <dig>  singletons  to the zebrafish genome assembly .
the common carp transcript map coordinates are available from ensembl version  <dig> as a das track  <cit> .
the  <dig> percentile of all intron lengths within the zebrafish ensembl database is  <dig>  nucleotides.
there were  <dig> cases, where two common carp clusters/singletons mapped to the zebrafish genome within  <dig>  nucleotides.
these represent cases where the clusters and/or singletons potentially correspond to the same gene but were partitioned into separate clusters because of the absence of sequence data in the est database.
interestingly, there were  <dig> cases, where at least two clusters and/or singletons overlapped the same zebrafish locus.
these represent potential gene family expansions in the common carp relative to zebrafish, but would require experimental validation in the future.
these cases provide support for the incorporation of est sequences from closely related "sequence-poor" species into the analysis pipeline of  completely sequenced genomes.
common carp ests map to regions lacking expressed sequence information in the zebrafish genome
nearly 40% of ests obtained from genbank and those sequenced in our lab are bi-directional due to the est sequencing protocol used.
as a result, the strandedness of the genome-aligned common carp ests were obtained using the splice-site orientation as defined in the est2genome algorithm  <cit> .
to identify un-annotated regions in the zebrafish genome, we required both plus and minus strands of the zebrafish genome be free of any sequence similarity features to non-common carp cdna and proteins.
of the  <dig>  common carp transcripts mapped to the zebrafish genome assembly ,  <dig>  overlapped zebrafish cdnas supported by genes and/or ests.
the remaining  <dig> "mapped common carp transcripts" showed sequence identity to regions overlapping zebrafish introns , ab-initio predictions , non-zebrafish exons , intergenic regions  and non-zebrafish introns  .
five of the  <dig> common carp transcripts that map to intergenic regions are located less than  <dig> kb from the 5' end of the nearest neighbouring gene.
considering their close proximity to an annotated gene, these common carp transcripts represent potential untranslated regions .
in fact, the five neighbouring genes are annotated as developmental genes .
developmental genes are highly conserved among species and very often the sequence conservation extends to their regulatory regions  <cit> .
furthermore, each of the common carp transcripts mapped to the zebrafish genome have sequence identity in excess of 80%, suggesting that the use of a lower threshold for common carp est mapping might retrieve many more utr sequences that could be subjected to similar utr analyses as described in the materials and methods.
the remaining eight common carp transcripts that map to intergenic regions are located between  <dig> and  <dig> kb away from the nearest zebrafish locus, suggesting the presence of novel gene loci that require experimental verification in the future.
forty-two of the  <dig> mapped common carp transcripts have not been identified in the zebrafish and fathead minnow est collections so far, therefore they represented novel cyprinid sequences.
another  <dig> of the  <dig> mapped common carp transcripts showed significant sequence similarity to the zebrafish and fathead minnow unigene collection .
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the remaining  <dig> common carp transcripts shared very weak sequence similarity  with either zebrafish or fathead minnow, thus might point to genes that diverged from their orthologs.
alternatively, these transcripts could represent sequences orthologous to zebrafish utrs that are yet to be assigned to the annotated zebrafish genome.
the above cases illustrate the potential advantages of utilizing partial transcriptomes from related species in order to provide information on the functional properties of  un-annotated parts of genomes to be assembled as well as  regions annotated with distantly related species.
alternative exon usage identified by comparing cyprinid transcripts
est-based analysis of alternative splicing has been performed earlier in mammals; the results suggest that 40–60% of the genes produce alternatively spliced transcripts  <cit> .
only a few studies have been performed on fish sequences  resulting in a limited amount of data on splicing from teleosts.
interestingly, out of  <dig>  common carp transcripts that map to coding regions, there were  <dig> cases where the exon structure showed evidence for a missing exon compared to the overlapping zebrafish ensembl gene .
similar comparisons yielded  <dig> cases where an exon that was present in the overlapping common carp ests was missing from the zebrafish transcript .
there are four possibilities to explain such differences: i) the exon in question is missing from one of the two genomes; ii) exclusive usage of different splice products in the two related species; iii) different preferences of alternative splice products; and iv) virtual difference due to partial transcriptomes.
at the time of submission genbank contained over  <dig>  million ests for zebrafish, fathead minnow and common carp.
we propose that the broad mrna diversity contained in teleost est resources could be leveraged to understand the extent of alternative splicing within this diverse group of teleosts using analyses similar to those reported for human ests  <cit> .
comparison of the partial cyprinid gonad transcriptomes identifies  <dig> novel testis-derived transcripts
the unigene collection  contains datasets for two cyprinid species namely the zebrafish and fathead minnow.
the common carp est data reported in this study, sampled by nearly  <dig>  unique transcripts, represent an additional cyprinid species that will be included in subsequent unigene releases.
the new est data for common carp has also provided an opportunity to examine the value of tissue-specific sequencing on the existing gene collections.
the common carp est data were compared to the zebrafish unigene collection  and subsequently to the fathead minnow data set using a blast e-value <1e- <dig> and sequence identity over 40% of the sequence length.
a total of  <dig> testis-derived common carp singletons and  <dig> clusters containing exclusively testis-derived common carp ests  did not overlap any of the zebrafish and fathead minnow unigene transcripts.
this dataset added  <dig> potentially novel sequences to the combined testis transcriptome of cyprinid teleosts .
a total of  <dig> out of  <dig> testis-only transcripts contained an orf.
among these  <dig> transcripts, three testis-derived clusters contained an interleukin-8-like domain .
the absence of significant sequence identity to zebrafish and fathead minnow at the nucleotide level is partly due to cytokines representing rapidly diverging genes involved in regulation of the immune system.
another domain, tissue inhibitor of metalloproteinase , which was identified in a common carp testis-derived transcript was present in two zebrafish unigene clusters , but not sampled by any gonad-derived zebrafish sequences.
the remaining  <dig> common carp transcripts do not show the presence of any characterized domains.
these unique testis-derived transcripts could provide starting material for the isolation of their zebrafish orthologs, if any, and their potential application as markers for functional studies on gonad differentiation.
the potential homologs of  <dig> common carp clusters with at least one testis-derived est were identified in the zebrafish unigene data collection .
when compared to the fathead minnow est collection, six of these  <dig> common carp clusters showed sequence identity to adult testis-derived ests only .
the common carp data correspond to differentiating testis , whereas the testis-derived zebrafish and fathead minnow clones presently found in the public databases are all from an adult organ.
therefore our results have complemented the previously available knowledge about the expression of these genes with experimental data on their activity during testis differentiation, providing indications on potentially conserved aspects of cyprinid gonad development.
moreover, the fact that common carp transcripts help the identification or confirmation of these coding regions in zebrafish exemplifies the usefulness of sequences from closely related species for the annotation of model genomes.
comparing the overall architecture of utr regions for a set of orthologous genes from common carp and zebrafish
there is an average 82% sequence identity between the coding region of homologous gene pairs in zebrafish and common carp, whereas the same value for their 5' and 3' utrs is only 61% and 58%, respectively .
we set out to explore the extent to which common carp and zebrafish retained similarity in the 5'utr regions of their orthologous genes as this can reveal aspects of regulatory roles of these regions in both species.
this task was difficult for two reasons: i) the fact that only limited sequence information is available from common carp dramatically decreased our ability to identify large number of orthologs between these species; and ii) the usual approaches to evaluate similarity based on local alignments are not really suitable for the similarity assessment of regulatory regions as demonstrated by blanco and colleagues  <cit> .
by analyzing  <dig> pairs of orthologous sequences and an additional six paralogs, which contained at least  <dig> bp at their 5' utr  we identified motif families shared in the 5' utr of common carp and zebrafish mrnas.
analyzing each of the orthologous pairs individually , we determined the order of a maximum of  <dig> shared motifs between common carp and zebrafish.
the distribution of coverage for all orthologous pairs relative to the number of motifs in these arrangements is represented in additional file 9: figure s <dig>  about two-third of the orthologous 5'utr pairs tested shared 4– <dig> motifs in the conserved positional arrangement, whereas most of the rest shared 7– <dig>  the distribution of identified motifs together with the conserved arrangement in the zebrafish caudal type homeobox transcription factor  <dig>   and its common carp ortholog, cdx <dig>  are shown in figure  <dig> as an example.
a detailed utr analysis is not within the scope of the present manuscript, therefore we propose a large-scale analysis to find out whether 5'utr regions from different orthologous pairs share motifs from the same family.
the presence of such shared motif families would suggest the existence of regulatory components common to both species suitable for further evaluation.
CONCLUSIONS
in this study, we have demonstrated the value of using ests for comparative analysis of transcriptomes from species with vastly different amount of sequence information.
for example, common carp ests were successfully mapped to un-annotated regions of the zebrafish genome demonstrating the value of using closely related species for sequence comparison.
the existing cyprinid ests represent a useful resource for comparative genomics to understand the evolution of this family.
sequenced genomes are being integrated with functional information  to improve the efficiency of data mining.
however, integrating fragmented genomic data for non-sequenced genomes remain a challenge for scientists who want to leverage inter-species comparisons.
we suggest that there is a need to co-ordinate the isolated "in-house" integration attempts across laboratories in order to maximize and improve the quality of the information content that is currently under-utilized.
materials and methods
isolation of differentiating testis from common carp individuals
androgenetic common carp "supermales"  have been crossed with wild type females  to give rise to an all-male offspring population.
the gonad has been isolated from a minimum of  <dig> individuals at 59/ <dig> ,  <dig>   <dig> and  <dig> dpf, respectively.
one of the two gonads from each individual has been processed for histological analyses , while the other one has been stored in rnalater  for the use of rna isolation.
construction of cdna libraries from the differentiating carp testis
total rna was isolated from the testis of 59/ <dig>   <dig> and  <dig> days-old individuals, respectively.
full-length cdna was synthesized using creator smart library construction kit  according to the manufacturer's instruction.
after sfii restriction enzyme digestion the adaptors and short cdnas were removed by chromaspin  <dig> column .
the size fractionated cdna pool was then cloned into a pbluescript based vector  and transformed into e. coli xl10-gold cells.
clones were picked into thirty, twenty and ten 96-well plates from the libraries generated from testes collected at  <dig>   <dig> and  <dig> dpf, respectively, and their insert was sequenced using m <dig> forward primer as described in  <cit> .
total rna was isolated from the testis of  <dig> and  <dig> day old individuals, respectively.
two sets of subtractive hybridizations were performed:  <dig> dpf male gonad  from  <dig> dpf testis , and  <dig> dpf testis  from  <dig> dpf male gonad .
the pcr-select™ cdna subtraction kit  was used to enrich for developmental stage-specific fragments from the smart cdna template according to the recommendations of the manufacturer.
the selectively amplified cdna fragments  were ligated into pgem-t  cloning vector.
in total  <dig>  clones have been picked from the two libraries and their insert was sequenced using m <dig> forward primer.
sequence acquisition and est clustering
a total of  <dig>  common carp ests, sequenced from a range of tissues other than gonad, were downloaded from genbank ;  <dig>  of those sequences are also available from carpbase  <cit>  .
they were combined with  <dig> mrnas from genbank and with  <dig>  gonad-derived common carp ests generated in our labs within the framework of this project .
low quality regions were trimmed at the 3' end of ests prior to masking against libraries of repeats, mitochondrial and ribosomal sequences using repeatmasker  <cit> .
sequences that comprised at least 70% unmasked nucleotides  were retained for further analysis.
the combined est data set was clustered using the stackpack clustering tools  <cit>  on hpcompaq alpha es <dig> architecture.
functional characterization of common carp transcripts
common carp transcripts  were partitioned into orf- and nonorf-containing sequences using estscan  <cit> .
the orf-containing transcripts were annotated for protein domains and functional sites by matching them against the pfam, prosite and prints databases  <cit>  using hmmpfam, a program within the hmmer package that uses hidden markov models to do sensitive searching of a protein database  <cit> .
the protein domains were mapped to gene ontology categories using go tables  <cit> .
mapping of common carp transcripts to the zebrafish genome
in order to further categorize the common carp transcripts they were searched against the zebrafish genome assembly .
the possibility of multigene families within est clusters allow for common carp clusters to map to multiple zebrafish genomic locations.
a single high quality zebrafish genomic location was identified for each mapped common cluster in order to screen for novel genes and potential alternative splice variants.
transcripts that map to the zebrafish genome with blast e-value of at least 1e- <dig> where passed through a set of stringent filters as defined in additional file 8: figure s <dig> in order to identify a single zebrafish genomic location for each of the mapped common carp clusters.
the best zebrafish chromosome location for each est in a common carp cluster was considered: the zebrafish chromosome locus shared by all ests within a cluster was chosen as the mapped genomic locus for the corresponding common carp cluster.
est clusters that represented best hits to different chromosome locations for constituent ests were screened for a common zebrafish chromosome hit by considering the top five best hits for each est in a cluster.
a common zebrafish chromosome hit identified in the top five best hits was assigned as the unique map location for the common carp cluster.
mapped common carp clusters were not considered if there was not at least one zebrafish chromosome hit shared among all the ests in a cluster.
all common carp transcripts that passed these filtering criteria were aligned to the specific segment of the overlapping zebrafish genome using est2genome  <cit> .
exon-intron boundaries were extracted from the est2genome results and served as a das track on the ensembl browser  <cit> .
genome-aligned common carp ests were classified according to one of five criteria that were satisfied on the plus or minus strand.
common carp ests overlapped  zebrafish coding regions: exons corresponding to an ensembl gene or zebrafish est; or  zebrafish introns: the entire genome-aligned common carp est was contained within the intronic region of a zebrafish gene; or  non-zebrafish exons: common carp ests mapped to regions of the zebrafish genome that overlapped non-zebrafish cdna;  non-zebrafish introns: the non-coding portions of cdna or proteins aligned to the zebrafish genome;  intergenic: regions of the zebrafish genome void of any annotations;  ab initio predictions: common carp ests mapped to regions of the zebrafish genome with an in silico gene prediction only.
comparing testis derived common carp sequences with zebrafish and fathead minnow est data
testis-only transcripts for common carp were defined as clusters or singletons represented by ests obtained exclusively from common carp testis cdna libraries.
gonad derived genes for zebrafish were sampled from the unigene zebrafish collection  where unigene clusters contained ests that were sampled from zebrafish testis or ovary cdna libraries.
common carp testis-derived transcripts were searched against the zebrafish gonad-derived unigene dataset using blastn with  an e-value < 1e-04; and  sequence overlap where 40% of the query sequence overlapped the matching database sequence.
the common carp transcripts without identity to zebrafish gonad derived sequences were searched against the remainder of the zebrafish unigene build  <dig> using an e-value < 1e- <dig> but without the requirement for 50% of the query sequence overlapping the database sequence.
this relaxed criteria resulted in the identification of fewer common carp ests without homologous zebrafish ests in unigene .
however, these common carp ests provide a minimum dataset of testis-derived sequences not sampled by the zebrafish est collection.
the resulting "unique" common carp transcripts were searched against the fathead minnow unigene  est data using the same criteria as used for zebrafish.
acquisition of sequence data for common carp and zebrafish orthologs and paralogs
a total  <dig> common carp mrna sequences were downloaded from genbank.
about  <dig> mrnas represented partial mrna sequences and were removed.
the sequences corresponding to the remaining  <dig> mrna records in genbank were searched against ncbi's non-redundant database using protein-protein blast .
the blast results were filtered for a significant sequence match to zebrafish  and matching zebrafish mrnas that were partial sequences was filtered.
the remaining  <dig> common carp and zebrafish homologous pairs were screened manually for orthologous relationships using cross-linked information including publications, curated annotations and filtering for redundant genbank records.
eventually  <dig> pairs of orthologous genes were selected for sequence comparison between coding and non-coding regions  and a subset containing  <dig> pairs, plus six additional paralogs  was used for motif searches .
first we analyzed the sequence similarity among the coding regions and the utrs for the orthologous gene set.
at the nucleotide level, sequence conservation was observed more often in the cds regions, followed by the 5' utr and 3' utr regions, respectively .
specifically, 75% of the orthologous pairs are captured when we set a sequence identity threshold of 80% at the cds and protein levels.
in comparison, only 25% of the 5' utr sequences are captured under the same conditions .
the threshold of 80% sequence identity was implemented for subsequent blast searches of common carp ests against the zebrafish genome assembly.
ab-initio motif identification, motif arrangement and 5'utr sequence similarity
for the identification of motifs in 5'utr regions, we compared the efficiency of dragon motif builder system  <cit>  with a local alignment method, clustalw  <cit> .
with the dragon motif builder we searched for any motif with the length between  <dig> bp and  <dig> bp, used the matrix score threshold of  <dig> , and searched for up to  <dig> motifs in the two sequences of a given orthologous pair .
for clustalw the common motifs were manually identified and restricted to the same criteria as those used by dragon motif builder .
a significant difference was observed showing that clustalw was not able to identify sufficient similarity between the ortholog sequences in the 5'utr regions  as the segments that contain similar arrangement of common motifs between the two species were not residing at similar genomic locations.
once the motifs were identified, we analyzed the motif arrangements.
we selected the group of motifs that contained the largest number of common motifs, but retained the same positional arrangement in the two species .
thus each of the ortholog pairs was screened for such a representative motif arrangement.
we used the number of motifs in the representative arrangements as a possible measure of similarity between the 5'utr regions.
in most cases, the regions where this arrangement had been spotted, was found at significantly different distances from the starting codon.
authors' contributions
ac had designed the bioinformatics pathway, performed most computational analyses, generated most tables and figures and took part in the writing of the manuscript.
rb has contributed to the experimental design, constructed the full-length libraries and took part in the writing of the manuscript.
hs has participated in the bioinformatics analysis of the data and in the maintenance of the cyprinid est database of tll.
hk has supervised the generation of yy androgenic common carp line, generated the monosex populations, isolated the testis samples from them and contributed to the experimental design.
lo initiated the project on comparative analysis of cyprinid ests, contributed to the experimental design and took part in the writing of the manuscript.
supplementary material
additional file 1
the description of common carp cdna libraries analyzed in this study.
details include tissue, developmental stage and source of cdna libraries.
click here for file
additional file 2
distribution of functional categories identified in the partial transcriptome of common carp.
click here for file
additional file 8
protocol to map common carp transcripts to the zebrafish genome assembly .
the flow chart depicts the pipeline implemented for mapping common carp transcripts to the zebrafish genome.
filter criteria are denoted in the decision tree.
total number of clusters and singletons are indicated in square brackets.
click here for file
additional file 3
classification of  <dig> common carp ests that map to intergenic, intronic, ab initio predictions and non-zebrafish supported annotations.
click here for file
additional file 4
list of hyperlinks to potential common carp splice variants.
overlapping common carp and zebrafish transcripts are presented on a gbrowse viewer to highlight the missing exons in one of the two species.
click here for file
additional file 5
list of  <dig> testis-only transcripts that do not overlap any of the zebrafish and fathead minnow ests.
click here for file
additional file 6
list of  <dig> testis-derived clusters that show sequence identity to  <dig> zebrafish and  <dig> fathead minnow unigene clusters.
testis-expression information was added to the adult-stage zebrafish expression data.
click here for file
additional file 7
homologous gene pairs identified through manual curation of common carp and zebrafish genes.
the table includes dna and protein accession numbers and corresponding gene descriptions.
genes in rows highlighted with yellow contain 5' utr sequence  and were used in utr analysis.
click here for file
additional file 9
distribution of orthologous clusters with given number of common motifs using dragon motif builder  and clustalw .
click here for file
additional file 10
percent sequence identity between common carp and zebrafish orthologous proteins, cds, 5' utr and 3' utr regions.
click here for file
acknowledgements
the authors thank aarti giri, james a. hill, mei yin ho, balamuragan kumarasamy, yang li and tina eyre for their technical help.
they also acknowledge the help of oliver bezuidt, cameron macpherson and vladimir bajic for the comparative analysis of homologous 5'utrs from common carp and zebrafish as well as vladimir bajic's helpful comments and corrections on an earlier version of the manuscript.
this project was supported from internal funding by temasek life sciences laboratory.
this article has been published as part of bmc bioinformatics volume  <dig>  supplement  <dig>  2006: apbionet – fifth international conference on bioinformatics .
the full contents of the supplement are available online at .
figures and tables

 eukaryotic gene prediction has been a challenging problem, explored over the last two decades and driven by the availability of large volumes of genomic data. the development of gene prediction methods have traditionally included ab-initio approaches such as genscan that do not use any experimental evidence, alignment-based methods such as genewise that attempts to align an homologous protein sequence to a genomic sequence and more recently, hybrid approaches that incorporate cdna-defined splice junctions into ab-initio and protein alignment information. such hybrid approaches for automatic annotation of genome sequences have been implemented within the ensembl annotation project. ensembl represents a bioinformatics project aimed at annotating sequenced genomes and integrating biological data that can be mapped or assigned to features described in the genomic data. at present, twenty fully or near-fully sequenced vertebrate genomes have been included in ensembl. teleosts, comprising about half the number of all extant vertebrate species, are represented by only five species, namely japanese fugu, green spotted pufferfish, zebrafish, japanese medaka and three-spined stickleback, within the ensembl data. the zebrafish is a representative of the most abundant and widespread primary freshwater fish family, cyprinidae with ample genomic resources including a nearly fully sequenced genome and over a million expressed sequence tags. however, genomic data for the rest of the cyprinids are quite scarce, partly due to polyploidy that represents a characteristic feature of several members of the cyprinidae family. in the absence of genome projects from closely related species, the automatic annotation of genomes relies heavily on available cdna and protein sequences of other vertebrates for sequence comparisons. for example, mammalian and teleost genome comparisons have been used successfully to identify conserved protein-coding genes and regulatory elements despite the million years that elapsed since their last common ancestor. in contrast, a recent study by thomas and colleagues concluded that fish-mammal comparisons were unable to detect most non-coding regions that were conserved between amniotes. theoretically, the annotation of the zebrafish genome could benefit from sequence data for a closely related species excluding the annotated genomes of japanese fugu and the green spotted pufferfish that share a common ancestor with zebrafish more than million years ago. the unigene collection represents a database of species-specific mrna and ests that are grouped into clusters or genes based on stringent sequence identity. currently two cyprinid species are present in the unigene collection, namely the zebrafish and fathead minnow. zebrafish belongs to the subfamily rasborinae, whereas fathead minnow is a member of leuciscinae. nearly ests are present in dbest for a third cyprinid species, common carp, however they were not sampled in the recent unigene collection.. common carp is the most important fish species of freshwater aquaculture, probably with the earliest domestication records among fishes. it has been used in fish biology and aquaculture research quite extensively. common carp is a close relative of the zebrafish, they both belong to the same family. the ancestors of common carp and zebrafish have split about million years ago, whereas the corresponding divergence data for fathead minnow is not available. the wealth of est data for these three cyprinid species and the recent speciation event provides a valuable resource to aid the ongoing zebrafish genome annotation project. in order to facilitate the comparative genomic analysis of gonad development in cyprinid teleosts, primarily the zebrafish and common carp, we set out to complement the non-gonadal common carp transcriptome data by sequencing clones from testis-derived cdna libraries. we then performed a cross-species analysis of cyprinids by comparing common carp ests sequences to those originating from zebrafish and fathead minnow, as well as to the partially sequenced zebrafish genome. we mapped common carp ests to un-annotated regions of the zebrafish genome. our results identified novel testis-expressed transcripts in cyprinids and new splice variants in the common carp transcriptome. we were able to show that the two species share a significant level of similarity in the 5'utr regions. collectively, these results indicate that such a comparative approach, based on the usage of closely related species, could add value to the current ongoing improvements to the zebrafish genome assembly and annotation by the genomic community.  testis-derived common carp cdnas add nearly unique sequences to the public est collection at the start of our work genbank and carpbase together contained common carp ests, all of which originated from non-gonadal cdna libraries. we enriched the existing transcriptome dataset for common carp, by generating an additional ests by random sequencing of clones from five different cdna libraries derived from differentiating common carp testis. we also added an additional common carp mrnas extracted from genbank in order to assist the assembly of ests. following cleaning and quality control, over ests { from genbank plus carpbase and from our own data } were retained and clustered. the clustered dataset of unique sequences contained "testis-only" sequences, including clusters with exclusively testis-derived ests and singletons isolated from one of the testis cdna libraries. in order to initiate functional annotation of the partial transcriptome of common carp, we identified open reading frames in our clustered est set using estscan. an orf prediction was obtained for 81% of the clusters and % of the singletons, yielding a total of sequences. the orf-containing common carp transcripts were classified into functional categories using protein domain databases. mapping of common carp ests to the zebrafish genome in the zebrafish ensembl annotation genes were annotated using mrna and proteins from the target species as well as a range of other vertebrates, the closest to zebrafish being japanese fugu and green spotted pufferfish. we mapped our common carp est data to the zebrafish genome assembly according to a multi-step protocol. a total of common carp clusters and singletons showed sequence similarity to the zebrafish genome with a blast e-value cutoff of 1e- after stringent filtering – selecting a unique zebrafish genomic location for each mapped common carp cluster and sequence identity of 80% over 70% of the est length – we assigned clusters and singletons to the zebrafish genome assembly. the common carp transcript map coordinates are available from ensembl version as a das track. the percentile of all intron lengths within the zebrafish ensembl database is nucleotides. there were cases, where two common carp clusters/singletons mapped to the zebrafish genome within nucleotides. these represent cases where the clusters and/or singletons potentially correspond to the same gene but were partitioned into separate clusters because of the absence of sequence data in the est database. interestingly, there were cases, where at least two clusters and/or singletons overlapped the same zebrafish locus. these represent potential gene family expansions in the common carp relative to zebrafish, but would require experimental validation in the future. these cases provide support for the incorporation of est sequences from closely related "sequence-poor" species into the analysis pipeline of completely sequenced genomes. common carp ests map to regions lacking expressed sequence information in the zebrafish genome nearly 40% of ests obtained from genbank and those sequenced in our lab are bi-directional due to the est sequencing protocol used. as a result, the strandedness of the genome-aligned common carp ests were obtained using the splice-site orientation as defined in the est2genome algorithm. to identify un-annotated regions in the zebrafish genome, we required both plus and minus strands of the zebrafish genome be free of any sequence similarity features to non-common carp cdna and proteins. of the common carp transcripts mapped to the zebrafish genome assembly, overlapped zebrafish cdnas supported by genes and/or ests. the remaining "mapped common carp transcripts" showed sequence identity to regions overlapping zebrafish introns, ab-initio predictions, non-zebrafish exons, intergenic regions and non-zebrafish introns. five of the common carp transcripts that map to intergenic regions are located less than kb from the 5' end of the nearest neighbouring gene. considering their close proximity to an annotated gene, these common carp transcripts represent potential untranslated regions. in fact, the five neighbouring genes are annotated as developmental genes. developmental genes are highly conserved among species and very often the sequence conservation extends to their regulatory regions. furthermore, each of the common carp transcripts mapped to the zebrafish genome have sequence identity in excess of 80%, suggesting that the use of a lower threshold for common carp est mapping might retrieve many more utr sequences that could be subjected to similar utr analyses as described in the materials and methods. the remaining eight common carp transcripts that map to intergenic regions are located between and kb away from the nearest zebrafish locus, suggesting the presence of novel gene loci that require experimental verification in the future. forty-two of the mapped common carp transcripts have not been identified in the zebrafish and fathead minnow est collections so far, therefore they represented novel cyprinid sequences. another of the mapped common carp transcripts showed significant sequence similarity to the zebrafish and fathead minnow unigene collection.. the remaining common carp transcripts shared very weak sequence similarity with either zebrafish or fathead minnow, thus might point to genes that diverged from their orthologs. alternatively, these transcripts could represent sequences orthologous to zebrafish utrs that are yet to be assigned to the annotated zebrafish genome. the above cases illustrate the potential advantages of utilizing partial transcriptomes from related species in order to provide information on the functional properties of un-annotated parts of genomes to be assembled as well as regions annotated with distantly related species. alternative exon usage identified by comparing cyprinid transcripts est-based analysis of alternative splicing has been performed earlier in mammals; the results suggest that 40–60% of the genes produce alternatively spliced transcripts. only a few studies have been performed on fish sequences resulting in a limited amount of data on splicing from teleosts. interestingly, out of common carp transcripts that map to coding regions, there were cases where the exon structure showed evidence for a missing exon compared to the overlapping zebrafish ensembl gene. similar comparisons yielded cases where an exon that was present in the overlapping common carp ests was missing from the zebrafish transcript. there are four possibilities to explain such differences: i) the exon in question is missing from one of the two genomes; ii) exclusive usage of different splice products in the two related species; iii) different preferences of alternative splice products; and iv) virtual difference due to partial transcriptomes. at the time of submission genbank contained over million ests for zebrafish, fathead minnow and common carp. we propose that the broad mrna diversity contained in teleost est resources could be leveraged to understand the extent of alternative splicing within this diverse group of teleosts using analyses similar to those reported for human ests. comparison of the partial cyprinid gonad transcriptomes identifies novel testis-derived transcripts the unigene collection contains datasets for two cyprinid species namely the zebrafish and fathead minnow. the common carp est data reported in this study, sampled by nearly unique transcripts, represent an additional cyprinid species that will be included in subsequent unigene releases. the new est data for common carp has also provided an opportunity to examine the value of tissue-specific sequencing on the existing gene collections. the common carp est data were compared to the zebrafish unigene collection and subsequently to the fathead minnow data set using a blast e-value <1e- and sequence identity over 40% of the sequence length. a total of testis-derived common carp singletons and clusters containing exclusively testis-derived common carp ests did not overlap any of the zebrafish and fathead minnow unigene transcripts. this dataset added potentially novel sequences to the combined testis transcriptome of cyprinid teleosts. a total of out of testis-only transcripts contained an orf. among these transcripts, three testis-derived clusters contained an interleukin-8-like domain. the absence of significant sequence identity to zebrafish and fathead minnow at the nucleotide level is partly due to cytokines representing rapidly diverging genes involved in regulation of the immune system. another domain, tissue inhibitor of metalloproteinase, which was identified in a common carp testis-derived transcript was present in two zebrafish unigene clusters, but not sampled by any gonad-derived zebrafish sequences. the remaining common carp transcripts do not show the presence of any characterized domains. these unique testis-derived transcripts could provide starting material for the isolation of their zebrafish orthologs, if any, and their potential application as markers for functional studies on gonad differentiation. the potential homologs of common carp clusters with at least one testis-derived est were identified in the zebrafish unigene data collection. when compared to the fathead minnow est collection, six of these common carp clusters showed sequence identity to adult testis-derived ests only. the common carp data correspond to differentiating testis, whereas the testis-derived zebrafish and fathead minnow clones presently found in the public databases are all from an adult organ. therefore our results have complemented the previously available knowledge about the expression of these genes with experimental data on their activity during testis differentiation, providing indications on potentially conserved aspects of cyprinid gonad development. moreover, the fact that common carp transcripts help the identification or confirmation of these coding regions in zebrafish exemplifies the usefulness of sequences from closely related species for the annotation of model genomes. comparing the overall architecture of utr regions for a set of orthologous genes from common carp and zebrafish there is an average 82% sequence identity between the coding region of homologous gene pairs in zebrafish and common carp, whereas the same value for their 5' and 3' utrs is only 61% and 58%, respectively. we set out to explore the extent to which common carp and zebrafish retained similarity in the 5'utr regions of their orthologous genes as this can reveal aspects of regulatory roles of these regions in both species. this task was difficult for two reasons: i) the fact that only limited sequence information is available from common carp dramatically decreased our ability to identify large number of orthologs between these species; and ii) the usual approaches to evaluate similarity based on local alignments are not really suitable for the similarity assessment of regulatory regions as demonstrated by blanco and colleagues. by analyzing pairs of orthologous sequences and an additional six paralogs, which contained at least bp at their 5' utr we identified motif families shared in the 5' utr of common carp and zebrafish mrnas. analyzing each of the orthologous pairs individually, we determined the order of a maximum of shared motifs between common carp and zebrafish. the distribution of coverage for all orthologous pairs relative to the number of motifs in these arrangements is represented in additional file 9: figure s about two-third of the orthologous 5'utr pairs tested shared 4– motifs in the conserved positional arrangement, whereas most of the rest shared 7– the distribution of identified motifs together with the conserved arrangement in the zebrafish caudal type homeobox transcription factor and its common carp ortholog, cdx are shown in figure as an example. a detailed utr analysis is not within the scope of the present manuscript, therefore we propose a large-scale analysis to find out whether 5'utr regions from different orthologous pairs share motifs from the same family. the presence of such shared motif families would suggest the existence of regulatory components common to both species suitable for further evaluation.  in this study, we have demonstrated the value of using ests for comparative analysis of transcriptomes from species with vastly different amount of sequence information. for example, common carp ests were successfully mapped to un-annotated regions of the zebrafish genome demonstrating the value of using closely related species for sequence comparison. the existing cyprinid ests represent a useful resource for comparative genomics to understand the evolution of this family. sequenced genomes are being integrated with functional information to improve the efficiency of data mining. however, integrating fragmented genomic data for non-sequenced genomes remain a challenge for scientists who want to leverage inter-species comparisons. we suggest that there is a need to co-ordinate the isolated "in-house" integration attempts across laboratories in order to maximize and improve the quality of the information content that is currently under-utilized. materials and methods isolation of differentiating testis from common carp individuals androgenetic common carp "supermales" have been crossed with wild type females to give rise to an all-male offspring population. the gonad has been isolated from a minimum of individuals at 59/, and dpf, respectively. one of the two gonads from each individual has been processed for histological analyses, while the other one has been stored in rnalater for the use of rna isolation. construction of cdna libraries from the differentiating carp testis total rna was isolated from the testis of 59/ and days-old individuals, respectively. full-length cdna was synthesized using creator smart library construction kit according to the manufacturer's instruction. after sfii restriction enzyme digestion the adaptors and short cdnas were removed by chromaspin column. the size fractionated cdna pool was then cloned into a pbluescript based vector and transformed into e. coli xl10-gold cells. clones were picked into thirty, twenty and ten 96-well plates from the libraries generated from testes collected at and dpf, respectively, and their insert was sequenced using m forward primer as described in. total rna was isolated from the testis of and day old individuals, respectively. two sets of subtractive hybridizations were performed: dpf male gonad from dpf testis, and dpf testis from dpf male gonad. the pcr-select™ cdna subtraction kit was used to enrich for developmental stage-specific fragments from the smart cdna template according to the recommendations of the manufacturer. the selectively amplified cdna fragments were ligated into pgem-t cloning vector. in total clones have been picked from the two libraries and their insert was sequenced using m forward primer. sequence acquisition and est clustering a total of common carp ests, sequenced from a range of tissues other than gonad, were downloaded from genbank; of those sequences are also available from carpbase. they were combined with mrnas from genbank and with gonad-derived common carp ests generated in our labs within the framework of this project. low quality regions were trimmed at the 3' end of ests prior to masking against libraries of repeats, mitochondrial and ribosomal sequences using repeatmasker. sequences that comprised at least 70% unmasked nucleotides were retained for further analysis. the combined est data set was clustered using the stackpack clustering tools on hpcompaq alpha es architecture. functional characterization of common carp transcripts common carp transcripts were partitioned into orf- and nonorf-containing sequences using estscan. the orf-containing transcripts were annotated for protein domains and functional sites by matching them against the pfam, prosite and prints databases using hmmpfam, a program within the hmmer package that uses hidden markov models to do sensitive searching of a protein database. the protein domains were mapped to gene ontology categories using go tables. mapping of common carp transcripts to the zebrafish genome in order to further categorize the common carp transcripts they were searched against the zebrafish genome assembly. the possibility of multigene families within est clusters allow for common carp clusters to map to multiple zebrafish genomic locations. a single high quality zebrafish genomic location was identified for each mapped common cluster in order to screen for novel genes and potential alternative splice variants. transcripts that map to the zebrafish genome with blast e-value of at least 1e- where passed through a set of stringent filters as defined in additional file 8: figure s in order to identify a single zebrafish genomic location for each of the mapped common carp clusters. the best zebrafish chromosome location for each est in a common carp cluster was considered: the zebrafish chromosome locus shared by all ests within a cluster was chosen as the mapped genomic locus for the corresponding common carp cluster. est clusters that represented best hits to different chromosome locations for constituent ests were screened for a common zebrafish chromosome hit by considering the top five best hits for each est in a cluster. a common zebrafish chromosome hit identified in the top five best hits was assigned as the unique map location for the common carp cluster. mapped common carp clusters were not considered if there was not at least one zebrafish chromosome hit shared among all the ests in a cluster. all common carp transcripts that passed these filtering criteria were aligned to the specific segment of the overlapping zebrafish genome using est2genome. exon-intron boundaries were extracted from the est2genome results and served as a das track on the ensembl browser. genome-aligned common carp ests were classified according to one of five criteria that were satisfied on the plus or minus strand. common carp ests overlapped zebrafish coding regions: exons corresponding to an ensembl gene or zebrafish est; or zebrafish introns: the entire genome-aligned common carp est was contained within the intronic region of a zebrafish gene; or non-zebrafish exons: common carp ests mapped to regions of the zebrafish genome that overlapped non-zebrafish cdna; non-zebrafish introns: the non-coding portions of cdna or proteins aligned to the zebrafish genome; intergenic: regions of the zebrafish genome void of any annotations; ab initio predictions: common carp ests mapped to regions of the zebrafish genome with an in silico gene prediction only. comparing testis derived common carp sequences with zebrafish and fathead minnow est data testis-only transcripts for common carp were defined as clusters or singletons represented by ests obtained exclusively from common carp testis cdna libraries. gonad derived genes for zebrafish were sampled from the unigene zebrafish collection where unigene clusters contained ests that were sampled from zebrafish testis or ovary cdna libraries. common carp testis-derived transcripts were searched against the zebrafish gonad-derived unigene dataset using blastn with an e-value < 1e-04; and sequence overlap where 40% of the query sequence overlapped the matching database sequence. the common carp transcripts without identity to zebrafish gonad derived sequences were searched against the remainder of the zebrafish unigene build using an e-value < 1e- but without the requirement for 50% of the query sequence overlapping the database sequence. this relaxed criteria resulted in the identification of fewer common carp ests without homologous zebrafish ests in unigene. however, these common carp ests provide a minimum dataset of testis-derived sequences not sampled by the zebrafish est collection. the resulting "unique" common carp transcripts were searched against the fathead minnow unigene est data using the same criteria as used for zebrafish. acquisition of sequence data for common carp and zebrafish orthologs and paralogs a total common carp mrna sequences were downloaded from genbank. about mrnas represented partial mrna sequences and were removed. the sequences corresponding to the remaining mrna records in genbank were searched against ncbi's non-redundant database using protein-protein blast. the blast results were filtered for a significant sequence match to zebrafish and matching zebrafish mrnas that were partial sequences was filtered. the remaining common carp and zebrafish homologous pairs were screened manually for orthologous relationships using cross-linked information including publications, curated annotations and filtering for redundant genbank records. eventually pairs of orthologous genes were selected for sequence comparison between coding and non-coding regions and a subset containing pairs, plus six additional paralogs was used for motif searches. first we analyzed the sequence similarity among the coding regions and the utrs for the orthologous gene set. at the nucleotide level, sequence conservation was observed more often in the cds regions, followed by the 5' utr and 3' utr regions, respectively. specifically, 75% of the orthologous pairs are captured when we set a sequence identity threshold of 80% at the cds and protein levels. in comparison, only 25% of the 5' utr sequences are captured under the same conditions. the threshold of 80% sequence identity was implemented for subsequent blast searches of common carp ests against the zebrafish genome assembly. ab-initio motif identification, motif arrangement and 5'utr sequence similarity for the identification of motifs in 5'utr regions, we compared the efficiency of dragon motif builder system with a local alignment method, clustalw. with the dragon motif builder we searched for any motif with the length between bp and bp, used the matrix score threshold of, and searched for up to motifs in the two sequences of a given orthologous pair. for clustalw the common motifs were manually identified and restricted to the same criteria as those used by dragon motif builder. a significant difference was observed showing that clustalw was not able to identify sufficient similarity between the ortholog sequences in the 5'utr regions as the segments that contain similar arrangement of common motifs between the two species were not residing at similar genomic locations. once the motifs were identified, we analyzed the motif arrangements. we selected the group of motifs that contained the largest number of common motifs, but retained the same positional arrangement in the two species. thus each of the ortholog pairs was screened for such a representative motif arrangement. we used the number of motifs in the representative arrangements as a possible measure of similarity between the 5'utr regions. in most cases, the regions where this arrangement had been spotted, was found at significantly different distances from the starting codon. authors' contributions ac had designed the bioinformatics pathway, performed most computational analyses, generated most tables and figures and took part in the writing of the manuscript. rb has contributed to the experimental design, constructed the full-length libraries and took part in the writing of the manuscript. hs has participated in the bioinformatics analysis of the data and in the maintenance of the cyprinid est database of tll. hk has supervised the generation of yy androgenic common carp line, generated the monosex populations, isolated the testis samples from them and contributed to the experimental design. lo initiated the project on comparative analysis of cyprinid ests, contributed to the experimental design and took part in the writing of the manuscript. supplementary material additional file 1 the description of common carp cdna libraries analyzed in this study. details include tissue, developmental stage and source of cdna libraries. click here for file additional file 2 distribution of functional categories identified in the partial transcriptome of common carp. click here for file additional file 8 protocol to map common carp transcripts to the zebrafish genome assembly. the flow chart depicts the pipeline implemented for mapping common carp transcripts to the zebrafish genome. filter criteria are denoted in the decision tree. total number of clusters and singletons are indicated in square brackets. click here for file additional file 3 classification of common carp ests that map to intergenic, intronic, ab initio predictions and non-zebrafish supported annotations. click here for file additional file 4 list of hyperlinks to potential common carp splice variants. overlapping common carp and zebrafish transcripts are presented on a gbrowse viewer to highlight the missing exons in one of the two species. click here for file additional file 5 list of testis-only transcripts that do not overlap any of the zebrafish and fathead minnow ests. click here for file additional file 6 list of testis-derived clusters that show sequence identity to zebrafish and fathead minnow unigene clusters. testis-expression information was added to the adult-stage zebrafish expression data. click here for file additional file 7 homologous gene pairs identified through manual curation of common carp and zebrafish genes. the table includes dna and protein accession numbers and corresponding gene descriptions. genes in rows highlighted with yellow contain 5' utr sequence and were used in utr analysis. click here for file additional file 9 distribution of orthologous clusters with given number of common motifs using dragon motif builder and clustalw. click here for file additional file 10 percent sequence identity between common carp and zebrafish orthologous proteins, cds, 5' utr and 3' utr regions. click here for file acknowledgements the authors thank aarti giri, james a. hill, mei yin ho, balamuragan kumarasamy, yang li and tina eyre for their technical help. they also acknowledge the help of oliver bezuidt, cameron macpherson and vladimir bajic for the comparative analysis of homologous 5'utrs from common carp and zebrafish as well as vladimir bajic's helpful comments and corrections on an earlier version of the manuscript. this project was supported from internal funding by temasek life sciences laboratory. this article has been published as part of bmc bioinformatics volume supplement 2006: apbionet – fifth international conference on bioinformatics. the full contents of the supplement are available online at. figures and tables
automatic annotation of sequenced eukaryotic genomes integrates a combination of methodologies such as ab-initio methods and alignment of homologous genes and/or proteins. we have generated ests from the differentiating testis of common carp and clustered them with non-gonadal ests from carpbase as well as ests and common carp mrnas from genbank. we also demonstrate that 5' utr sequences of common carp and zebrafish orthologs share a significant level of similarity based on preservation of motif arrangements for as many as ab-initio motifs. moreover, of these transcripts did not match any sequence in the zebrafish or fathead minnow est collection. a total of unique common carp sequences could be stringently mapped to the zebrafish genome, of which matched coding sequences of zebrafish genes with or without potential splice variants. for example, annotation of the zebrafish genome within ensembl relies heavily on available cdna and protein sequences from two distantly related fish species and other vertebrates that have diverged several hundred million years ago. the fact that common carp transcripts help the identification or confirmation of these coding regions in zebrafish exemplifies the usefulness of sequences from closely related species for the annotation of model genomes. we show that common carp transcripts map to intergenic and intronic regions on the zebrafish genome assembly and regions annotated with non-teleost sequences. interestingly, an additional common carp transcripts indicate the potential presence of new splicing variants not found in zebrafish databases so far. our data show that there is sufficient homology between the transcribed sequences of common carp and zebrafish to warrant an even deeper cyprinid transcriptome comparison. we highlight the need for integrated resources to leverage the wealth of fragmented genomic data. 18– december international conference in bioinformatics – incob new dehli, india over 28% of the resulting unique transcripts are exclusively testis-derived ests.
 automatic annotation of sequenced eukaryotic genomes integrates a combination of methodologies such as ab-initio methods and alignment of homologous genes and/or proteins. for example, annotation of the zebrafish genome within ensembl relies heavily on available cdna and protein sequences from two distantly related fish species and other vertebrates that have diverged several hundred million years ago. the scarcity of genomic information from other cyprinids provides the impetus to leverage est collections to understand gene structures in this diverse teleost group.  we have generated ests from the differentiating testis of common carp and clustered them with non-gonadal ests from carpbase as well as ests and common carp mrnas from genbank. over 28% of the resulting unique transcripts are exclusively testis-derived ests. moreover, of these transcripts did not match any sequence in the zebrafish or fathead minnow est collection. a total of unique common carp sequences could be stringently mapped to the zebrafish genome, of which matched coding sequences of zebrafish genes with or without potential splice variants. we show that common carp transcripts map to intergenic and intronic regions on the zebrafish genome assembly and regions annotated with non-teleost sequences. interestingly, an additional common carp transcripts indicate the potential presence of new splicing variants not found in zebrafish databases so far. the fact that common carp transcripts help the identification or confirmation of these coding regions in zebrafish exemplifies the usefulness of sequences from closely related species for the annotation of model genomes. we also demonstrate that 5' utr sequences of common carp and zebrafish orthologs share a significant level of similarity based on preservation of motif arrangements for as many as ab-initio motifs.  our data show that there is sufficient homology between the transcribed sequences of common carp and zebrafish to warrant an even deeper cyprinid transcriptome comparison. on the other hand, the comparative analysis illustrates the value in utilizing partially sequenced transcriptomes to understand gene structure in this diverse teleost group. we highlight the need for integrated resources to leverage the wealth of fragmented genomic data. 18– december international conference in bioinformatics – incob new dehli, india 