BACKGROUND
the gilthead sea bream  is a member of the sparidae family widely and successfully cultured in the mediterranean region.
during the last decade more than  <dig>  scientific papers have focused on gilthead sea bream nutrition, immune response, physiology and genetics.
this high valuable fish for aquaculture industry becomes, thereby, an interesting animal model, and the development of molecular and genomic tools for research is highly desirable.
previous attempts have been made in this way and the assembly and annotation of more than  <dig>  expressed sequence tags  allowed the development of specific gilthead sea bream microarrays that have been used in transcriptomic studies of crowding stress
<cit>  and parasite and nutritional challenges
<cit> .
microarray approaches have also been done in this species to assess transcriptome differences between two early larval stages
<cit> , to underline the liver transcriptomic response after cortisol administration
<cit>  or to identify the key genes involved in the re-epithelialization process after scale removal
<cit> .
nevertheless, genomic tools still remain relatively scarce in gilthead sea bream and need to be further improved in comparison to other cultured fish, such as atlantic salmon
<cit> , common carp
<cit> , nile tilapia
<cit>  or turbot
<cit> , for which large ests collections generated by sanger sequencing of cdna libraries are currently available.
with the advent of the next generation sequencing technologies, the gathering of large amounts of sequence data for a given organism at affordable costs is more feasible
<cit> , and high-throughput  <dig> pyrosequencing has been used to explore the transcriptome of rainbow trout
<cit> , atlantic cod
<cit>  and turbot
<cit> .
in the case of gilthead sea bream, two deep sequencing studies have been reported from whole larval tissues
<cit>  and skeletal muscle
<cit> , yielding  <dig>  and  <dig>  assembled sequences, respectively.
nevertheless, the assembly of high-throughput sequences from one unique tissue usually results in relatively short sequences.
as the annotation success of a sequencing project is highly dependent on the size of the assembled sequences, approaches conducted to obtain longer sequences become desirable.
at this respect, it has been proved in some animal models, including rainbow trout
<cit> , that the use of two or more tissues for sequencing and assembly increases the number of annotated genes.
on this basis, the primary goal of the present study was to generate a large amount of gilthead sea bream transcriptomic reads from metabolically and immunologically relevant tissues by means of the construction of five  <dig> pyrosequencing libraries, combining them with sanger sequences from public repositories and our own published data.
it comprised nine previously constructed suppression subtractive hybridization  libraries that were obtained from a variety of tissues  from animals exposed to confinement stress, parasite infection or a nutritional stress
<cit> .
having this into account, tissues selected for the improvement of the current knowledge on gilthead sea bream transcriptome by means of high-throughput sequencing were related to animal growth , nutrient digestion  and immune response .
because of the importance of the development of non-lethal diagnostic methods, blood was also considered for  <dig> sequencing.
the second goal of this work was to build a reliable assembly database, with a high confidence of functional annotation by means of similarity searches, gene ontology  terms and detailed insights on pathway analysis for its use as a practical tool by the scientific community.
this will assist gene discovery and studies on functional genomics, as well as future genome projects of this important fish species.
RESULTS
five normalized cdna libraries from skeletal muscle, intestine, blood and head kidney  were constructed and sequenced in separate half-runs using 454-titanium flx technology.
table
<dig> summarizes the results for each one of the five normalized  <dig> libraries.
collectively, more than  <dig> , <dig> high-quality reads averaging  <dig> nt each were produced, representing  <dig>  gb of expressed sequence information.
for each library, an independent assembly was performed with the newbler software, and the number of assembled contigs ranged within libraries from  <dig>  to  <dig>  with an average length of 831– <dig>  nt.
at this stage and regardless of the tissue library, the number of singletons that failed to assemble remained relatively high.
as a result of this, the 67-77% of the information derived from each individual library was supported only by short singleton reads , which is particularly critical given that most assembly software programs include default steps that discard them to give more reliable results, with the unadvised loss of potentially valuable data
<cit> .
this is a complex issue and to solve it, we considered a unique global assembly with the already available mrna sequences and est collections in combination with all the unassembled high-quality reads derived from the five  <dig> libraries .
sequence characteristics of the five  <dig> normalized libraries derived from skeletal muscle, intestine, blood and head kidney .
as a result of the above integrative approach, the average number of reads composing each contig  increased from  <dig> - <dig>  in the individual tissue assemblies to  <dig>  in the global assembly .
this large improvement increased the reliability of the transcriptomic database, yielding  <dig>  assembled contigs with an average length of  <dig> nt that cover the  <dig> % of the consensus megabases .
this finding agrees with the observation that the total number of assembled contigs increased from  <dig>  to  <dig>  when five skeletal muscle libraries of gilthead sea bream were globally and not individually assembled
<cit> .
both in that and the present study, the number of  <dig> reads used for the assembly is of the same order of magnitude , but in our case they are derived from four different tissues and after combination with  <dig>  sanger reads from public repositories the final number of contigs was more than the double  of that reported in
<cit> .
of a total number of  <dig>  sequences , the  <dig> %  were annotated using nucleotide and protein reference databases.
this annotation ratio is quite higher than those reported in high-throughput sequencing studies conducted in other fish species of interest in aquaculture, such as turbot
<cit> , blunt snout bream
<cit>  or silver carp
<cit> .
this annotation improvement can be explained by the high length  of the contigs derived from the present study , which is also inferred when comparisons are made with previous studies in the same species, rendering a contig size of  <dig> nt and  <dig> nt in length for  <dig> libraries of skeletal muscle
<cit>  and whole larvae
<cit> , respectively.
the contig depth was increased in parallel and the best blastx hits for the potentially transcribed proteins yielded  <dig>  different swissprot accessions.
this finding suggests that our nucleotide database would contain more than the 75% of the protein coding transcripts of gilthead sea bream, assuming an average size of  <dig> - <dig>  genes in a fish with a non-recently duplicated genome
<cit> .
with independence of a high coverage of protein-coding transcripts, more than  <dig>  assemblies still remain without annotation.
this relatively high number of unknown sequences could correspond to more divergently evolved genes through vertebrate evolution, though we cannot exclude that some of these sequences may result from assembly artifacts.
alternatively, some of these sequences might correspond to long non-coding rnas , which are now emerging as an important class of regulatory transcripts with an extent that increases much more with organism complexity
<cit> .
otherwise, high-throughput sequencing is prone to sequencing errors at homopolymer regions, even when assembled at high coverage, which often give rise to a range of artificial sequences
<cit> .
to overcome this issue, an in silico correction step was introduced in the pipeline procedure, which allowed to obtain continuous open reading frames for annotated sequences avoiding frameshifting by edition  of single nucleotides at homopolymer regions.
with this newly developed tool, up to 34% of annotated sequences  were detected to carry one or more frameshifts.
among them,  <dig>  were satisfactorily corrected with the pipeline and only  <dig> needed a manual curation.
quality and reliability of the assembled gilthead sea bream database was assessed by blastx comparison of  <dig> randomly chosen transcripts of the stickleback transcriptome.
of note, the 71% of these stickleback transcripts  were originally annotated by the stickleback consortium, and for 94% of them  a positive result with the same annotation was found using our database as a transcriptome reference .
besides, reliable annotations were found for  <dig> sequences out of  <dig> that were not previously annotated in the stickleback transcriptome .
again this successful result can be explained by the high average length of the assembled gilthead sea bream sequences, obtained by de novo global assembly of sanger sequences and high-throughput sequences of four metabolically and immunologically relevant tissues.
blast2go analysis of the different annotations  reveals that the most abundant go terms related to biological processes were transport , protein modification process , response to stress , regulation of biological quality  and negative regulation of cellular process .
other highly represented terms are immune system process , haemopoiesis  and coagulation , which is not surprising given that  <dig> out of five  <dig> libraries were derived from blood and head kidney.
regarding skeletal muscle, tissue-specific biological processes like muscle structure development , muscle cell differentiation  or muscle contraction  were also highly represented in the annotated gilthead sea bream transcriptome.
likewise, the intestine participates in nutrient digestion and absorption with also an important role in xenobiotic metabolism
<cit> , and accordingly genes related to the molecular functions hydrolase activity  and oxidoreductase activity  were highly abundant in our database.
go terms define gene sets, but the relevance of a given metabolic process or biological pathway in a dataset is determined not only by the number of identified genes, but also by their relative abundance ratio considering the total number of genes involved in each pathway.
for this reason, precise mapping of metabolic pathways becomes necessary, and the usefulness of pathway analysis software is largely increasing
<cit> .
to date, the only fish metabolic network available, the public online tool metafishnet
<cit> , is still far from completion, so pathway analysis of the gilthead sea bream annotated sequences was performed by means of the ingenuity pathway analysis  software.
conversion of the annotated gilthead sea bream sequences to ipa elements assumed that the functional classification of genes did not vary too much widely between teleostean fish and model species .
in this scenario, from the initial list of  <dig>  annotated gilthead sea bream descriptions, the ipa software identified  <dig>  uniprot codes with model species equivalences, and  <dig>  of them were eligible for function/pathway analysis.
these sequences were represented in  <dig> ipa canonical pathways mapped from a total of  <dig>  which reinforces the idea that the present gilthead sea bream transcriptome is of high coverage and, thereby, very close to completion of the protein-coding transcriptome.
the most representative high-level functional categories are listed in table
<dig> with precise information of statistic values and number of represented molecules.
the top five canonical pathways were regulation of eif- <dig> and p70s6k signaling, purine metabolism, eif <dig> signaling, protein ubiquitination pathway, and glucocorticoid receptor signaling.
as a corollary, the complete list of canonical pathways with the ratio of gilthead sea bream genes present in each one is provided as additional file
<dig>  and the graphical representation of representative canonical pathways of processes of interest  are presented in additional file
<dig>
in order to construct a reliable database of protein-coding transcripts with a minimum of frameshift errors or redundancies, a strict filtering was applied to the annotated sequences after the in silico homopolymer correction step.
sequences with a significant hit against swissprot were selected, and then sequences encoding for a same annotation regardless of the species were clustered.
filtering selected for each annotation non-overlaping sequences and putative complete open reading frames, that made a total of  <dig>  sequences that were uploaded in our nucleotide database .
this online resource is intended to become a dynamic and useful tool for scientific community.
with this interface, data can be queried using different strategies, such as several blast options or direct word search for annotation or go terms.
search results provide additional information for each sequence, like its length, depth or the related swissprot accession for the assigned annotation, among others.
CONCLUSIONS
a gilthead sea bream transcriptome database has been constructed by de novo assembly of five normalized  <dig> libraries from metabolically and immunologically relevant tissues combined with public sequences that included nine ssh libraries.
this approach yielded  <dig>  different sequences, and for  <dig>  a reliable annotation was found, resulting in  <dig>  different gene descriptions that comprised a vast array of functional categories and biological pathways.
this constitutes the largest and most complete transcriptome reported to date for gilthead sea bream, having a size and depth equivalent to those reported in the ensembl genome database for atlantic cod and other cultured fish species.
the information of annotated contigs has been semi-automatically corrected and filtered for redundancies, and is stored in a web database  that has been provided with blast and other search options for the scientific community.
this represents a valuable tool to assist fish phenotyping and the concomitant development of molecular biomarker panels  of prognostic and diagnostic value to cope with developmental-, nutritional-, environmental- and disease-related stressors.

 the gilthead sea bream is a member of the sparidae family widely and successfully cultured in the mediterranean region. during the last decade more than scientific papers have focused on gilthead sea bream nutrition, immune response, physiology and genetics. this high valuable fish for aquaculture industry becomes, thereby, an interesting animal model, and the development of molecular and genomic tools for research is highly desirable. previous attempts have been made in this way and the assembly and annotation of more than expressed sequence tags allowed the development of specific gilthead sea bream microarrays that have been used in transcriptomic studies of crowding stress and parasite and nutritional challenges. microarray approaches have also been done in this species to assess transcriptome differences between two early larval stages, to underline the liver transcriptomic response after cortisol administration or to identify the key genes involved in the re-epithelialization process after scale removal. nevertheless, genomic tools still remain relatively scarce in gilthead sea bream and need to be further improved in comparison to other cultured fish, such as atlantic salmon, common carp, nile tilapia or turbot, for which large ests collections generated by sanger sequencing of cdna libraries are currently available. with the advent of the next generation sequencing technologies, the gathering of large amounts of sequence data for a given organism at affordable costs is more feasible, and high-throughput pyrosequencing has been used to explore the transcriptome of rainbow trout, atlantic cod and turbot. in the case of gilthead sea bream, two deep sequencing studies have been reported from whole larval tissues and skeletal muscle, yielding and assembled sequences, respectively. nevertheless, the assembly of high-throughput sequences from one unique tissue usually results in relatively short sequences. as the annotation success of a sequencing project is highly dependent on the size of the assembled sequences, approaches conducted to obtain longer sequences become desirable. at this respect, it has been proved in some animal models, including rainbow trout, that the use of two or more tissues for sequencing and assembly increases the number of annotated genes. on this basis, the primary goal of the present study was to generate a large amount of gilthead sea bream transcriptomic reads from metabolically and immunologically relevant tissues by means of the construction of five pyrosequencing libraries, combining them with sanger sequences from public repositories and our own published data. it comprised nine previously constructed suppression subtractive hybridization libraries that were obtained from a variety of tissues from animals exposed to confinement stress, parasite infection or a nutritional stress. having this into account, tissues selected for the improvement of the current knowledge on gilthead sea bream transcriptome by means of high-throughput sequencing were related to animal growth, nutrient digestion and immune response. because of the importance of the development of non-lethal diagnostic methods, blood was also considered for sequencing. the second goal of this work was to build a reliable assembly database, with a high confidence of functional annotation by means of similarity searches, gene ontology terms and detailed insights on pathway analysis for its use as a practical tool by the scientific community. this will assist gene discovery and studies on functional genomics, as well as future genome projects of this important fish species.  five normalized cdna libraries from skeletal muscle, intestine, blood and head kidney were constructed and sequenced in separate half-runs using 454-titanium flx technology. table  summarizes the results for each one of the five normalized libraries. collectively, more than, high-quality reads averaging nt each were produced, representing gb of expressed sequence information. for each library, an independent assembly was performed with the newbler software, and the number of assembled contigs ranged within libraries from to with an average length of 831– nt. at this stage and regardless of the tissue library, the number of singletons that failed to assemble remained relatively high. as a result of this, the 67-77% of the information derived from each individual library was supported only by short singleton reads, which is particularly critical given that most assembly software programs include default steps that discard them to give more reliable results, with the unadvised loss of potentially valuable data. this is a complex issue and to solve it, we considered a unique global assembly with the already available mrna sequences and est collections in combination with all the unassembled high-quality reads derived from the five libraries. sequence characteristics of the five normalized libraries derived from skeletal muscle, intestine, blood and head kidney. as a result of the above integrative approach, the average number of reads composing each contig increased from - in the individual tissue assemblies to in the global assembly. this large improvement increased the reliability of the transcriptomic database, yielding assembled contigs with an average length of nt that cover the % of the consensus megabases. this finding agrees with the observation that the total number of assembled contigs increased from to when five skeletal muscle libraries of gilthead sea bream were globally and not individually assembled. both in that and the present study, the number of reads used for the assembly is of the same order of magnitude, but in our case they are derived from four different tissues and after combination with sanger reads from public repositories the final number of contigs was more than the double of that reported in. of a total number of sequences, the % were annotated using nucleotide and protein reference databases. this annotation ratio is quite higher than those reported in high-throughput sequencing studies conducted in other fish species of interest in aquaculture, such as turbot, blunt snout bream or silver carp. this annotation improvement can be explained by the high length of the contigs derived from the present study, which is also inferred when comparisons are made with previous studies in the same species, rendering a contig size of nt and nt in length for libraries of skeletal muscle and whole larvae, respectively. the contig depth was increased in parallel and the best blastx hits for the potentially transcribed proteins yielded different swissprot accessions. this finding suggests that our nucleotide database would contain more than the 75% of the protein coding transcripts of gilthead sea bream, assuming an average size of - genes in a fish with a non-recently duplicated genome. with independence of a high coverage of protein-coding transcripts, more than assemblies still remain without annotation. this relatively high number of unknown sequences could correspond to more divergently evolved genes through vertebrate evolution, though we cannot exclude that some of these sequences may result from assembly artifacts. alternatively, some of these sequences might correspond to long non-coding rnas, which are now emerging as an important class of regulatory transcripts with an extent that increases much more with organism complexity. otherwise, high-throughput sequencing is prone to sequencing errors at homopolymer regions, even when assembled at high coverage, which often give rise to a range of artificial sequences. to overcome this issue, an in silico correction step was introduced in the pipeline procedure, which allowed to obtain continuous open reading frames for annotated sequences avoiding frameshifting by edition of single nucleotides at homopolymer regions. with this newly developed tool, up to 34% of annotated sequences were detected to carry one or more frameshifts. among them, were satisfactorily corrected with the pipeline and only needed a manual curation. quality and reliability of the assembled gilthead sea bream database was assessed by blastx comparison of randomly chosen transcripts of the stickleback transcriptome. of note, the 71% of these stickleback transcripts were originally annotated by the stickleback consortium, and for 94% of them a positive result with the same annotation was found using our database as a transcriptome reference. besides, reliable annotations were found for sequences out of that were not previously annotated in the stickleback transcriptome. again this successful result can be explained by the high average length of the assembled gilthead sea bream sequences, obtained by de novo global assembly of sanger sequences and high-throughput sequences of four metabolically and immunologically relevant tissues. blast2go analysis of the different annotations reveals that the most abundant go terms related to biological processes were transport, protein modification process, response to stress, regulation of biological quality and negative regulation of cellular process. other highly represented terms are immune system process, haemopoiesis and coagulation, which is not surprising given that out of five libraries were derived from blood and head kidney. regarding skeletal muscle, tissue-specific biological processes like muscle structure development, muscle cell differentiation or muscle contraction were also highly represented in the annotated gilthead sea bream transcriptome. likewise, the intestine participates in nutrient digestion and absorption with also an important role in xenobiotic metabolism, and accordingly genes related to the molecular functions hydrolase activity and oxidoreductase activity were highly abundant in our database. go terms define gene sets, but the relevance of a given metabolic process or biological pathway in a dataset is determined not only by the number of identified genes, but also by their relative abundance ratio considering the total number of genes involved in each pathway. for this reason, precise mapping of metabolic pathways becomes necessary, and the usefulness of pathway analysis software is largely increasing. to date, the only fish metabolic network available, the public online tool metafishnet, is still far from completion, so pathway analysis of the gilthead sea bream annotated sequences was performed by means of the ingenuity pathway analysis software. conversion of the annotated gilthead sea bream sequences to ipa elements assumed that the functional classification of genes did not vary too much widely between teleostean fish and model species. in this scenario, from the initial list of annotated gilthead sea bream descriptions, the ipa software identified uniprot codes with model species equivalences, and of them were eligible for function/pathway analysis. these sequences were represented in ipa canonical pathways mapped from a total of which reinforces the idea that the present gilthead sea bream transcriptome is of high coverage and, thereby, very close to completion of the protein-coding transcriptome. the most representative high-level functional categories are listed in table  with precise information of statistic values and number of represented molecules. the top five canonical pathways were regulation of eif- and p70s6k signaling, purine metabolism, eif signaling, protein ubiquitination pathway, and glucocorticoid receptor signaling. as a corollary, the complete list of canonical pathways with the ratio of gilthead sea bream genes present in each one is provided as additional file and the graphical representation of representative canonical pathways of processes of interest are presented in additional file in order to construct a reliable database of protein-coding transcripts with a minimum of frameshift errors or redundancies, a strict filtering was applied to the annotated sequences after the in silico homopolymer correction step. sequences with a significant hit against swissprot were selected, and then sequences encoding for a same annotation regardless of the species were clustered. filtering selected for each annotation non-overlaping sequences and putative complete open reading frames, that made a total of sequences that were uploaded in our nucleotide database. this online resource is intended to become a dynamic and useful tool for scientific community. with this interface, data can be queried using different strategies, such as several blast options or direct word search for annotation or go terms. search results provide additional information for each sequence, like its length, depth or the related swissprot accession for the assigned annotation, among others.  a gilthead sea bream transcriptome database has been constructed by de novo assembly of five normalized libraries from metabolically and immunologically relevant tissues combined with public sequences that included nine ssh libraries. this approach yielded different sequences, and for a reliable annotation was found, resulting in different gene descriptions that comprised a vast array of functional categories and biological pathways. this constitutes the largest and most complete transcriptome reported to date for gilthead sea bream, having a size and depth equivalent to those reported in the ensembl genome database for atlantic cod and other cultured fish species. the information of annotated contigs has been semi-automatically corrected and filtered for redundancies, and is stored in a web database that has been provided with blast and other search options for the scientific community. this represents a valuable tool to assist fish phenotyping and the concomitant development of molecular biomarker panels of prognostic and diagnostic value to cope with developmental-, nutritional-, environmental- and disease-related stressors. 
the newly assembled gilthead sea bream transcriptome represents a progress in genomic resources for this species, as it probably contains more than 75% of actively transcribed genes, constituting a valuable tool to assist studies on functional genomics and future genome projects. a transcriptome database was constructed by de novo assembly of gilthead sea bream sequences derived from public repositories of mrna and collections of expressed sequence tags together with new high-quality reads from five cdna normalized libraries of skeletal muscle, intestine, head kidney and blood. the high representation of randomly selected stickleback transcripts by blast search in the nucleotide gilthead sea bream database evidenced its high coverage of protein-coding transcripts. the gilthead sea bream is the main fish species cultured in the mediterranean area and constitutes an interesting model of research. blast analysis directed to protein and nucleotide databases annotated sequences encoding for gene descriptions, that were curated for redundancies and frameshifting at the homopolymer regions of open reading frames, and hosted at http://www.nutrigroup-iats.org/seabreamdb. sequencing of the new normalized libraries produced, high-quality reads and the de novo global assembly yielded unique sequences with an average length of nt.
 the gilthead sea bream is the main fish species cultured in the mediterranean area and constitutes an interesting model of research. nevertheless, transcriptomic and genomic data are still scarce for this highly valuable species. a transcriptome database was constructed by de novo assembly of gilthead sea bream sequences derived from public repositories of mrna and collections of expressed sequence tags together with new high-quality reads from five cdna normalized libraries of skeletal muscle, intestine, head kidney and blood.  sequencing of the new normalized libraries produced, high-quality reads and the de novo global assembly yielded unique sequences with an average length of nt. blast analysis directed to protein and nucleotide databases annotated sequences encoding for gene descriptions, that were curated for redundancies and frameshifting at the homopolymer regions of open reading frames, and hosted at http://www.nutrigroup-iats.org/seabreamdb. among the annotated gene descriptions, were mapped in the ingenuity pathway analysis database, and were eligible for functional analysis with a representation in out of ipa canonical pathways. the high representation of randomly selected stickleback transcripts by blast search in the nucleotide gilthead sea bream database evidenced its high coverage of protein-coding transcripts.  the newly assembled gilthead sea bream transcriptome represents a progress in genomic resources for this species, as it probably contains more than 75% of actively transcribed genes, constituting a valuable tool to assist studies on functional genomics and future genome projects. sparus auratanext-generation sequencingde novo assemblytranscriptomedatabase 