BACKGROUND
the term “soles” refers to a wide group of flatfish species belonging to the soleidae  and cynoglossidae  families.
common sole  and senegalese sole  are two economically important species highly appreciated worldwide due to the excellent quality of their flesh  and heavily exploited in industrial fisheries.
as a result, sole overfishing has had a profound effect on some life-history traits observing a shift towards earlier sexual maturation and a decline of the spawning biomass  <cit> .
therefore, aquaculture efforts have focused on s. senegalensis as one of the most promising species for diversification in southern europe due to its fast growth rates .
however, solea aquaculture is facing several bottlenecks such as the production of high-quality larvae, the improvement and optimization of nutrition for better growth rates and the development of strategies for the control of infectious diseases.
in addition, the failure to reproduce successfully soles of the f <dig> generation in captivity precludes the development of selection programs  <cit> .
moreover, soles are an excellent model to study development and metamorphosis in fish.
soles undergo drastic morphological, physiological and ethological changes early during development for a short period of time .
therefore, sole species have become a suitable model to study larval ontogeny, skin pigmentation, hormonal and epigenetic mechanisms controlling development, sex differentiation, nutritional requirements, asymmetrical development as well as comparative genomics in flatfish .
in this context, development of large-scale genomic resources is a priority to facilitate the implementation of new technological approaches such as rna-seq and genome-wide mapping studies, that can assist in the identification of signalling networks controlling metamorphosis, growth, reproduction or disease resistance to advance in the knowledge of their biology and to improve rearing techniques and selective breeding  <cit> .
several studies have focused previously on the development of genomic resources in s. senegalensis and s. solea species.
molecular markers, including microsatellites  and single nucleotide polymorphisms , and bacterial artificial chromosomes  libraries have been developed .
being scarce, more polymorphic markers are required for population management and breeding programs.
moreover, a limited set of expressed sequence tags  has been described in each species that were used for the design of species-specific oligo-dna microarrays  <cit> .
consequently, the number of available ests is still far to conform a representative transcriptome as described in other teleosts and further sequencing efforts are required.
next generation sequencing technologies  have drastically transformed the way researchers can address genomic questions on non-model species, including soles.
the ngs platforms are able to generate quickly an enormous bulk of cost-effective genomic information, even for those species with limited or no previous genomic information available  <cit> .
although ngs offers different applications, cdna/rna sequencing  has become very popular for genome-wide transcriptome profiling and de novo sequencing of transcriptomes.
the high-volume of transcriptomic reads generated constitute a rich and important source of potential molecular markers, including ssrs and snps, as well as for transcript processing, making possible the design and implementation of other techniques such as microarray hybridization or qpcr  <cit> .
in aquaculture, several studies have described the implementation of  <dig> pyrosequencing for de novo transcriptome sequencing of some mediterranean species such as seabream  <cit> , common sole  <cit>  or seabass  <cit> .
most of these studies have focused on the characterization of transcriptomes under specific developmental stages, pathogen challenge or tissue-specific profiles.
nevertheless, the design of a dedicated species-specific database would be useful for easier management of genomic information  and development of complementary techniques such as microarrays or rna-seq.
the main aim of this work was the generation of a representative transcriptome for s. senegalensis and s. solea after processing an important large set of transcriptomic information produced by roche/ <dig> and illumina paired-end ngs technologies covering a large number of developmental stages associated to physiological challenges.
the main transcriptome features and characteristics were identified.
comparative analysis between soles allowed for the identification of orthologs, new genes and molecular markers.
finally, a browseable database referred to as soleadb was constructed and a new microarray tool was designed and validated.
RESULTS
pre-processing and assembly of ngs data
a total of  <dig> and  <dig> illumina libraries  were prepared for s. senegalensis and s. solea, respectively, and additional roche/ <dig> sequences  were prepared for s. senegalensis .
most paired reads were useful after pre-processing .
the most important source of reads removed were ribosomal and mitochondrial sequences .
other contaminants less represented in the illumina data were reads from live preys and microalgae used in larvae feeding , rotifers  and t-iso  and some microorganisms, mainly fungi .
assuming that the putative number of genes occurring in solea might range between  <dig>  and  <dig>  protein-coding genes, as reported in cynoglossus semilaevis and danio rerio, respectively , with an average transcript length of  <dig>  nt, the estimated transcriptome coverage of useful reads ranged from  <dig> × to  <dig> × for s. senegalensis, and from  <dig> × to  <dig> × for s. solea.
therefore, the number of high quality reads was sufficient to assemble a reliable transcriptome in both species.table  <dig>
pre-processing summary of raw reads
1
s. senegalensis
s. solea
s. senegalensis
76
100
757
66
89
184
the s. senegalensis transcriptome was initially assembled using only the roche/ <dig> reads, following the workflow depicted in figure 1a .
interestingly, the assembly resulted in a large number of transcripts longer than  <dig> bp, slightly higher than expected for a teleost  <cit> .
the addition of illumina libraries to improve transcriptome assembly  required the workflow depicted in figure 1b, where the long read strategy was slightly modified .
the s. solea transcriptome  was assembled using the short read strategy depicted in figure 1b, except that k-mers used were  <dig> and  <dig> due to longer raw reads .
s. solea v <dig> and s. senegalensis v <dig> transcriptomes were comparable with respect to  the frequency distribution of transcript length ,  the total number of transcripts  and  the number of transcripts longer than  <dig> bp .
however, mean length and n <dig> were clearly longer in s. solea , which may be explained by the longer input reads  and a low relative contribution of roche/ <dig> reads in s. senegalensis v <dig>  this low contribution may be explained by the fact that roche/ <dig> libraries were normalized to reduce highly abundant transcripts which might have led to more fragmented assemblies limiting the contribution of roche/ <dig> reads to the final transcriptome  <cit> .figure  <dig>
schematic representation of the pre-processing, assembling and reconciliation approach to obtain the final transcriptome.
a, strategy for a set of only long reads.
b, strategy combining long and short reads.
the numbers with “#” serve as a reference for table  <dig> description.
representation of transcript abundance with respect of their lengths in the
s. senegalensis
and
s. solea
transcriptomes.
previous reports that focused on de novo transcriptome assembly used a variable number of input reads .
however, a total of 20– <dig> million reads were considered sufficient to generate a de novo transcriptome, even though assembly is highly influenced by factors such as species or type of sample   <cit> .
for example, the number of transcripts in six different marine invertebrates varied from  <dig>  to  <dig>  in spite of the fact that the number of input of reads was similar .
moreover, more transcripts were discovered using whole-body samples  <cit> .
in this study, a joint analysis of the complete set of samples comprising a wide range of organs and developmental stages was performed to facilitate a maximal gene representation into the final transcriptome assembly.
as a whole, more than  <dig>  million reads were assembled in each sole species, representing the highest number of reads reported to date for any organism.
transcriptome characteristics
the main characteristics of the assembled transcriptomes are depicted in table  <dig>  a detailed analysis of the assembled transcriptomes reveals, first, that the number of artifacts was very low in spite of the extraordinarily large number of transcripts assembled.
second, that the s. senegalensis v <dig> transcriptome was improved over the s. senegalensis v <dig> transcriptome in terms of transcript length and number of complete orfs, but both transcriptome versions presented a similar number of different orthologous ids, suggesting that the high number of juvenile tissues pooled to prepare the roche/ <dig> libraries could have importantly contributed to obtain a better gene representation.
third, that the number of different complete orfs was high and similar between the transcriptomes of the two sole species , suggesting that sole transcriptomes are equally reliable.
finally, that s. senegalensis v <dig> had a higher number of orthologous ids and a lower number of different complete orfs than s. solea v <dig>  which may be due to a greater transcript fragmentation in s. senegalensis because roche/ <dig> data only contributed to increase the orthology in the final assembly.table  <dig>
overview of assembled transcriptomes in
s. senegalensis
and
s. solea
s. senegalensis
s. solea
artifacts2
transcripts with ortholog1
transcripts without ortholog1
reference transcriptome
v <dig> :  <dig>
v <dig> :  <dig>
the values were calculated using fulllengthernext software.
the minimum number of transcripts that can be considered as a reference transcriptome is shown in bold.
1percentages for subclassifications of this category were calculated using this line as 100% reference.
2artifacts refer mainly to misassemblies and chimeric contigs.
nc: non-calculated.
the extremely large number of reads used to assemble the two sole transcriptomes could favour the accumulation of assembly errors  <cit> .
evaluation of transcript accuracy was initially based on mapping useful reads of two randomly selected libraries of each sole transcriptome using bowtie <dig> .
since  <dig> - <dig> % of the reads mapped onto assembled transcripts, the assembly errors can be considered negligible.
interestingly, the longest transcript in s. senegalensis v <dig> and s. solea v <dig> transcriptomes  is clearly not an artifact: in both species, it corresponded to a titin-like protein highly similar to a long mrna  previously assembled in tilapia .
titin is a giant filamentous protein highly abundant in muscle that forms a separate myofilament system in both skeletal and cardiac muscle  <cit> .
the fact that this transcript is 6-fold longer in s. senegalensis v <dig> than in s. senegalensis v <dig> supports the significant contribution of illumina short-reads to the final assembly.
soleadb, a database for browsing soleatranscriptomes
genomic databases are extremely useful for target sequence retrieval, blast comparisons, sequence management and compilation of all information that can help to a better understanding of the function and roles of genes.
a preliminary database devoted to host sanger est information, microarray data and ish pictures was developed for s. senegalensis
<cit> .
nevertheless, this database was not suitable to manage the important volume of information generated using ngs.
hence, a new database, soleadb, was built to host all the information for s. senegalensis and s. solea transcriptomes, following the same architecture as reported for europinedb  <cit>  and sustainpine  <cit> .
soleadb was structured and designed in a user-friendly manner showing all information regarding experimental conditions, ngs libraries characteristics and processing pipelines to clean, assemble and annotate the transcriptomes.
navigation is very intuitive with information structured by assemblies including global assemblies  and by experimental conditions.
a search tool to find specific transcript information by different fields and a blast tool was also incorporated.in the “assemblies” tab, different transcriptome versions can be browsed .
for each assembly , there is a brief description  of experimental conditions as well as assembly, markers and annotation statistics.
raw sequencing reads, pre-processing reports, fasta and ace files, as well as annotations for functional analysis can be downloaded from the same tab .
in the “unigenes” section, users can search for specific transcripts and browse their specific information.
information available for each transcript includes the consensus sequence, the corresponding contig, functional annotations, full-length status, orf prediction, and putative markers.
by means of kegg and ec annotations, genes in specific pathways can also be retrieved.
all annotations incorporated in soleadb are freely downloadable for the scientific community.figure  <dig>
screen captures of soleadb interface.
a, illustration of the “assemblies” tab containing all information about all transcriptome versions and subversions.
b, capture of the part of the screen corresponding to the “assembly info” tab where general information about the transcriptome as well as downloadable files and other useful tools can be found.
therefore, soleadb can be extremely useful for data comparison across experiments allowing for the identification of paralogues, alternative spliced transcripts and novel genes.
it represents a new, easy-to-use, valuable tool to host ngs data and for sharing genomic information between users applying these techniques.
moreover, whole and reference transcriptomes will be a useful tool for downstream applications such as rna-seq.
examples of usefulness of soleadb can be found in the next sections.
transcripts without ortholog as a source of putative new sole transcripts
the high number of transcripts without ortholog  deserved a deeper analysis.
based on their low testcode index, >91% of the unknown transcripts did not encode for proteins , which could explain in part the lack of orthology.
to check the accuracy of these unknown transcripts, genome reads  were mapped onto the unknown transcripts of s. senegalensis v <dig>  resulting in  <dig>   unknown transcripts mapped .
these high mapping percentages indicate that these sequences were not assembly artifacts and that they might correspond to co-purified genomic fragments or immature transcripts.
among the  <dig> % and  <dig> % of transcripts without ortholog in s. senegalensis v <dig> and s. solea v <dig>  respectively, some showed a testcode index > <dig>   and, therefore, are likely coding transcripts.
those transcripts referred to as non-redundant new transcripts , based on the absence of orthologs in uniprotkb database, may represent “new” proteins  in sole.
a reference transcriptome for each sole species
the high number of assembled transcripts indicated an overestimation of sole transcriptomes when compared to other teleosts .
probably, a certain number of transcripts could actually represent alleles, paralogs, fragmented transcripts, spliced forms, immature mrnas and even a combination of them.
therefore, identification of representative transcripts from these transcriptomes would be a useful tool to be used as a reference for future gene expression studies.
for this purpose, representative transcripts were selected from  the longest transcripts with unique, different orthologous id and  the putative, non-redundant new transcripts .
hence, the reference transcriptome for s. senegalensis  consisted of  <dig>  transcripts and the s. solea  reference transcriptome contained  <dig>  transcripts .
when useful reads  were mapped onto these reference transcriptomes,  <dig> - <dig> % of reads were mapped onto transcripts, while  <dig> - <dig> % of transcripts received more than one read, suggesting that they represent adequately the transcriptome.
additional verification of the reference transcriptomes  was based on an orthology analysis using zebrafish .
in s. senegalensis v <dig> ,  <dig>  reference transcripts were found to be orthologs to  <dig>  refseq and to  <dig>  ensembl zebrafish entries, and in s. solea v <dig>   <dig>  reference transcripts were found to be orthologs to  <dig>  refseq and to  <dig>  ensembl zebrafish entries .
these numbers suggest that a certain number of alleles, immature mrnas and lineage-specific genes  may have been included in the reference transcriptome.
moreover, since the number of refseq and ensembl ids nearly corresponds to half the number of sole transcripts, it is likely that both alleles for each gene were included in the sole reference transcriptomes.
this hypothesis is also supported by the fact that most of the samples analyzed corresponded to wild animals or larvae, being mostly heterozygous.
it is worth noting that the number of different zebrafish ids in refseq or ensembl  is close to the ~ <dig>  genes recently reported for half-smooth tongue sole  <cit>  and not so different from the  <dig>  genes that have been recently reported in zebrafish  <cit> .
therefore, it can be suggested that most sole genes have been covered in the sole reference transcriptomes.
s. soleaand s. senegalensisshow clear functional similarity, are highly orthologous, and contain sole- and flatfish-specific transcripts
s. solea and s. senegalensis are two closely-related species with similar morphology , ecology  and feeding habits and preys .
in silico genome comparisons previously performed among fish species to identify orthologous gene groups identified a high percentage of shared genes  and only a small number of species-specific gene families   <cit> .
therefore, a comparative analysis of sole transcriptomes could reveal new clues about their biology and evolution and also can provide supporting evidence of reference transcriptome accuracies.
the  <dig> % and  <dig> % of annotation success in the s. senegalensis v <dig> and s. solea v <dig> transcriptomes, respectively, is in concordance with the analysis of full-lengthernext .
comparison of go terms between the two sole species revealed that they were similar .
the highest number of annotated transcripts by biological process was associated with metabolic  and cellular  processes.
by cellular components, the most represented categories were cell  and organelle .
by molecular function, the highest number of annotated transcripts was within the catalytic activity category .
interestingly, the channel regulator and antioxidant activity categories were only represented in s. senegalensis.
in conclusion, both sole transcriptomes appear to be very similar from a biological and functional point of view.
another comparison of sole transcriptomes was based on orthology with zebrafish.
figure  <dig> shows that  <dig> % of orthologous transcripts in soles with an identified ortholog in zebrafish had a similarity ≥95% at the nucleotide level with  <dig>  transcripts being 100% identical.
as expected, transcripts without zebrafish orthology had a lesser degree of identity, as evidenced by the plateau at 92-96% identity .
interestingly,  <dig> transcripts without ortholog had the same sequence and  <dig> transcripts showed a 99% identity between the two sole species.
all these data evidenced a high level of similarity between the two sole transcriptomes.
assuming that sole transcripts that share the same zebrafish ortholog could also be considered sole orthologs, a total of  <dig>  reference transcripts in s. senegalensis and  <dig>  reference transcripts in s. solea shared  <dig>  ids for refseq and  <dig>  ids for ensembl, which clearly reflects the high level of orthology between these two sole species.figure  <dig>
distribution of the level of similarity between both sole reference transcriptomes for those transcripts with  or without  a zebrafish ortholog.
true orthologs between both sole species were obtained after performing a reciprocal blast using the reference transcriptomes.
in this analysis, two transcripts were considered as blast-based, true orthologs when a highly restrictive reciprocal blast  always resulted with their sequences giving the best score and e values  <cit> .
a total of  <dig>  reference transcripts of s. senegalensis were highly homologous to reference transcripts of s. solea, and  <dig>  reference transcripts of s. solea were highly homologous to reference transcripts of s. senegalensis.
of these homologous sequences, only  <dig>  could be considered as true, blast-based orthologs .
these include  <dig> unannotated transcripts with an average length of  <dig> nt in s. senegalensis and  <dig>  nt in s. solea.
more interestingly,  <dig> of them had a testcode ≥ <dig> , indicating that they likely code for a specialized protein .
to confirm this hypothesis, they were compared to other fish proteins  and only  <dig>  failed to find any orthology .
moreover,  <dig> transcripts  showed a clear ortholog only in the flatfish c. semilaevis, suggesting that these they might represent flatfish-specific transcripts.
in conclusion,  <dig>  transcripts were identified as true, blast-based orthologs between both sole species, from which  <dig> are likely to be flatfish-specific transcripts and  <dig> are putative new sole transcripts.
transcriptome comparison across teleosts revealed a set of lineage-specific genes
the  <dig>  sole blast-based orthologs with annotation  were investigated based on their refseq and/or ensembl ortholog for zebrafish.
as shown in figure  <dig>  most blast-based orthologs  had a zebrafish ortholog.
however, the most interesting finding is the small subset of sole orthologs lacking zebrafish similarity .
some of these transcripts without zebrafish ortholog were related to the immune system such as hepcidin antimicrobial peptides and some interleukins .
hepcidins appear as a highly diversified family in acanthopterygians  that favoured the radiation of teleosts in marine and brackish environments  <cit> .
similarly, il11b duplication appeared later during evolution not occurring in zebrafish  <cit> .
these data suggest that this subset of sole orthologs without zebrafish orthology might represent lineage-specific genes that have appeared, subfunctionalized or neofunctionalized later during teleost evolution.
to check the presence of these transcripts in other teleosts, proteins deduced from reference transcripts were compared , observing that most of them were also present in the c. semilaevis genome .
only  <dig> transcripts  lacked any orthology in the teleosts analyzed confirming that this collection of transcripts could correspond to genes acquired or fixed during fish evolution .figure  <dig>
venn’s diagrams reflecting coincidences by
solea
species among sole, blast-based orthologs and transcripts with refseq/ensembl ortholog for zebrafish.
diagrams are comparing the  <dig>  blast-based orthologs with the unique zebrafish refseq identifiers in soleadb for s. senegalensis  and s. solea  and with the unique zebrafish ensembl identifiers in soleadb for s. senegalensis  and s. solea .
within the venn’s diagrams, the numbers refer to the amount of transcripts in soleadb for s. senegalensis  and s. solea , the number of transcript in soleadb with a zebrafish refseq identifier  of with a zebrafish ensembl identifier .
sole transcriptomes confirmed the retention of crystallin genes
recently, it has been suggested that the visual system had evolved in relation to their benthic way of life  <cit> .
this observation is based on the loss of genes related with vision such as crystallins crybb <dig> and crybb <dig> in c. semilaevis
<cit> .
five crybb orthologs have been identified in s. senegalensis and s. solea transcriptomes that grouped into crybb <dig> and crybb <dig> clusters  and none to the crybb <dig> clade .
moreover, additional crybb-like transcripts could be grouped into two crybb1-related clusters that seem to be fish-specific sequences.
extension of the analysis to closely related crybb-like sequences revealed that additional crybb sequences exist in all three flatfish c. semilaevis , s. solea  and s. senegalensis .
moreover, est sequence analysis from atlantic halibut  suggested that this flatfish also possesses crybb <dig>  crybb <dig> and several crybb1-like sequences .
taken together these results suggest that flatfish have indeed lost and retained specifically some crybb genes likely as a consequence of independent events indicative of divergent evolution and do not support a decay of the visual system as previously hypothesized in flatfish based on the set of crystallin-encoding genes  <cit> .figure  <dig>
phylogenetic tree of crybb and crybb-like proteins in vertebrates.
a neighbor-joining tree based on the alignment of vertebrates crybb and crybb-like sequences was built.
species are indicated as sse , sso  dre , tni , oni , ola , cse , xla  and gga .
solea sequences are indicated according to the transcript name assigned in soleadb.
clusters are indicated as arcs of a circle.
the tree obtained was rooted using xenopus laevis cryga.
numbers adjacent to nodes indicate percentage bootstrap support; only values larger than 70%  are shown.
sole transcriptomes as a source of molecular markers
a transcriptome represents an important source of molecular markers, mainly ssrs and snps.
a total of  <dig> ssr markers in senegalese sole derived from ests in public databases were applied to evaluate ssr evolution in flatfish species  <cit> .
sole transcriptomes described here will surely add more molecular markers suitable for a wide range of genetic applications.
ssrs were determined in the whole sole transcriptomes , the reference transcriptomes  and the set of sole blast-based orthologs .
it is noteworthy that in whole transcriptomes, dinucleotide repeats were the most abundant ssrs followed by tri-nucleotide repeats and tetra-nucleotide repeats.
nevertheless, the reference transcriptome was enriched in ssrs bearing a tri-nucleotide motif .
this difference in motif abundance can be explained by the selective constraints imposed by protein-coding dna, more abundant in the latter transcriptome.
in fact, di-nucleotide and tetra-nucleotide ssrs were mainly located in the utr, whereas tri-nucleotides were in the orf .
these results agree with genome-wide analyses that identified a bias distribution of tri- and hexa-nucleotide repeats in protein-coding exons of vertebrate, invertebrates, plants and fungi  <cit> .
the most common ssr motifs were ac/gt for di-nucleotides , agg/cct in s. senegalensis and agc/gct in s. solea for tri-nucleotides , and aaac/gttt for tetra-nucleotides .
similar percentages were estimated for the reference transcriptome except for the fact that agc/gct was more abundant than agg/cct.
fish analysis using gt, gtta and gata probes confirmed the relative abundance of these ssrs on the genome .
the ac/gt motif has been reported as the most frequent ssr repeat in the intergenic regions and introns of vertebrates  <cit> .
the ac/tg motif has been identified as the most frequent ssr in roche/ <dig> assembled sequences of s. solea
<cit> .
also, g + c-rich tri-nucleotides  and tetra-nucleotides with <50% of g + c  are characteristic of vertebrates  <cit> .
it is worth mentioning the low representation of gata repeats  confirmed by fish analysis .
comparison of ssrs blast-based orthologs in soles  identified  <dig>  sole-conserved ssrs in s. senegalensis and  <dig>  in s. solea, out of which  <dig>  and  <dig>  ssrs in s. senegalensis and s. solea, respectively, can be considered species-specific as they were only found in the orthologs of only one species.
this analysis provides new and very useful ssr markers for development of multiplex assays, genetic mapping and deciphering genome evolution in flatfish as well as species identification in processed fish.table  <dig>
ssr summary statistics for whole and reference transcriptomes
s. senegalensis
s. solea
species-specific ssr1
same repeat motif2
total number of ssrs and frequency according to their repeat motif are indicated.
ssrs present in one species but not in orthologs of the other species.
exactly the same ssr repeat motif was found in both orthologs; in a few cases, ssr occurs once in one ortholog and twice in the other.
a total of  <dig>  snps were identified in s. senegalensis and  <dig>  in s. solea transcriptomes.
a significant proportion of snps occurred in transcripts containing an orf  with approximately half occurring within the orf .
these figures for snp location in coding regions are similar to those found in other fish species ranging from  <dig>  to  <dig> % .
although snp prediction is only based on bioinformatic analysis and requires empirical validation to eliminate false positives and sequencing errors  <cit> , these snps can also be used as a source of putative molecular markers.
design and validation of an oligonucleotide microarray for s. senegalensis
microarrays have become a cost-effective technique for gene expression profiling and whole-transcriptome studies  <cit> .
two species-specific oligo-dna microarrays have been reported in s. senegalensis and s. solea containing a limited number of unique transcripts due to the limited number of ests available in soles  <cit> .
this limitation was compensated to some extent using heterologous microarrays  <cit> .
the sole transcriptomes described in this study have overcome these restrictions.
the strategy to select sole-specific probes is depicted in figure  <dig>  briefly, the  <dig>  complete non-redundant transcripts were added to the  <dig>  longest, non-redundant, incomplete transcripts.
clustering them resulted in  <dig>  longest, non-redundant transcripts  that were combined with  <dig>  selected “coding” transcripts, providing a total of  <dig>  probes.
the final panel of probes included genes related to reproduction, cell differentiation, response to stress, growth, biosynthetic and catabolic processes, transport, embryonic development and immune system, among other functions.figure  <dig>
schematic representation of the probe selection strategy for the construction of the senegalese sole oligonucleotide microarray.
the number of transcripts that resulted after the described filtration is indicated.
the microarray was tested with larvae incubated at two salinities .
hybridization signal was detected for  <dig>  probes.
a total of  <dig>  probes were found differentially expressed  of which  <dig>  were up-regulated and  <dig> down-regulated at  <dig> ppt compared to  <dig> ppt.
application of a cut-off value of log <dig>  > ± <dig> filtered  <dig>  up-regulated and  <dig> down-regulated probes.
the differentially expressed genes  were involved in osmoregulation , inflammation and innate immune system , chaperones, antioxidant enzymes, catabolic enzymes , vitamin a and retinoic acid metabolism and scavenging and bone and cartilage metabolism .
to validate these results, a panel of  <dig> genes involved in different physiological functions were selected: ace <dig>  agt and nkcc <dig> in osmoregulation; tf and fth in iron metabolism, hsp90aa, a stress chaperone; try1a, try <dig>  ctr <dig>  cela <dig> and cela <dig> in digestion; c <dig> and lyg related to the innate immune system; tshb, a pituitary regulator of pituitary-thyroid axis; and taldo, an enzyme of the pentose phosphate pathway .
all the selected  <dig> degs selected in the microarray analysis were validated using qpcr.
a correlation analysis between average absolute fold-changes for all  <dig> genes showed a low pearson’s correlation coefficient  probably due to differences in fold-change between the two techniques used, but if c <dig>  was removed from the correlation analysis, the r value raised to  <dig> .
as a whole, these data indicate that the developed microarray was suitable to be applied in gene expression studies in s. senegalensis.
moreover, at least  <dig>  probes of the microarray provided repetitive and consistent positive hybridization signals.table  <dig>
validation of microarray data using qpcr
ace2
agt
nkcc2
tf
fth
hsp90aa
try1
try2
ctr2
cela1
cela4
c3
lyg
tshb
taldo
fold-changes  and p-values obtained for target genes by microarray and qpcr are indicated.
moreover, the transcript code in the soleadb for s. senegalensis v <dig> transcriptome is also shown.
for qpcr, data were normalized to those of gapdh <dig> and referred to the calibrator group .
CONCLUSIONS
de novo transcriptomes of s. solea and s. senegalensis covering their main developmental stages and organs were described based on a combined assembly approach that can be applied to other transcriptomic studies.
the huge volume of reads processed in each species  produced a high number of transcripts that were mined to obtain a representative reference transcriptome for each species.
the organization and deposit of all this information at soleadb offers the scientific community a new powerful resource for the management of genomic information in soles.
transcriptome comparisons and orthology analyses showed that both species are highly homologous and even contain transcripts with the same sequence.
moreover, comparisons across teleost transcriptomes allowed for the identification of some subsets of transcripts considered as new, species-specific and flatfish-specific transcripts.
transcriptome analysis followed by a phylogenetic analysis confirmed the retention of crystallins crybb <dig> and crybb <dig> confirming species-specific events during flatfish evolution.
in conclusion, this study not only provided functional information about soles, but also provides new tools to the scientific community in the form of a database, ssr and snp markers, and a new microarray with  <dig>  features in senegalese sole.

 the term “soles” refers to a wide group of flatfish species belonging to the soleidae and cynoglossidae families. common sole and senegalese sole are two economically important species highly appreciated worldwide due to the excellent quality of their flesh and heavily exploited in industrial fisheries. as a result, sole overfishing has had a profound effect on some life-history traits observing a shift towards earlier sexual maturation and a decline of the spawning biomass. therefore, aquaculture efforts have focused on s. senegalensis as one of the most promising species for diversification in southern europe due to its fast growth rates. however, solea aquaculture is facing several bottlenecks such as the production of high-quality larvae, the improvement and optimization of nutrition for better growth rates and the development of strategies for the control of infectious diseases. in addition, the failure to reproduce successfully soles of the f generation in captivity precludes the development of selection programs. moreover, soles are an excellent model to study development and metamorphosis in fish. soles undergo drastic morphological, physiological and ethological changes early during development for a short period of time. therefore, sole species have become a suitable model to study larval ontogeny, skin pigmentation, hormonal and epigenetic mechanisms controlling development, sex differentiation, nutritional requirements, asymmetrical development as well as comparative genomics in flatfish. in this context, development of large-scale genomic resources is a priority to facilitate the implementation of new technological approaches such as rna-seq and genome-wide mapping studies, that can assist in the identification of signalling networks controlling metamorphosis, growth, reproduction or disease resistance to advance in the knowledge of their biology and to improve rearing techniques and selective breeding. several studies have focused previously on the development of genomic resources in s. senegalensis and s. solea species. molecular markers, including microsatellites and single nucleotide polymorphisms, and bacterial artificial chromosomes libraries have been developed. being scarce, more polymorphic markers are required for population management and breeding programs. moreover, a limited set of expressed sequence tags has been described in each species that were used for the design of species-specific oligo-dna microarrays. consequently, the number of available ests is still far to conform a representative transcriptome as described in other teleosts and further sequencing efforts are required. next generation sequencing technologies have drastically transformed the way researchers can address genomic questions on non-model species, including soles. the ngs platforms are able to generate quickly an enormous bulk of cost-effective genomic information, even for those species with limited or no previous genomic information available. although ngs offers different applications, cdna/rna sequencing has become very popular for genome-wide transcriptome profiling and de novo sequencing of transcriptomes. the high-volume of transcriptomic reads generated constitute a rich and important source of potential molecular markers, including ssrs and snps, as well as for transcript processing, making possible the design and implementation of other techniques such as microarray hybridization or qpcr. in aquaculture, several studies have described the implementation of pyrosequencing for de novo transcriptome sequencing of some mediterranean species such as seabream, common sole or seabass. most of these studies have focused on the characterization of transcriptomes under specific developmental stages, pathogen challenge or tissue-specific profiles. nevertheless, the design of a dedicated species-specific database would be useful for easier management of genomic information and development of complementary techniques such as microarrays or rna-seq. the main aim of this work was the generation of a representative transcriptome for s. senegalensis and s. solea after processing an important large set of transcriptomic information produced by roche/ and illumina paired-end ngs technologies covering a large number of developmental stages associated to physiological challenges. the main transcriptome features and characteristics were identified. comparative analysis between soles allowed for the identification of orthologs, new genes and molecular markers. finally, a browseable database referred to as soleadb was constructed and a new microarray tool was designed and validated.  pre-processing and assembly of ngs data a total of and illumina libraries were prepared for s. senegalensis and s. solea, respectively, and additional roche/ sequences were prepared for s. senegalensis. most paired reads were useful after pre-processing. the most important source of reads removed were ribosomal and mitochondrial sequences. other contaminants less represented in the illumina data were reads from live preys and microalgae used in larvae feeding, rotifers and t-iso and some microorganisms, mainly fungi. assuming that the putative number of genes occurring in solea might range between and protein-coding genes, as reported in cynoglossus semilaevis and danio rerio, respectively, with an average transcript length of nt, the estimated transcriptome coverage of useful reads ranged from × to × for s. senegalensis, and from × to × for s. solea. therefore, the number of high quality reads was sufficient to assemble a reliable transcriptome in both species.table pre-processing summary of raw reads 1 s. senegalensis s. solea s. senegalensis 76 100 757 66 89 184 the s. senegalensis transcriptome was initially assembled using only the roche/ reads, following the workflow depicted in figure 1a. interestingly, the assembly resulted in a large number of transcripts longer than bp, slightly higher than expected for a teleost. the addition of illumina libraries to improve transcriptome assembly required the workflow depicted in figure 1b, where the long read strategy was slightly modified. the s. solea transcriptome was assembled using the short read strategy depicted in figure 1b, except that k-mers used were and due to longer raw reads. s. solea v and s. senegalensis v transcriptomes were comparable with respect to the frequency distribution of transcript length, the total number of transcripts and the number of transcripts longer than bp. however, mean length and n were clearly longer in s. solea, which may be explained by the longer input reads and a low relative contribution of roche/ reads in s. senegalensis v this low contribution may be explained by the fact that roche/ libraries were normalized to reduce highly abundant transcripts which might have led to more fragmented assemblies limiting the contribution of roche/ reads to the final transcriptome.figure schematic representation of the pre-processing, assembling and reconciliation approach to obtain the final transcriptome. a, strategy for a set of only long reads. b, strategy combining long and short reads. the numbers with “#” serve as a reference for table  description. representation of transcript abundance with respect of their lengths in the s. senegalensis and s. solea transcriptomes. previous reports that focused on de novo transcriptome assembly used a variable number of input reads. however, a total of 20– million reads were considered sufficient to generate a de novo transcriptome, even though assembly is highly influenced by factors such as species or type of sample. for example, the number of transcripts in six different marine invertebrates varied from to in spite of the fact that the number of input of reads was similar. moreover, more transcripts were discovered using whole-body samples. in this study, a joint analysis of the complete set of samples comprising a wide range of organs and developmental stages was performed to facilitate a maximal gene representation into the final transcriptome assembly. as a whole, more than million reads were assembled in each sole species, representing the highest number of reads reported to date for any organism. transcriptome characteristics the main characteristics of the assembled transcriptomes are depicted in table  a detailed analysis of the assembled transcriptomes reveals, first, that the number of artifacts was very low in spite of the extraordinarily large number of transcripts assembled. second, that the s. senegalensis v transcriptome was improved over the s. senegalensis v transcriptome in terms of transcript length and number of complete orfs, but both transcriptome versions presented a similar number of different orthologous ids, suggesting that the high number of juvenile tissues pooled to prepare the roche/ libraries could have importantly contributed to obtain a better gene representation. third, that the number of different complete orfs was high and similar between the transcriptomes of the two sole species, suggesting that sole transcriptomes are equally reliable. finally, that s. senegalensis v had a higher number of orthologous ids and a lower number of different complete orfs than s. solea v which may be due to a greater transcript fragmentation in s. senegalensis because roche/ data only contributed to increase the orthology in the final assembly.table overview of assembled transcriptomes in s. senegalensis and s. solea s. senegalensis s. solea artifacts2 transcripts with ortholog1 transcripts without ortholog1 reference transcriptome v: v: the values were calculated using fulllengthernext software. the minimum number of transcripts that can be considered as a reference transcriptome is shown in bold. 1percentages for subclassifications of this category were calculated using this line as 100% reference. 2artifacts refer mainly to misassemblies and chimeric contigs. nc: non-calculated. the extremely large number of reads used to assemble the two sole transcriptomes could favour the accumulation of assembly errors. evaluation of transcript accuracy was initially based on mapping useful reads of two randomly selected libraries of each sole transcriptome using bowtie. since - % of the reads mapped onto assembled transcripts, the assembly errors can be considered negligible. interestingly, the longest transcript in s. senegalensis v and s. solea v transcriptomes is clearly not an artifact: in both species, it corresponded to a titin-like protein highly similar to a long mrna previously assembled in tilapia. titin is a giant filamentous protein highly abundant in muscle that forms a separate myofilament system in both skeletal and cardiac muscle. the fact that this transcript is 6-fold longer in s. senegalensis v than in s. senegalensis v supports the significant contribution of illumina short-reads to the final assembly. soleadb, a database for browsing soleatranscriptomes genomic databases are extremely useful for target sequence retrieval, blast comparisons, sequence management and compilation of all information that can help to a better understanding of the function and roles of genes. a preliminary database devoted to host sanger est information, microarray data and ish pictures was developed for s. senegalensis. nevertheless, this database was not suitable to manage the important volume of information generated using ngs. hence, a new database, soleadb, was built to host all the information for s. senegalensis and s. solea transcriptomes, following the same architecture as reported for europinedb and sustainpine. soleadb was structured and designed in a user-friendly manner showing all information regarding experimental conditions, ngs libraries characteristics and processing pipelines to clean, assemble and annotate the transcriptomes. navigation is very intuitive with information structured by assemblies including global assemblies and by experimental conditions. a search tool to find specific transcript information by different fields and a blast tool was also incorporated.in the “assemblies” tab, different transcriptome versions can be browsed. for each assembly, there is a brief description of experimental conditions as well as assembly, markers and annotation statistics. raw sequencing reads, pre-processing reports, fasta and ace files, as well as annotations for functional analysis can be downloaded from the same tab. in the “unigenes” section, users can search for specific transcripts and browse their specific information. information available for each transcript includes the consensus sequence, the corresponding contig, functional annotations, full-length status, orf prediction, and putative markers. by means of kegg and ec annotations, genes in specific pathways can also be retrieved. all annotations incorporated in soleadb are freely downloadable for the scientific community.figure screen captures of soleadb interface. a, illustration of the “assemblies” tab containing all information about all transcriptome versions and subversions. b, capture of the part of the screen corresponding to the “assembly info” tab where general information about the transcriptome as well as downloadable files and other useful tools can be found. therefore, soleadb can be extremely useful for data comparison across experiments allowing for the identification of paralogues, alternative spliced transcripts and novel genes. it represents a new, easy-to-use, valuable tool to host ngs data and for sharing genomic information between users applying these techniques. moreover, whole and reference transcriptomes will be a useful tool for downstream applications such as rna-seq. examples of usefulness of soleadb can be found in the next sections. transcripts without ortholog as a source of putative new sole transcripts the high number of transcripts without ortholog deserved a deeper analysis. based on their low testcode index, >91% of the unknown transcripts did not encode for proteins, which could explain in part the lack of orthology. to check the accuracy of these unknown transcripts, genome reads were mapped onto the unknown transcripts of s. senegalensis v resulting in unknown transcripts mapped. these high mapping percentages indicate that these sequences were not assembly artifacts and that they might correspond to co-purified genomic fragments or immature transcripts. among the % and % of transcripts without ortholog in s. senegalensis v and s. solea v respectively, some showed a testcode index > and, therefore, are likely coding transcripts. those transcripts referred to as non-redundant new transcripts, based on the absence of orthologs in uniprotkb database, may represent “new” proteins in sole. a reference transcriptome for each sole species the high number of assembled transcripts indicated an overestimation of sole transcriptomes when compared to other teleosts. probably, a certain number of transcripts could actually represent alleles, paralogs, fragmented transcripts, spliced forms, immature mrnas and even a combination of them. therefore, identification of representative transcripts from these transcriptomes would be a useful tool to be used as a reference for future gene expression studies. for this purpose, representative transcripts were selected from the longest transcripts with unique, different orthologous id and the putative, non-redundant new transcripts. hence, the reference transcriptome for s. senegalensis consisted of transcripts and the s. solea reference transcriptome contained transcripts. when useful reads were mapped onto these reference transcriptomes, - % of reads were mapped onto transcripts, while - % of transcripts received more than one read, suggesting that they represent adequately the transcriptome. additional verification of the reference transcriptomes was based on an orthology analysis using zebrafish. in s. senegalensis v, reference transcripts were found to be orthologs to refseq and to ensembl zebrafish entries, and in s. solea v reference transcripts were found to be orthologs to refseq and to ensembl zebrafish entries. these numbers suggest that a certain number of alleles, immature mrnas and lineage-specific genes may have been included in the reference transcriptome. moreover, since the number of refseq and ensembl ids nearly corresponds to half the number of sole transcripts, it is likely that both alleles for each gene were included in the sole reference transcriptomes. this hypothesis is also supported by the fact that most of the samples analyzed corresponded to wild animals or larvae, being mostly heterozygous. it is worth noting that the number of different zebrafish ids in refseq or ensembl is close to the ~ genes recently reported for half-smooth tongue sole and not so different from the genes that have been recently reported in zebrafish. therefore, it can be suggested that most sole genes have been covered in the sole reference transcriptomes. s. soleaand s. senegalensisshow clear functional similarity, are highly orthologous, and contain sole- and flatfish-specific transcripts s. solea and s. senegalensis are two closely-related species with similar morphology, ecology and feeding habits and preys. in silico genome comparisons previously performed among fish species to identify orthologous gene groups identified a high percentage of shared genes and only a small number of species-specific gene families. therefore, a comparative analysis of sole transcriptomes could reveal new clues about their biology and evolution and also can provide supporting evidence of reference transcriptome accuracies. the % and % of annotation success in the s. senegalensis v and s. solea v transcriptomes, respectively, is in concordance with the analysis of full-lengthernext. comparison of go terms between the two sole species revealed that they were similar. the highest number of annotated transcripts by biological process was associated with metabolic and cellular processes. by cellular components, the most represented categories were cell and organelle. by molecular function, the highest number of annotated transcripts was within the catalytic activity category. interestingly, the channel regulator and antioxidant activity categories were only represented in s. senegalensis. in conclusion, both sole transcriptomes appear to be very similar from a biological and functional point of view. another comparison of sole transcriptomes was based on orthology with zebrafish. figure  shows that % of orthologous transcripts in soles with an identified ortholog in zebrafish had a similarity ≥95% at the nucleotide level with transcripts being 100% identical. as expected, transcripts without zebrafish orthology had a lesser degree of identity, as evidenced by the plateau at 92-96% identity. interestingly, transcripts without ortholog had the same sequence and transcripts showed a 99% identity between the two sole species. all these data evidenced a high level of similarity between the two sole transcriptomes. assuming that sole transcripts that share the same zebrafish ortholog could also be considered sole orthologs, a total of reference transcripts in s. senegalensis and reference transcripts in s. solea shared ids for refseq and ids for ensembl, which clearly reflects the high level of orthology between these two sole species.figure distribution of the level of similarity between both sole reference transcriptomes for those transcripts with or without a zebrafish ortholog. true orthologs between both sole species were obtained after performing a reciprocal blast using the reference transcriptomes. in this analysis, two transcripts were considered as blast-based, true orthologs when a highly restrictive reciprocal blast always resulted with their sequences giving the best score and e values. a total of reference transcripts of s. senegalensis were highly homologous to reference transcripts of s. solea, and reference transcripts of s. solea were highly homologous to reference transcripts of s. senegalensis. of these homologous sequences, only could be considered as true, blast-based orthologs. these include unannotated transcripts with an average length of nt in s. senegalensis and nt in s. solea. more interestingly, of them had a testcode ≥, indicating that they likely code for a specialized protein. to confirm this hypothesis, they were compared to other fish proteins and only failed to find any orthology. moreover, transcripts showed a clear ortholog only in the flatfish c. semilaevis, suggesting that these they might represent flatfish-specific transcripts. in conclusion, transcripts were identified as true, blast-based orthologs between both sole species, from which are likely to be flatfish-specific transcripts and are putative new sole transcripts. transcriptome comparison across teleosts revealed a set of lineage-specific genes the sole blast-based orthologs with annotation were investigated based on their refseq and/or ensembl ortholog for zebrafish. as shown in figure  most blast-based orthologs had a zebrafish ortholog. however, the most interesting finding is the small subset of sole orthologs lacking zebrafish similarity. some of these transcripts without zebrafish ortholog were related to the immune system such as hepcidin antimicrobial peptides and some interleukins. hepcidins appear as a highly diversified family in acanthopterygians that favoured the radiation of teleosts in marine and brackish environments. similarly, il11b duplication appeared later during evolution not occurring in zebrafish. these data suggest that this subset of sole orthologs without zebrafish orthology might represent lineage-specific genes that have appeared, subfunctionalized or neofunctionalized later during teleost evolution. to check the presence of these transcripts in other teleosts, proteins deduced from reference transcripts were compared, observing that most of them were also present in the c. semilaevis genome. only transcripts lacked any orthology in the teleosts analyzed confirming that this collection of transcripts could correspond to genes acquired or fixed during fish evolution.figure venn’s diagrams reflecting coincidences by solea species among sole, blast-based orthologs and transcripts with refseq/ensembl ortholog for zebrafish. diagrams are comparing the blast-based orthologs with the unique zebrafish refseq identifiers in soleadb for s. senegalensis and s. solea and with the unique zebrafish ensembl identifiers in soleadb for s. senegalensis and s. solea. within the venn’s diagrams, the numbers refer to the amount of transcripts in soleadb for s. senegalensis and s. solea, the number of transcript in soleadb with a zebrafish refseq identifier of with a zebrafish ensembl identifier. sole transcriptomes confirmed the retention of crystallin genes recently, it has been suggested that the visual system had evolved in relation to their benthic way of life. this observation is based on the loss of genes related with vision such as crystallins crybb and crybb in c. semilaevis. five crybb orthologs have been identified in s. senegalensis and s. solea transcriptomes that grouped into crybb and crybb clusters and none to the crybb clade. moreover, additional crybb-like transcripts could be grouped into two crybb1-related clusters that seem to be fish-specific sequences. extension of the analysis to closely related crybb-like sequences revealed that additional crybb sequences exist in all three flatfish c. semilaevis, s. solea and s. senegalensis. moreover, est sequence analysis from atlantic halibut suggested that this flatfish also possesses crybb crybb and several crybb1-like sequences. taken together these results suggest that flatfish have indeed lost and retained specifically some crybb genes likely as a consequence of independent events indicative of divergent evolution and do not support a decay of the visual system as previously hypothesized in flatfish based on the set of crystallin-encoding genes.figure phylogenetic tree of crybb and crybb-like proteins in vertebrates. a neighbor-joining tree based on the alignment of vertebrates crybb and crybb-like sequences was built. species are indicated as sse, sso dre, tni, oni, ola, cse, xla and gga. solea sequences are indicated according to the transcript name assigned in soleadb. clusters are indicated as arcs of a circle. the tree obtained was rooted using xenopus laevis cryga. numbers adjacent to nodes indicate percentage bootstrap support; only values larger than 70% are shown. sole transcriptomes as a source of molecular markers a transcriptome represents an important source of molecular markers, mainly ssrs and snps. a total of ssr markers in senegalese sole derived from ests in public databases were applied to evaluate ssr evolution in flatfish species. sole transcriptomes described here will surely add more molecular markers suitable for a wide range of genetic applications. ssrs were determined in the whole sole transcriptomes, the reference transcriptomes and the set of sole blast-based orthologs. it is noteworthy that in whole transcriptomes, dinucleotide repeats were the most abundant ssrs followed by tri-nucleotide repeats and tetra-nucleotide repeats. nevertheless, the reference transcriptome was enriched in ssrs bearing a tri-nucleotide motif. this difference in motif abundance can be explained by the selective constraints imposed by protein-coding dna, more abundant in the latter transcriptome. in fact, di-nucleotide and tetra-nucleotide ssrs were mainly located in the utr, whereas tri-nucleotides were in the orf. these results agree with genome-wide analyses that identified a bias distribution of tri- and hexa-nucleotide repeats in protein-coding exons of vertebrate, invertebrates, plants and fungi. the most common ssr motifs were ac/gt for di-nucleotides, agg/cct in s. senegalensis and agc/gct in s. solea for tri-nucleotides, and aaac/gttt for tetra-nucleotides. similar percentages were estimated for the reference transcriptome except for the fact that agc/gct was more abundant than agg/cct. fish analysis using gt, gtta and gata probes confirmed the relative abundance of these ssrs on the genome. the ac/gt motif has been reported as the most frequent ssr repeat in the intergenic regions and introns of vertebrates. the ac/tg motif has been identified as the most frequent ssr in roche/ assembled sequences of s. solea. also, g + c-rich tri-nucleotides and tetra-nucleotides with <50% of g + c are characteristic of vertebrates. it is worth mentioning the low representation of gata repeats confirmed by fish analysis. comparison of ssrs blast-based orthologs in soles identified sole-conserved ssrs in s. senegalensis and in s. solea, out of which and ssrs in s. senegalensis and s. solea, respectively, can be considered species-specific as they were only found in the orthologs of only one species. this analysis provides new and very useful ssr markers for development of multiplex assays, genetic mapping and deciphering genome evolution in flatfish as well as species identification in processed fish.table ssr summary statistics for whole and reference transcriptomes s. senegalensis s. solea species-specific ssr1   same repeat motif2 total number of ssrs and frequency according to their repeat motif are indicated. ssrs present in one species but not in orthologs of the other species. exactly the same ssr repeat motif was found in both orthologs; in a few cases, ssr occurs once in one ortholog and twice in the other. a total of snps were identified in s. senegalensis and in s. solea transcriptomes. a significant proportion of snps occurred in transcripts containing an orf with approximately half occurring within the orf. these figures for snp location in coding regions are similar to those found in other fish species ranging from to %. although snp prediction is only based on bioinformatic analysis and requires empirical validation to eliminate false positives and sequencing errors, these snps can also be used as a source of putative molecular markers. design and validation of an oligonucleotide microarray for s. senegalensis microarrays have become a cost-effective technique for gene expression profiling and whole-transcriptome studies. two species-specific oligo-dna microarrays have been reported in s. senegalensis and s. solea containing a limited number of unique transcripts due to the limited number of ests available in soles. this limitation was compensated to some extent using heterologous microarrays. the sole transcriptomes described in this study have overcome these restrictions. the strategy to select sole-specific probes is depicted in figure  briefly, the complete non-redundant transcripts were added to the longest, non-redundant, incomplete transcripts. clustering them resulted in longest, non-redundant transcripts that were combined with selected “coding” transcripts, providing a total of probes. the final panel of probes included genes related to reproduction, cell differentiation, response to stress, growth, biosynthetic and catabolic processes, transport, embryonic development and immune system, among other functions.figure schematic representation of the probe selection strategy for the construction of the senegalese sole oligonucleotide microarray. the number of transcripts that resulted after the described filtration is indicated. the microarray was tested with larvae incubated at two salinities. hybridization signal was detected for probes. a total of probes were found differentially expressed of which were up-regulated and down-regulated at ppt compared to ppt. application of a cut-off value of log  > ± filtered up-regulated and down-regulated probes. the differentially expressed genes were involved in osmoregulation, inflammation and innate immune system, chaperones, antioxidant enzymes, catabolic enzymes, vitamin a and retinoic acid metabolism and scavenging and bone and cartilage metabolism. to validate these results, a panel of genes involved in different physiological functions were selected: ace agt and nkcc in osmoregulation; tf and fth in iron metabolism, hsp90aa, a stress chaperone; try1a, try ctr cela and cela in digestion; c and lyg related to the innate immune system; tshb, a pituitary regulator of pituitary-thyroid axis; and taldo, an enzyme of the pentose phosphate pathway. all the selected degs selected in the microarray analysis were validated using qpcr. a correlation analysis between average absolute fold-changes for all genes showed a low pearson’s correlation coefficient probably due to differences in fold-change between the two techniques used, but if c was removed from the correlation analysis, the r value raised to. as a whole, these data indicate that the developed microarray was suitable to be applied in gene expression studies in s. senegalensis. moreover, at least probes of the microarray provided repetitive and consistent positive hybridization signals.table validation of microarray data using qpcr ace2 agt nkcc2 tf fth hsp90aa try1 try2 ctr2 cela1 cela4 c3 lyg tshb taldo fold-changes and p-values obtained for target genes by microarray and qpcr are indicated. moreover, the transcript code in the soleadb for s. senegalensis v transcriptome is also shown. for qpcr, data were normalized to those of gapdh and referred to the calibrator group.  de novo transcriptomes of s. solea and s. senegalensis covering their main developmental stages and organs were described based on a combined assembly approach that can be applied to other transcriptomic studies. the huge volume of reads processed in each species produced a high number of transcripts that were mined to obtain a representative reference transcriptome for each species. the organization and deposit of all this information at soleadb offers the scientific community a new powerful resource for the management of genomic information in soles. transcriptome comparisons and orthology analyses showed that both species are highly homologous and even contain transcripts with the same sequence. moreover, comparisons across teleost transcriptomes allowed for the identification of some subsets of transcripts considered as new, species-specific and flatfish-specific transcripts. transcriptome analysis followed by a phylogenetic analysis confirmed the retention of crystallins crybb and crybb confirming species-specific events during flatfish evolution. in conclusion, this study not only provided functional information about soles, but also provides new tools to the scientific community in the form of a database, ssr and snp markers, and a new microarray with features in senegalese sole. 
a reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. a comprehensive characterization of the transcriptome for each species was carried out using a large set of illumina data and reads, providing coverages ranging from x to x. after a de novo assembly, and different transcripts were obtained, comprising and full-length cdnas in s. senegalensis and s. solea, respectively. although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. senegalese sole and common sole are two economically and evolutionary important flatfish species both in fisheries and aquaculture. a small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. moreover, the integration of transcriptomic data in the soleadb will facilitate the management of genomic information in these important species. moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution. the design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. transcriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. transcriptome information was applied to the design of a microarray tool in s. senegalensis that was successfully tested and validated by qpcr. furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by fish to be used in further genetic and expression studies.
 senegalese sole and common sole are two economically and evolutionary important flatfish species both in fisheries and aquaculture. although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution.  a comprehensive characterization of the transcriptome for each species was carried out using a large set of illumina data and reads, providing coverages ranging from x to x. after a de novo assembly, and different transcripts were obtained, comprising and full-length cdnas in s. senegalensis and s. solea, respectively. a reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. a subset of reference transcripts was qualified as highly reliable orthologs between both species. a small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by fish to be used in further genetic and expression studies. moreover, evidences on the retention of crystallins crybb crybb1-like and crybb in the two species of soles are also presented. transcriptome information was applied to the design of a microarray tool in s. senegalensis that was successfully tested and validated by qpcr. finally, transcriptomic data were hosted and structured at soleadb.  transcriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. the design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. moreover, the integration of transcriptomic data in the soleadb will facilitate the management of genomic information in these important species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords solestranscriptomemicroarrayorthologymolecular markerssoleadbissue-copyright-statement© the author 2014 