BACKGROUND
zoarces viviparus, or common eelpout, lives in the shallow waters along the coasts in northern europe  <cit> .
in contrast to many other fish species living in these regions, the eelpout has a number of distinct characteristics which makes this fish particularly interesting for ecological and ecotoxicological studies.
the relatively stationary behavior makes it possible to correlate physiological changes to the local exposure situation in the area where the fish were sampled  <cit> .
this makes the eelpout a good candidate for environmental monitoring since it is possible to associate adverse effect to specific pollutants or pollution sources.
another beneficial characteristic is the internal fertilization where the females, after several months of pregnancy, give birth to relatively well-developed young.
the possibility to link the mother with her offspring makes the eelpout suited for investigations of reproductive success, both in general and under the exposure of pollutants  <cit> .
the value of the eelpout as an important species for coastal environmental monitoring in the field has been demonstrated in a number of studies.
exposure to effluent from scandinavia's largest pulp mill results in skewed sex ratio of eelpout embryos  <cit> .
excessive proportions of malformed and/or dead eelpout embryos have revealed the impact of environmental pollution at several locations along the coast of the baltic sea  <cit> .
climate-induced temperature shifts have also been linked to reduced growth of eelpout  <cit> .
as a species high on the food web of the marine ecosystems, the eelpout is known to bioaccumulate toxic substances such as heavy metals  <cit>  and the polycyclic aromatic hydrocarbons  <cit> .
z. viviparus is therefore a prime target for the german environmental specimen bank , who monitors and identifies local and regional trends of the aquatic ecosystems along the german coasts.
genomic-based techniques are today important tools for investigating molecular level effects caused by exposure of toxicants and other substances.
large-scale measurements of gene expression using dna microarrays have become particular popular since they provide both quantitative data for individual biomarkers  <cit>  as well as a genome-wide snapshot of the current state of the cells  <cit> .
as a consequence, groups of genes and entire pathways can be associated with different types of toxicity  <cit> , which leads to both improved ability to detect exposures and an increased knowledge of the mechanisms of action and subsequent adverse effects  <cit> .
however, microarray-based gene expression measurements require a substantial amount of knowledge about the targeted species' transcriptome, and these valuable tools are therefore unavailable for many ecologically important species.
during the last years there has been a remarkable progress in dna sequencing technology with several novel solutions on the market  <cit> .
the performance has increased multiple orders of magnitude and millions of bases can today be sequenced in a matter of hours  <cit> .
within the fields of ecology and ecotoxicology, this development is particularly welcome due to the large number of model organisms, of which few have a well-characterized genome.
the new fast and cost-efficient methods for dna sequencing hold therefore great promise to change the current situation and pave the way for modern genomic tools such as gene expression microarrays  <cit>  and genome-wide association population studies  <cit> .
for transcriptome sequencing, the superior performance of high-throughput techniques have already been demonstrated in several studies, including the plant medicago truncatula  <cit> , the butterfly melitaea cinxia  <cit> , the tree eucalyptus grandis  <cit>  and the freshwater fish micropterus salmoides  <cit> .
the novel sequencing techniques also comes with a number of weaknesses where the limited sequence length is most pronounced  <cit> .
among the currently available techniques, the flx pyrosequencer from  <dig> life science/roche generates the longest reads, currently 250– <dig> bases depending on the protocol, and is therefore most well-suited for de novo transcriptome sequencing  <cit> .
even though hundreds of thousands reads are generated simultaneous, the limited length will severely restrain the assembly of full length transcripts  <cit> .
in this study we present the first comprehensive characterization of the z. viviparus liver transcriptome using massively parallel pyrosequencing.
based on data corresponding to one single run on the flx gene sequencer from  <dig> life science, almost  <dig> million bases were assembled into ~ <dig>  pieces of putative transcripts and the majority of these have been annotated and functionally classified.
furthermore, an oligonucleotide microarray for large-scale gene expression measurement in z. viviparus has been designed.
our results, including the sequence data and the validated microarray probes are publicly available and will provide means for future genome-wide research in z. viviparus.
RESULTS
sequence analysis and quality assessment
the polymer chain reaction  amplification adapters used in the sequencing process were first identified and removed; the start tag was found for 99% of the reads while the end tag only was found for 42%, indicating that the majority of the reads have reached their maximum length and less than half stopped prematurely.
a substantial part of the genome of higher eukaryotes consists of stretches with low-complexity such as the short and long interspaced repetitive elements  and other transposons  <cit>  and these regions makes gene assembly treacherous since reads can be conjoined because they share a similar repetitive region.
this is also true for the poly-a-tail, the a/t rich region that is attached to the 3' end of the mrna molecule after transcription.
the entire dataset was therefore screened for low-complexity regions and poly-a tails.
as expected, the number of identified poly-a tails was low  compared to traditional cloned est library due to the uniform distribution of read positions exhibited by parallel pyrosequencing  <cit> .
in total,  <dig>  million bases were classified as low-complex, and were either substituted for ambiguity bases or removed whether their location was intermediate or in the ends.
assembly of transcripts
to assemble the reads into transcripts, we applied the tgi clustering tools , which is a set of utilities originally developed for assembly of data from cloned est libraries.
the assembly resulted in  <dig>  contiguous sequences , while  <dig>  reads could not be matched against any other , resulting in  <dig>  putative eelpout transcripts consisting of more than  <dig> million bases in total .
the average length of the contigs was ~ <dig> base pairs, with more than  <dig>  contigs with a length over  <dig> base pairs .
a using a blast e-value cut-off of 10-5
b gasterosteus aculeatus, tetraodon nigroviridis, takifugu rubripes, oryzias latipes and danio rerio.
the number of reads per contig ranged up to ~ <dig>  with an average of eight reads  and, even though the cdna library was normalized prior to sequencing, the distribution was highly asymmetric and right-tailed.
the coverage, i.e.
the average number of supporting reads per base pair, was estimated to  <dig>  times .
with the low error rate of massively parallel pyrosequencing in mind  <cit> , these results suggest that the assembly should be of high quality with very few technical errors.
annotation
the assembled transcripts were annotated against swiss-prot/uniprotkb  <cit> , a manually curated database containing ~ <dig>  known protein from more than  <dig>  different species.
using a modest e-value cut-off of 10- <dig>  73% of the contigs and 70% of the singlets had a homologous hit, resulting in  <dig>  annotated eelpout transcripts.
analogously, 89% of the contigs and 87% of the singles had a hit in at least one of the five available fish genomes.
a closer examination of the annotations revealed several genes that are of particular interest for environmental monitoring and ecological research .
several proteins within the cytochrome p <dig> protein superfamily catalyzes biotransformation of many xenobiotics and therefore contains a number of pivotal biomarkers for a large number of different compounds.
among the sequenced z. viviparus transcripts,  <dig> different cytochrome p <dig> variants from  <dig> different families could be identified .
other interesting classes of proteins, such as heat shock proteins  and genes related to oxidative stress  were also present, as well as several other common biomarkers, such as vitellogenin, the zona pellucida and metalothionein proteins.
alength of longest contiguous fragment
comparative genomics
among the five fish species with sequence genome, gasterosteus aculeatus  is evolutionarily closest to z. viviparus and the genome encompasses  <dig>  protein coding genes which have a total size of  <dig> million base pairs.
assuming similar numbers in eelpout, the assembled transcripts would cover roughly 40% of the transcriptome and each gene would on average be represented by more than two contigs or singlets.
the gc-content differed between the two species, with g. aculeatus at 55% while z. viviparus had 48% for the contigs but only 43% for the singlets.
the  <dig>  putative z. viviparus transcripts matched  <dig>  unique genes in g. aculeatus and among these, 72% corresponded to hits from contigs and 3% from singlets while more than 25% had hits from both contigs and singlets .
hence, most singlet are therefore from genes already represented by a least one contig and very few genes are represented only as singlets.
even though the number of unique genes decreased slightly, the conclusions were consistent for the other four fish species with sequenced genome .
not surprisingly, the number of human genes with a match from z. viviparus transcripts was lower than all the fish but slightly higher than the frog  .
to investigate the presence of novel previously undescribed genes, all contigs and singlets were aligned against the five available fish genomes.
in total  <dig>  of the transcripts could be found in at least three of the five fish species with sequenced genome, and among those,  <dig>  aligned in regions without previous annotation.
not surprisingly, only  <dig>  of these could not be annotated against uniprot, which is lower than the transcripts aligning in annotated regions .
the transcripts in the regions without annotation had a slightly higher coverage  and a similar number of reads per transcript .
more interestingly, these unknown transcripts aligned in regions with a higher evolutionary conservation  compared to transcripts aligning in annotated regions .
microarray analysis
a 50-mer oligonucleotide microarray was designed for large-scale gene expression analysis in z. viviparus.
to measure the expression of the probes and the corresponding assembled transcripts, the pool of mrna used for the sequencing was hybridized.
for  <dig> transcripts with reliable annotation , the gene expression measurement according to the probes in the annotated direction was highly correlated to the number of reads produced by the sequencing .
thus, the data from the gene expression microarray is consistent with the massively parallel pyrosequencing reads which validates both the assembled transcripts as well as the corresponding probe .
note however, that when additional transcripts with less reliable annotation are included the correlation dropped .
data availability
the raw sequence data from the massively parallel pyrosequencing is available at ncbi short read archive , the assembled transcripts and the generated probes are available at .
the data from the gene expression microarray are available as accession number gse <dig> at gene expression omnibus  <cit> .
discussion
we have used massively parallel pyrosequencing to characterize the transcriptome of zoarces viviparus.
however, sequencing and assembly of a eukaryotic transcriptome without any genomic data a priori is a notoriously difficult undertaking  <cit> .
based on  <dig>  reads we assembled ~ <dig>  putative transcripts, a strikingly high number compared to amount of genes found in the five sequenced fish genomes .
one obvious reason for the high number of transcripts is the short length of the reads, which may result in several assembled contigs and singlets for each gene.
another important factor is the process of alternative splicing, where one single gene can result in several transcripts by combining and removing different parts .
this mechanism is common in higher eukaryotes  <cit> , including in fish  <cit> , and the number of expressed transcripts is therefore substantially higher than the number of genes in the genome.
another issue is the limited sequence length exhibited by massively parallel pyrosequencing.
even though the length is substantially longer than other high-throughput sequencing techniques, such as solid and illumina sequencing  <cit> , the difference compared to sanger-based sequencing is notable.
in this study, we used a flx gene sequencer, which generates  <dig> bases long reads, which is only a fourth of the  <dig> bases long sequences which can be produced by sanger sequencing  <cit> .
the short sequence reads makes the assembly more difficult and thus the resulting contigs shorter.
however, the average contig length  from our assembly is slightly longer compared to previous studies  <cit>  and it is likely that this difference is due to the single tissue targeted in this study, but also due to the advance processing pipeline where genomic uninteresting regions, such as repeats, are removed.
for transcriptome sequencing, massively parallel pyrosequencing exhibits a high sensitivity compared to traditional sanger-based approaches  <cit> .
the large number of generated reads allows detection of low-abundant transcripts and the cdna cloning step where mrna molecules are inserted into bacterial vectors is no longer required  <cit> .
transcripts that previously have been hard to sequence can therefore be detected and previous studies have reported a considerable amount of reads aligning in genomic regions previously not annotated  <cit> .
among our assembled transcripts, ~ <dig>  could be aligned to at least three out of five available fish genomes and among these 3% matched regions without any prior annotation.
the expression levels for these unknown transcripts were at least as high as those aligning in annotated regions and they aligned in regions with evolutionary conservation between distant species.
hence, these transcripts are likely to represent novel transcribed elements such as not yet described fish-specific genes.
massively parallel pyrosequencing can currently only process dna stretches of limited length.
thus, a fragmentation step is applied and as a consequence the reading direction, traditionally indicated by poly-a tail primers, is lost.
hence, the assembly pipeline therefore needs to be flexible and build contigs based on reads from both directions.
nevertheless, the direction of transcription will still be unknown for the resulting contigs.
for blast-based sequence alignments, the knowledge of the direction of transcription is not necessary since the comparison can easily be done for both strands.
however, when constructing gene expression microarrays, the direction of transcription is crucial for proper probe design, both for placing the probe on the correct strand and for evaluating probe specificity.
unfortunately, estimating the direction of transcription is intricate for short stretches of mrna, especially for sequences without a satisfactory annotation.
even though we complemented our blast-based annotations with estimates from framefinder, an application used to identify open reading frames, a substantial part of our transcripts lacks reliable estimates of their direction of transcriptions.
these transcripts may therefore be represented by probes in both directions in future gene expression assays, and thereby circumvent the problem at the cost of space on the microarray.
it is finally worth to mention that the gene expression levels on our evaluation microarray are not good predictors of the direction of transcription.
only ~60% of the well-annotated transcripts have probes that had a higher expression levels in the correct direction compared to the erroneous direction .
this result is, however, far from surprising since the probes for the two directions will have different thermodynamic properties and specificity and therefore different intensity levels  <cit> .
the correlation between the developed eelpout microarray and the number of reads from the massively parallel pyrosequencing was estimated to 62%.
this number is strikingly high considering both the normalization of the cdna library prior sequencing and the non-linearity typically exhibited by microarrays.
previous studies comparing hybridization- and high-throughput sequencing-based gene expression measurement techniques have reported a correlation between 46%–72% .
in contrast to these studies, which use established previously evaluated microarrays from well-sequenced species, the eelpout microarray is designed from scratch based on less than half of the transcriptome.
thus, the correlation shows that that there is a good concordance between the developed eelpout microarray and the transcriptome sequence data, which validates both the assembled transcripts and the corresponding oligonucleotide probes.
the present study demonstrates the relative ease by which de novo transcriptome sequencing can be done today.
the performance of the modern high-throughput sequencers is high enough that one single run is enough to cover a substantial part of the trancsriptome of a higher eukaryote.
as a consequence, the assembled contigs becomes long enough to design reliable microarray probes for large-scale gene expression analysis.
this development will undoubtedly spur genome-based research in many biological disciplines, especially in fields where a wide range of wild-life species are studied  <cit> .
one prime example is ecology, where tools such as gene expression assays are needed for efficiently study the low-level mechanistic of ecosystems while other related techniques such as single nucleotide polymorphism  analysis can be used to unravel parts of the complex interplay between genetic variation and fitness in wild populations  <cit> .
the zoarces viviparus microarray developed in this study will constitute a valuable resource for marine environmental research in the northern europe.
the platform will allow correlation of molecular-level responses, caused by exposure to various toxicants, to the unique morphological and physiological characters of z. viviparus.
in particular, the reproductive output in the form of size, number and quality of the embryos, can be related to gene expression changes at an individual basis.
the stationary behavior of this fish species in combination with the sensitivity of gene expression biomarkers will form a unique instrument for accurate and robust monitoring of the aquatic ecosystems.
the generated sequence data for the z. viviparus transcriptome will also form a base to study molecular and physiological adaptations to viviparity in fish.
CONCLUSIONS
the present study describes the first comprehensive sequencing effort of zoarces viviparus, an important model organism for marine ecology research and environmental monitoring.
the sequence data was generated by massively parallel pyrosequencing, a high-throughput technique with a hundred-fold higher performance than traditional capillary sequencing.
the sequenced nucleotides were estimated to cover roughly 40% of the transcriptome and more than  <dig>  pieces of putative transcripts were assembled.
finally, an oligonucleotide microarray were constructed and used to evaluate expression of our assembled transcripts.

 zoarces viviparus, or common eelpout, lives in the shallow waters along the coasts in northern europe. in contrast to many other fish species living in these regions, the eelpout has a number of distinct characteristics which makes this fish particularly interesting for ecological and ecotoxicological studies. the relatively stationary behavior makes it possible to correlate physiological changes to the local exposure situation in the area where the fish were sampled. this makes the eelpout a good candidate for environmental monitoring since it is possible to associate adverse effect to specific pollutants or pollution sources. another beneficial characteristic is the internal fertilization where the females, after several months of pregnancy, give birth to relatively well-developed young. the possibility to link the mother with her offspring makes the eelpout suited for investigations of reproductive success, both in general and under the exposure of pollutants. the value of the eelpout as an important species for coastal environmental monitoring in the field has been demonstrated in a number of studies. exposure to effluent from scandinavia's largest pulp mill results in skewed sex ratio of eelpout embryos. excessive proportions of malformed and/or dead eelpout embryos have revealed the impact of environmental pollution at several locations along the coast of the baltic sea. climate-induced temperature shifts have also been linked to reduced growth of eelpout. as a species high on the food web of the marine ecosystems, the eelpout is known to bioaccumulate toxic substances such as heavy metals and the polycyclic aromatic hydrocarbons. z. viviparus is therefore a prime target for the german environmental specimen bank, who monitors and identifies local and regional trends of the aquatic ecosystems along the german coasts. genomic-based techniques are today important tools for investigating molecular level effects caused by exposure of toxicants and other substances. large-scale measurements of gene expression using dna microarrays have become particular popular since they provide both quantitative data for individual biomarkers as well as a genome-wide snapshot of the current state of the cells. as a consequence, groups of genes and entire pathways can be associated with different types of toxicity, which leads to both improved ability to detect exposures and an increased knowledge of the mechanisms of action and subsequent adverse effects. however, microarray-based gene expression measurements require a substantial amount of knowledge about the targeted species' transcriptome, and these valuable tools are therefore unavailable for many ecologically important species. during the last years there has been a remarkable progress in dna sequencing technology with several novel solutions on the market. the performance has increased multiple orders of magnitude and millions of bases can today be sequenced in a matter of hours. within the fields of ecology and ecotoxicology, this development is particularly welcome due to the large number of model organisms, of which few have a well-characterized genome. the new fast and cost-efficient methods for dna sequencing hold therefore great promise to change the current situation and pave the way for modern genomic tools such as gene expression microarrays and genome-wide association population studies. for transcriptome sequencing, the superior performance of high-throughput techniques have already been demonstrated in several studies, including the plant medicago truncatula, the butterfly melitaea cinxia, the tree eucalyptus grandis and the freshwater fish micropterus salmoides. the novel sequencing techniques also comes with a number of weaknesses where the limited sequence length is most pronounced. among the currently available techniques, the flx pyrosequencer from life science/roche generates the longest reads, currently 250– bases depending on the protocol, and is therefore most well-suited for de novo transcriptome sequencing. even though hundreds of thousands reads are generated simultaneous, the limited length will severely restrain the assembly of full length transcripts. in this study we present the first comprehensive characterization of the z. viviparus liver transcriptome using massively parallel pyrosequencing. based on data corresponding to one single run on the flx gene sequencer from life science, almost million bases were assembled into ~ pieces of putative transcripts and the majority of these have been annotated and functionally classified. furthermore, an oligonucleotide microarray for large-scale gene expression measurement in z. viviparus has been designed. our results, including the sequence data and the validated microarray probes are publicly available and will provide means for future genome-wide research in z. viviparus.  sequence analysis and quality assessment the polymer chain reaction amplification adapters used in the sequencing process were first identified and removed; the start tag was found for 99% of the reads while the end tag only was found for 42%, indicating that the majority of the reads have reached their maximum length and less than half stopped prematurely. a substantial part of the genome of higher eukaryotes consists of stretches with low-complexity such as the short and long interspaced repetitive elements and other transposons and these regions makes gene assembly treacherous since reads can be conjoined because they share a similar repetitive region. this is also true for the poly-a-tail, the a/t rich region that is attached to the 3' end of the mrna molecule after transcription. the entire dataset was therefore screened for low-complexity regions and poly-a tails. as expected, the number of identified poly-a tails was low compared to traditional cloned est library due to the uniform distribution of read positions exhibited by parallel pyrosequencing. in total, million bases were classified as low-complex, and were either substituted for ambiguity bases or removed whether their location was intermediate or in the ends. assembly of transcripts to assemble the reads into transcripts, we applied the tgi clustering tools, which is a set of utilities originally developed for assembly of data from cloned est libraries. the assembly resulted in contiguous sequences, while reads could not be matched against any other, resulting in putative eelpout transcripts consisting of more than million bases in total. the average length of the contigs was ~ base pairs, with more than contigs with a length over base pairs. a using a blast e-value cut-off of 10-5 b gasterosteus aculeatus, tetraodon nigroviridis, takifugu rubripes, oryzias latipes and danio rerio. the number of reads per contig ranged up to ~ with an average of eight reads and, even though the cdna library was normalized prior to sequencing, the distribution was highly asymmetric and right-tailed. the coverage, i.e. the average number of supporting reads per base pair, was estimated to times. with the low error rate of massively parallel pyrosequencing in mind, these results suggest that the assembly should be of high quality with very few technical errors. annotation the assembled transcripts were annotated against swiss-prot/uniprotkb, a manually curated database containing ~ known protein from more than different species. using a modest e-value cut-off of 10- 73% of the contigs and 70% of the singlets had a homologous hit, resulting in annotated eelpout transcripts. analogously, 89% of the contigs and 87% of the singles had a hit in at least one of the five available fish genomes. a closer examination of the annotations revealed several genes that are of particular interest for environmental monitoring and ecological research. several proteins within the cytochrome p protein superfamily catalyzes biotransformation of many xenobiotics and therefore contains a number of pivotal biomarkers for a large number of different compounds. among the sequenced z. viviparus transcripts, different cytochrome p variants from different families could be identified. other interesting classes of proteins, such as heat shock proteins and genes related to oxidative stress were also present, as well as several other common biomarkers, such as vitellogenin, the zona pellucida and metalothionein proteins. alength of longest contiguous fragment comparative genomics among the five fish species with sequence genome, gasterosteus aculeatus is evolutionarily closest to z. viviparus and the genome encompasses protein coding genes which have a total size of million base pairs. assuming similar numbers in eelpout, the assembled transcripts would cover roughly 40% of the transcriptome and each gene would on average be represented by more than two contigs or singlets. the gc-content differed between the two species, with g. aculeatus at 55% while z. viviparus had 48% for the contigs but only 43% for the singlets. the putative z. viviparus transcripts matched unique genes in g. aculeatus and among these, 72% corresponded to hits from contigs and 3% from singlets while more than 25% had hits from both contigs and singlets. hence, most singlet are therefore from genes already represented by a least one contig and very few genes are represented only as singlets. even though the number of unique genes decreased slightly, the conclusions were consistent for the other four fish species with sequenced genome. not surprisingly, the number of human genes with a match from z. viviparus transcripts was lower than all the fish but slightly higher than the frog. to investigate the presence of novel previously undescribed genes, all contigs and singlets were aligned against the five available fish genomes. in total of the transcripts could be found in at least three of the five fish species with sequenced genome, and among those, aligned in regions without previous annotation. not surprisingly, only of these could not be annotated against uniprot, which is lower than the transcripts aligning in annotated regions. the transcripts in the regions without annotation had a slightly higher coverage and a similar number of reads per transcript. more interestingly, these unknown transcripts aligned in regions with a higher evolutionary conservation compared to transcripts aligning in annotated regions. microarray analysis a 50-mer oligonucleotide microarray was designed for large-scale gene expression analysis in z. viviparus. to measure the expression of the probes and the corresponding assembled transcripts, the pool of mrna used for the sequencing was hybridized. for transcripts with reliable annotation, the gene expression measurement according to the probes in the annotated direction was highly correlated to the number of reads produced by the sequencing. thus, the data from the gene expression microarray is consistent with the massively parallel pyrosequencing reads which validates both the assembled transcripts as well as the corresponding probe. note however, that when additional transcripts with less reliable annotation are included the correlation dropped. data availability the raw sequence data from the massively parallel pyrosequencing is available at ncbi short read archive, the assembled transcripts and the generated probes are available at. the data from the gene expression microarray are available as accession number gse at gene expression omnibus. discussion we have used massively parallel pyrosequencing to characterize the transcriptome of zoarces viviparus. however, sequencing and assembly of a eukaryotic transcriptome without any genomic data a priori is a notoriously difficult undertaking. based on reads we assembled ~ putative transcripts, a strikingly high number compared to amount of genes found in the five sequenced fish genomes. one obvious reason for the high number of transcripts is the short length of the reads, which may result in several assembled contigs and singlets for each gene. another important factor is the process of alternative splicing, where one single gene can result in several transcripts by combining and removing different parts. this mechanism is common in higher eukaryotes, including in fish, and the number of expressed transcripts is therefore substantially higher than the number of genes in the genome. another issue is the limited sequence length exhibited by massively parallel pyrosequencing. even though the length is substantially longer than other high-throughput sequencing techniques, such as solid and illumina sequencing, the difference compared to sanger-based sequencing is notable. in this study, we used a flx gene sequencer, which generates bases long reads, which is only a fourth of the bases long sequences which can be produced by sanger sequencing. the short sequence reads makes the assembly more difficult and thus the resulting contigs shorter. however, the average contig length from our assembly is slightly longer compared to previous studies and it is likely that this difference is due to the single tissue targeted in this study, but also due to the advance processing pipeline where genomic uninteresting regions, such as repeats, are removed. for transcriptome sequencing, massively parallel pyrosequencing exhibits a high sensitivity compared to traditional sanger-based approaches. the large number of generated reads allows detection of low-abundant transcripts and the cdna cloning step where mrna molecules are inserted into bacterial vectors is no longer required. transcripts that previously have been hard to sequence can therefore be detected and previous studies have reported a considerable amount of reads aligning in genomic regions previously not annotated. among our assembled transcripts, ~ could be aligned to at least three out of five available fish genomes and among these 3% matched regions without any prior annotation. the expression levels for these unknown transcripts were at least as high as those aligning in annotated regions and they aligned in regions with evolutionary conservation between distant species. hence, these transcripts are likely to represent novel transcribed elements such as not yet described fish-specific genes. massively parallel pyrosequencing can currently only process dna stretches of limited length. thus, a fragmentation step is applied and as a consequence the reading direction, traditionally indicated by poly-a tail primers, is lost. hence, the assembly pipeline therefore needs to be flexible and build contigs based on reads from both directions. nevertheless, the direction of transcription will still be unknown for the resulting contigs. for blast-based sequence alignments, the knowledge of the direction of transcription is not necessary since the comparison can easily be done for both strands. however, when constructing gene expression microarrays, the direction of transcription is crucial for proper probe design, both for placing the probe on the correct strand and for evaluating probe specificity. unfortunately, estimating the direction of transcription is intricate for short stretches of mrna, especially for sequences without a satisfactory annotation. even though we complemented our blast-based annotations with estimates from framefinder, an application used to identify open reading frames, a substantial part of our transcripts lacks reliable estimates of their direction of transcriptions. these transcripts may therefore be represented by probes in both directions in future gene expression assays, and thereby circumvent the problem at the cost of space on the microarray. it is finally worth to mention that the gene expression levels on our evaluation microarray are not good predictors of the direction of transcription. only ~60% of the well-annotated transcripts have probes that had a higher expression levels in the correct direction compared to the erroneous direction. this result is, however, far from surprising since the probes for the two directions will have different thermodynamic properties and specificity and therefore different intensity levels. the correlation between the developed eelpout microarray and the number of reads from the massively parallel pyrosequencing was estimated to 62%. this number is strikingly high considering both the normalization of the cdna library prior sequencing and the non-linearity typically exhibited by microarrays. previous studies comparing hybridization- and high-throughput sequencing-based gene expression measurement techniques have reported a correlation between 46%–72%. in contrast to these studies, which use established previously evaluated microarrays from well-sequenced species, the eelpout microarray is designed from scratch based on less than half of the transcriptome. thus, the correlation shows that that there is a good concordance between the developed eelpout microarray and the transcriptome sequence data, which validates both the assembled transcripts and the corresponding oligonucleotide probes. the present study demonstrates the relative ease by which de novo transcriptome sequencing can be done today. the performance of the modern high-throughput sequencers is high enough that one single run is enough to cover a substantial part of the trancsriptome of a higher eukaryote. as a consequence, the assembled contigs becomes long enough to design reliable microarray probes for large-scale gene expression analysis. this development will undoubtedly spur genome-based research in many biological disciplines, especially in fields where a wide range of wild-life species are studied. one prime example is ecology, where tools such as gene expression assays are needed for efficiently study the low-level mechanistic of ecosystems while other related techniques such as single nucleotide polymorphism analysis can be used to unravel parts of the complex interplay between genetic variation and fitness in wild populations. the zoarces viviparus microarray developed in this study will constitute a valuable resource for marine environmental research in the northern europe. the platform will allow correlation of molecular-level responses, caused by exposure to various toxicants, to the unique morphological and physiological characters of z. viviparus. in particular, the reproductive output in the form of size, number and quality of the embryos, can be related to gene expression changes at an individual basis. the stationary behavior of this fish species in combination with the sensitivity of gene expression biomarkers will form a unique instrument for accurate and robust monitoring of the aquatic ecosystems. the generated sequence data for the z. viviparus transcriptome will also form a base to study molecular and physiological adaptations to viviparity in fish.  the present study describes the first comprehensive sequencing effort of zoarces viviparus, an important model organism for marine ecology research and environmental monitoring. the sequence data was generated by massively parallel pyrosequencing, a high-throughput technique with a hundred-fold higher performance than traditional capillary sequencing. the sequenced nucleotides were estimated to cover roughly 40% of the transcriptome and more than pieces of putative transcripts were assembled. finally, an oligonucleotide microarray were constructed and used to evaluate expression of our assembled transcripts. 
the teleost zoarces viviparus lives along the coasts of northern europe and has long been an established model organism for marine ecology and environmental monitoring. in the present study we present the first comprehensive characterization of the zoarces viviparus liver transcriptome. the generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in zoarces viviparus. from reads generated by massively parallel pyrosequencing, more than pieces of putative transcripts were assembled, annotated and functionally classified. the sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis. the scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays. our results show that one run using a genome sequencer flx from life science/roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. the data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of gasterosteus aculeatus were identified.
 the teleost zoarces viviparus lives along the coasts of northern europe and has long been an established model organism for marine ecology and environmental monitoring. the scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays.  in the present study we present the first comprehensive characterization of the zoarces viviparus liver transcriptome. from reads generated by massively parallel pyrosequencing, more than pieces of putative transcripts were assembled, annotated and functionally classified. the data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of gasterosteus aculeatus were identified. the sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis.  our results show that one run using a genome sequencer flx from life science/roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. the generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in zoarces viviparus. 