BACKGROUND
micrornas  have emerged as important post-transcriptional regulators of gene expression in a wide variety of physiological processes.
mirnas are short endogenous non-coding rna  present in a wide variety of organisms, including plants, animals, unicellular organisms, and viruses  <cit> .
mature mirnas derive from primary transcripts  forming hairpins that are cleaved into mirna precursor .
one strand of the resulting mirna duplex is loaded into a mirisc complex  by argonaute proteins.
most mature mirnas are 20–24 nt long while pre-mirna are 60–80 nt in length.
mainly, the mirisc complex can bind to the 3′utr of its target mrna and subsequently lead to degradation or repression of translation  <cit> .
several examples of non-canonical binding  have been reported lately for mirnas proven to be functional  <cit> .
the role of mirnas has been studied in several physiological and physiopathological processes.
they are involved in key animal developmental processes, such as maternal transcript clearance  <cit>  or axial patterning  <cit> .
moreover, mirnas are also involved in disease and  <dig> pathologies reported in mir2disease database have been associated with misregulation of  <dig> mirna genes or dysfunction of mirna/mrna target interaction  <cit> .
in addition, mirnas can be associated with economical traits.
in sheep, a mutation in myostatin 3′-utr creates a new target site for mirna and affects muscularity  <cit> .
mirnas are often expressed in a tissue-enriched manner  <cit> .
they can control both temporal and spatial gene expression and are believed to regulate  <dig> to 70 % of the protein-coding genes  <cit> .
before the development of high-throughput sequencing the number of mirnas discovered by cloning and sanger sequencing was dramatically underestimated and limited to approximately  <dig> per species.
with the advent of large-scale genomic analyses, even mirnas with low abundance have been successfully discovered in various species.
to date, at least  <dig>  mature mirnas sequence have been discovered, corresponding to a total of  <dig>  hairpins sequences in  <dig> species  .
mammals and insects are the most studied species with regards to mirna discovery but the most recent release  contains several entries for several fish species such as atlantic salmon , zebrafish , medaka , common carp , fugu , tetraodon , atlantic halibut , olive flounder , and channel catfish .
all above mirnas have been discovered by next-generation sequencing.
this database is however limited for salmonid species despite the reports of several mirnas in several tissues of atlantic salmon  <cit>  or rainbow trout  <cit>  and in rainbow trout eggs  <cit> .
salmonids are economically and environmentally important species for both wild fisheries and worldwide aquaculture production.
genomic resources for rainbow trout and atlantic salmon are advanced but our knowledge of salmonid mirnas repertoire remains incomplete.
the aim of the current study was to discover and characterize rainbow trout  mirnas in a large number of tissues using next-generation sequencing in order to provide an extensive repertoire of rainbow trout mirnas.
a total of  <dig> different samples corresponding to  <dig> different tissues were individually sequenced and analyzed independently in order to identify a large number of mirnas with high confidence.
differential expression analysis was performed in order to identify mirnas exhibiting specific or preferential expression among the  <dig> tissues analyzed.
the dataset was also used to discover novel mirnas that had not been previously reported in other species.
this mirna characterization is a first step to better understand the role of mirnas in gene regulation in rainbow trout.
RESULTS
overview of small rna sequencing
independent small rna libraries were constructed for  <dig> samples representing  <dig> different tissues including gonadal and somatic tissue.
a total of  <dig> , <dig>  reads were generated from the  <dig> libraries using a hiseq- <dig>  the number of reads per sample ranged from  <dig> , <dig> to  <dig> , <dig> .
the processing procedure applied to this dataset is described in fig.
<dig>  a cleaning procedure was first used that removed adapters sequences and eliminated reads of low complexity.
only the  <dig>  non redundant sequences, representing  <dig> , <dig>  reads with a size in the range of 16– <dig> nucleotides and a global expression level higher than  <dig> were kept for mapping.
the mapping procedure allowed to align  <dig>  % of the  <dig>  non redundant sequences in their full length at 100 % identity onto the rainbow trout genome  <cit> .
a maximum of  <dig> alignments was reported for each sequence.
from the  <dig>  alignments that were generated  <dig>  loci were built and submitted to the mirna prediction procedure.
<dig> loci were predicted as containing putative pre-mirna.
all the pre-mirna predicted loci were annotated against ncrna databases including mirbase  <dig>   <cit> , rfam  <dig>   <cit> , silva  <cit>  and gtrnadb  <cit> .
mirna from mirbase and rfam were merged before annotation in order to have a unique mirna database.
considering the  <dig> mirna predictions,  <dig> were not considered because they had a perfect match to known repeat elements in rainbow trout  or were annotated as other rna species than mirna , including for example snornas.
three of them were common to repeat elements and ncrna other than mirnas.
from the  <dig> remaining mirna predictions,  <dig> were annotated as mirna.
among them,  <dig> had multiple annotations in addition to mirna, including for example snornas and u <dig> snrna.
<dig> predictions were annotated only as mirna.
regarding the  <dig> mirna loci annotation,  <dig> mirna loci were annotated in both mirbase and rfam.
<dig> mirna predicted loci were only annotated in mirbase and  <dig> were only annotated as mirna in rfam.
therefore, a total of  <dig> mirna predictions corresponded to new putative mirna in rainbow trout.
the score of predicted mirna loci, given by the pipeline, ranged from  <dig> to  <dig>  the distribution of the score of predicted mirnas is not linear and a clear drop in score number is observed around  <dig> .
loci predicted and annotated as mirna  have mainly a score higher than  <dig> .
mis-predicted loci have mainly a score below 250: among the mis-predicted mirnas loci, only  <dig> have a score above  <dig>  this threshold of  <dig> will be further used to select high confidence novel mirnas among predicted loci.
considering predicted loci annotated as mirna only ,  <dig>  are located in intergenic regions,  <dig>  are in introns, and  <dig>  are in 5′utr, 3′utr or in exons.
we observe similar structural annotation percentages for loci predicted as mirna but not annotated in any database :  <dig> such loci are located in intergenic regions ,  <dig>  are in introns and  <dig>  are in exons.
a complete list of all mirna loci with structural and functional annotation is provided in additional file  <dig> fig.
<dig> identification and annotation pipeline of rainbow trout mirnas using srna-sequencing.
fastq files were first cleaned from low quality and adapter sequence.
the redundancy was removed from the  <dig> sample files for the annotation process .
unique sequences were then blasted against rrna, trna and rna database to remove already known rna sequences that are not mirna .
a blast against mirbase database predicts the already identified mirnas in other species .
in parallel, reads were mapped onto the rainbow trout genome to eliminate any contaminant sequences and characterize the mirna loci.
novel pre-mirna were also characterized with the specific mirnas features
fig.
<dig> mirna prediction and genomic localization.
the graph represents the distribution of the prediction score for the  <dig> mirnas predicted loci.
a clear drop in score numbers is observed around  <dig>  mirnas annotated loci are in red and unannotated predicted mirnas loci are in black
for most samples in the present study, the length distribution of the reads is as expected for a mirna study, with a peak at 22 nt, which corresponds to the expected mirna size .
for reproductive tissue samples, the distribution is quite different with a first peak at 22 nt and a second peak at 27 nt.
the number of reads belonging to the second peak is higher than the one belonging to the first peak.
both ovary  and testis  exhibit these two peaks in their small rnas population.
the first one is the expected size of mirnas and the size of the second is consistent with the size of pirnas  that are highly expressed in germ cells  <cit> .
in fact, pirnas are one of the main classes of non-coding rnas present in germ cells  <cit> .
ovarian and testicular samples contain a lot of germ cells and it is therefore not surprising to find this kind of non-coding rnas in these samples.fig.
<dig> srna-seq reads length repartition.
a the graph represents the read length repartition in a muscle sample .
b the graph represents the read length repartition in the egg sample.
c the graph represents the read length repartition in the testis stage i sample.
all the profiles are represented in additional file 3
fig.
<dig> pie charts of mirna abundance.
distribution of the mirna abundance by mirna family.
the top  <dig> mirna families are represented  and represents 70 % of the expressed mirnas.
all the others  represents 30 % of the overall expressed mirna
identification of conserved rainbow trout mirnas
many mirnas have been conserved during evolution and the sequence identity is extremely high even among evolutionary distant species .
in the present study, we identified rainbow trout mirnas based on their sequence similarity with other species and used mirbase database  and rfam database as reference datasets.
a total of  <dig> conserved putative precursors were discovered at unique genome locations that correspond to  <dig> mature conserved mirnas.
the annotation of mirnas showed that we have identified  <dig> evolutionary conserved mirna families in our dataset .
several isoforms in sequence and size were found for each mirna family.
this number of identified mirnas is consistent  with mirna repertoires characterized in other fish species such as salmon  <cit> , zebrafish  <cit> , and medaka  <cit> .
some mirnas are present at a unique locus but most of them are present at different positions in the genome .
the large presence of the same mirna at multiple genomic locations is directly linked to the last round of whole genome duplication  that occurred in salmonids species.
first, teleost wgd gave rise to several duplicated mirna genes located in multiple genomic loci as reported for several teleost fish species.
the fourth salmonid-specific event  further bolstered this condition in the genome of salmonid species.
as previously shown  <cit> , almost all mirna loci are retained in duplicated copies in the rainbow trout genome.
tissue distribution of rainbow trout mirnas
relative abundance of mirna in the dataset was characterized .
in the global dataset, two mirnas, mir- <dig> and mir- <dig>  represent almost half of the reads with  <dig> and 15 % of the total number of reads in the complete dataset, respectively.
gene expression datasets from several tissues usually exhibit a different pattern: each gene has a small contribution to the total but none of them are overrepresented like it is the case for mirnas.
mir- <dig> is a well studied mirna that is involved in different types of cancer  <cit>  and also in the development of heart disease  <cit> .
mir- <dig> is thought to be a mediator of inflammation and is upregulated by inflammatory factors such as interleukins and tumor necrosis factor-alpha  <cit> .
eight mirnas  account for more than 1 % each and collectively represent 70 % of the total number of reads.
when looking at the mirna distribution within the different organs , we found the same mirnas to be on the top of each list  but also some over-represented mirna are specific to one organ.
for instance, mir- <dig> accounts for respectively  <dig> and 10 % of the total reads in testis and ovary, mir- <dig> accounts for 42 % in muscles and 12 % in heart.
a similar distribution was reported in rainbow trout unfertilized eggs  <cit> , with slight differences.
let- <dig> was the most expressed mirna and accounts for 24 % of the total, followed by mir- <dig> .
in the present study, we confirm that a small subset of mirnas is highly expressed and accounts for approximately 70 % of total mirna counts.
this pattern has been observed in salmon fry, but the top expressed mirnas are not the same.
ssa-mir- <dig> and ssa-mir- <dig> account for more than 72 % of the total mirnas in salmon alevin  <cit> .
the two different studies in salmon do not include as many organs as we have in the present study, so that might be the reason why the top expressed mirnas are different.
in salmon, by looking at a subset of tissue or alevin, they are probably looking at mirna enrichment in those organs or developmental stages.
the strength of our study is that a large number of tissues were sequenced separately.
this allows us to perform analysis of mirna expression among the  <dig> tissues using unsupervised clustering analysis.
the heatmap in fig.
<dig> shows the tissue distribution of annotated mirnas including all isoforms .
a large number of mirnas are clearly overexpressed in a subset of organs/tissues or even only in a single organ/tissue.
in mammals, it was demonstrated that mirnas are expressed in a tissue-dependent manner  <cit>  and rainbow trout mirnas are clearly following the same rule.
this mirna distribution was validated by rt-qpcr for several selected mirnas.
as shown in fig.
<dig>  sequencing and rt-qpcr data are highly correlated for the  <dig> mirnas studied .
the  <dig> mirnas exhibit different patterns of expression  and different level of expression.
hereby, we show that srna-sequencing can be used for characterizing mirna repertoire and that expression profiles are confirmed by rt-qpcr.
this indicates that the over-representation of some mirnas does not bias the expression dataset.
another set of samples was used for the rt-qpcr analysis in order to exclude any rna-seq library preparation bias and to extend the validation to a larger set of samples.
some mirnas have a ubiquitous expression suggesting a role in common biological processes such as cell proliferation, differentiation or apoptosis.
in humans, mir- <dig> is involved in apoptosis by targeting bcl <dig>  <cit>  and mir- <dig> inhibits cell proliferation and induces cell cycle arrest  <cit> .
previous studies already reported the expression of those ubiquitous mirnas in rainbow trout.
salem et al.
and ramachandra et al.
<cit>  identified mirnas from an rna pool of nine tissues; muscle, heart, brain, kidney, liver, spleen, intestine, gill, and skin.
most of the ubiquitous mirnas identified in this study are present in their list.
in contrast, some mirnas have very specific tissue expression pattern.
for example , mir- <dig> is only expressed in liver and spleen and was shown to be involved in liver metabolism in rainbow trout  <cit> , and mir- <dig> is only expressed in gonads.
the function of mir- <dig> is not yet understood but it is involved in sex differentiation in chicken  <cit> .
this tissue-enriched expression is mostly conserved from one species to another.
for example, mir- <dig> was found to be gonad-specific in frog  <cit> , atlantic halibut  <cit> , human, mouse, and rat  <cit> .
thus, organ/tissue-enriched mirnas may have a specific role in the organ/tissue and therefore could be key players of evolutionary conserved biological processes.fig.
<dig> mirna expression in the  <dig> tissues.
unsupervised average linkage clustering analysis of mirna isoforms in rainbow trout tissue.
each row represents a mirna isoform  and each column a tissue rna sample.
data were median-centered prior to the clustering analysis.
for each mirna, the expression level within samples is indicated using a color density scale
fig.
<dig> mirna expression correlation between srna-seq and rt-qpcr.
pearson correlations between srna-seq and rt-qpcr data were calculated for  <dig> mirnas that show diverse expression profiles .
rna-seq data are on the y-axis and qpcr are on the x-axis
identification of novel rainbow trout mirnas
the remaining un-annotated small rnas mapped to the rainbow trout genome were subjected to novel mirna prediction.
reads that provided multiple  significant alignments against the rainbow trout genome were not considered as they are most likely interspersed repeats or long tandem repeats.
a total of  <dig> loci were predicted as mirna genes without any mirna annotation.
from those  <dig> loci,  <dig> of them had a prediction score greater than  <dig>  as seen previously in fig.
<dig>  a prediction score of  <dig> can be used as a threshold for high confidence prediction.
most of the already annoted mirnas have a prediction score above  <dig>  the above-mentioned  <dig> loci were therefore classified as high confidence loci while the  <dig> remaining loci are classified as low confidence loci.
further analyses were performed for high confidence loci .
sequencing data were used to build the novel mirna loci as illustrated in fig.
<dig> for one example .
information about the predicted locus are shown in fig.
7a: it includes structure and sequence data such as free-energy of the hairpin, hairpin sequence and nucleotides distribution.
in fig.
7b, the reads are shown as “stacks” below the precursor sequence and align to both ends of the precursor .
the count per base is shown in the top of the b panel.
as found in known mirnas, several isoforms in length are found in the sequencing dataset with a frame shift of  <dig> or  <dig> nucleotides in both ends.
expression profiles were also studied for the novel mirnas .
as expected most of them display a tissue-specific or tissue-predominant expression profile.
this is the case for instance for  <dig> testis-specific novel mirnas,  <dig> ovary-predominant novel mirnas ,  <dig> skin-predominant novel mirnas .
the novel rainbow trout mirnas may be found in close related species such as salmon but also in any other fish species with the development of srna sequencing in economical important species.fig.
<dig> novel mirnas in rainbow trout: prediction and tissue distribution.
a the illustration shows sequence and structure statistics of one predicted mirna precursor as an example.
b the graph shows, as an example, the 54 nt sequence  along with reads that aligned to this sequence.
the secondary structure of the precursor is shown with the orange parenthesis.
c unsupervised average linkage clustering analysis of new mirna in rainbow trout tissue.
each row represents a new mirna locus  and each column a tissue rna sample.
data were median-centered prior to the clustering analysis.
for each mirna, the expression level within sample set is indicated using a color density scale
conservation of identified rainbow trout mirnas
mirnas are known to be conserved among species to a certain extend  <cit>  and therefore we checked whether the identified rainbow trout mirnas were conserved among fish species.
we compared our rainbow trout dataset  to  <dig> fish genomes: atlantic salmon, atlantic cod, zebrafish, medaka, stickleback, sea bass, tetraodon and fugu and looked for mirna sequence conservation .
as shown in fig.
8a, 7 % of the rainbow trout mirnas are found in all the  <dig> other selected species, while 67 % of the trout mirnas are found in only one other species .
those 7 % of mirnas found in all  <dig> fish species are common mirnas also expressed in most vertebrates.
one to 4 % of the rainbow trout mirnas are found in either  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> of the selected species.
interestingly, 14 % of the rainbow trout mirnas are not found in any of those species, indicating that they might be rainbow trout-specific.
it was shown previously in other species that a subset of mirnas can be species-specific.
for instance,  <dig> mirnas identified in zebra finch were found to be avian specific and among them,  <dig> mirnas were found only in zebra finch  <cit> .
moreover, as shown in fig.
8b, most of the rainbow trout mirnas are found in the atlantic salmon genome  while only  <dig> to 13 % are found in the other species.
this could be partially explain by the fact that salmon and rainbow trout are closely related species that share the last round of whole genome duplication  while the  <dig> other species are more distant from rainbow trout and did not undergo the ss4r.
but, interestingly, the conservation pattern is very similar among these  <dig> species indicating that after the third round of whole genome duplication , the mirna repertoire in these fish species is quite stable.fig.
<dig> mirnas conservation among fish species.
conservation analysis of the rainbow trout mirnas repertoire among  <dig> fish species: atlantic salmon, atlantic cod, zebrafish, medaka, sea bass, stickleback, tetraodon and fugu  pie chart representing the percentage of rainbow trout mirnas that are found among the  <dig> fish genomes  stack histograms representing the conservation of rainbow trout mirnas in each fish species
CONCLUSIONS
in conclusion, this study represents the first characterization of rainbow trout mirna transcriptome from a wide variety of organs/tissues and describes an extensive repertoire of rainbow trout mirnas.
we identified  <dig> mirna loci in the rainbow trout genome including already known mirnas  and putative novel mirnas .
we also show that differential expression analysis in different tissues is possible using rna sequencing strategy by confirming the expression data by rt-qpcr with high percentage of correlation.
this study provides a starting point for future studies aimed at understanding the roles of mirnas in major physiological process such as growth, reproduction, nutrition, and adaptation.
this rainbow trout mirnas repertoire provides a novel resource to advance genomic research in salmonids species.

 micrornas have emerged as important post-transcriptional regulators of gene expression in a wide variety of physiological processes. mirnas are short endogenous non-coding rna present in a wide variety of organisms, including plants, animals, unicellular organisms, and viruses. mature mirnas derive from primary transcripts forming hairpins that are cleaved into mirna precursor. one strand of the resulting mirna duplex is loaded into a mirisc complex by argonaute proteins. most mature mirnas are 20–24 nt long while pre-mirna are 60–80 nt in length. mainly, the mirisc complex can bind to the 3′utr of its target mrna and subsequently lead to degradation or repression of translation. several examples of non-canonical binding have been reported lately for mirnas proven to be functional. the role of mirnas has been studied in several physiological and physiopathological processes. they are involved in key animal developmental processes, such as maternal transcript clearance or axial patterning. moreover, mirnas are also involved in disease and pathologies reported in mir2disease database have been associated with misregulation of mirna genes or dysfunction of mirna/mrna target interaction. in addition, mirnas can be associated with economical traits. in sheep, a mutation in myostatin 3′-utr creates a new target site for mirna and affects muscularity. mirnas are often expressed in a tissue-enriched manner. they can control both temporal and spatial gene expression and are believed to regulate to 70 % of the protein-coding genes. before the development of high-throughput sequencing the number of mirnas discovered by cloning and sanger sequencing was dramatically underestimated and limited to approximately per species. with the advent of large-scale genomic analyses, even mirnas with low abundance have been successfully discovered in various species. to date, at least mature mirnas sequence have been discovered, corresponding to a total of hairpins sequences in species. mammals and insects are the most studied species with regards to mirna discovery but the most recent release contains several entries for several fish species such as atlantic salmon, zebrafish, medaka, common carp, fugu, tetraodon, atlantic halibut, olive flounder, and channel catfish. all above mirnas have been discovered by next-generation sequencing. this database is however limited for salmonid species despite the reports of several mirnas in several tissues of atlantic salmon or rainbow trout and in rainbow trout eggs. salmonids are economically and environmentally important species for both wild fisheries and worldwide aquaculture production. genomic resources for rainbow trout and atlantic salmon are advanced but our knowledge of salmonid mirnas repertoire remains incomplete. the aim of the current study was to discover and characterize rainbow trout mirnas in a large number of tissues using next-generation sequencing in order to provide an extensive repertoire of rainbow trout mirnas. a total of different samples corresponding to different tissues were individually sequenced and analyzed independently in order to identify a large number of mirnas with high confidence. differential expression analysis was performed in order to identify mirnas exhibiting specific or preferential expression among the tissues analyzed. the dataset was also used to discover novel mirnas that had not been previously reported in other species. this mirna characterization is a first step to better understand the role of mirnas in gene regulation in rainbow trout.  overview of small rna sequencing independent small rna libraries were constructed for samples representing different tissues including gonadal and somatic tissue. a total of, reads were generated from the libraries using a hiseq- the number of reads per sample ranged from, to,. the processing procedure applied to this dataset is described in fig.  a cleaning procedure was first used that removed adapters sequences and eliminated reads of low complexity. only the non redundant sequences, representing, reads with a size in the range of 16– nucleotides and a global expression level higher than were kept for mapping. the mapping procedure allowed to align  % of the non redundant sequences in their full length at 100 % identity onto the rainbow trout genome. a maximum of alignments was reported for each sequence. from the alignments that were generated loci were built and submitted to the mirna prediction procedure. loci were predicted as containing putative pre-mirna. all the pre-mirna predicted loci were annotated against ncrna databases including mirbase, rfam, silva and gtrnadb. mirna from mirbase and rfam were merged before annotation in order to have a unique mirna database. considering the mirna predictions, were not considered because they had a perfect match to known repeat elements in rainbow trout or were annotated as other rna species than mirna, including for example snornas. three of them were common to repeat elements and ncrna other than mirnas. from the remaining mirna predictions, were annotated as mirna. among them, had multiple annotations in addition to mirna, including for example snornas and u snrna. predictions were annotated only as mirna. regarding the mirna loci annotation, mirna loci were annotated in both mirbase and rfam. mirna predicted loci were only annotated in mirbase and were only annotated as mirna in rfam. therefore, a total of mirna predictions corresponded to new putative mirna in rainbow trout. the score of predicted mirna loci, given by the pipeline, ranged from to the distribution of the score of predicted mirnas is not linear and a clear drop in score number is observed around. loci predicted and annotated as mirna have mainly a score higher than. mis-predicted loci have mainly a score below 250: among the mis-predicted mirnas loci, only have a score above this threshold of will be further used to select high confidence novel mirnas among predicted loci. considering predicted loci annotated as mirna only, are located in intergenic regions, are in introns, and are in 5′utr, 3′utr or in exons. we observe similar structural annotation percentages for loci predicted as mirna but not annotated in any database: such loci are located in intergenic regions, are in introns and are in exons. a complete list of all mirna loci with structural and functional annotation is provided in additional file fig. identification and annotation pipeline of rainbow trout mirnas using srna-sequencing. fastq files were first cleaned from low quality and adapter sequence. the redundancy was removed from the sample files for the annotation process. unique sequences were then blasted against rrna, trna and rna database to remove already known rna sequences that are not mirna. a blast against mirbase database predicts the already identified mirnas in other species. in parallel, reads were mapped onto the rainbow trout genome to eliminate any contaminant sequences and characterize the mirna loci. novel pre-mirna were also characterized with the specific mirnas features fig. mirna prediction and genomic localization. the graph represents the distribution of the prediction score for the mirnas predicted loci. a clear drop in score numbers is observed around mirnas annotated loci are in red and unannotated predicted mirnas loci are in black for most samples in the present study, the length distribution of the reads is as expected for a mirna study, with a peak at 22 nt, which corresponds to the expected mirna size. for reproductive tissue samples, the distribution is quite different with a first peak at 22 nt and a second peak at 27 nt. the number of reads belonging to the second peak is higher than the one belonging to the first peak. both ovary and testis exhibit these two peaks in their small rnas population. the first one is the expected size of mirnas and the size of the second is consistent with the size of pirnas that are highly expressed in germ cells. in fact, pirnas are one of the main classes of non-coding rnas present in germ cells. ovarian and testicular samples contain a lot of germ cells and it is therefore not surprising to find this kind of non-coding rnas in these samples.fig. srna-seq reads length repartition. a the graph represents the read length repartition in a muscle sample. b the graph represents the read length repartition in the egg sample. c the graph represents the read length repartition in the testis stage i sample. all the profiles are represented in additional file 3 fig. pie charts of mirna abundance. distribution of the mirna abundance by mirna family. the top mirna families are represented and represents 70 % of the expressed mirnas. all the others represents 30 % of the overall expressed mirna identification of conserved rainbow trout mirnas many mirnas have been conserved during evolution and the sequence identity is extremely high even among evolutionary distant species. in the present study, we identified rainbow trout mirnas based on their sequence similarity with other species and used mirbase database and rfam database as reference datasets. a total of conserved putative precursors were discovered at unique genome locations that correspond to mature conserved mirnas. the annotation of mirnas showed that we have identified evolutionary conserved mirna families in our dataset. several isoforms in sequence and size were found for each mirna family. this number of identified mirnas is consistent with mirna repertoires characterized in other fish species such as salmon, zebrafish, and medaka. some mirnas are present at a unique locus but most of them are present at different positions in the genome. the large presence of the same mirna at multiple genomic locations is directly linked to the last round of whole genome duplication that occurred in salmonids species. first, teleost wgd gave rise to several duplicated mirna genes located in multiple genomic loci as reported for several teleost fish species. the fourth salmonid-specific event further bolstered this condition in the genome of salmonid species. as previously shown, almost all mirna loci are retained in duplicated copies in the rainbow trout genome. tissue distribution of rainbow trout mirnas relative abundance of mirna in the dataset was characterized. in the global dataset, two mirnas, mir- and mir- represent almost half of the reads with and 15 % of the total number of reads in the complete dataset, respectively. gene expression datasets from several tissues usually exhibit a different pattern: each gene has a small contribution to the total but none of them are overrepresented like it is the case for mirnas. mir- is a well studied mirna that is involved in different types of cancer and also in the development of heart disease. mir- is thought to be a mediator of inflammation and is upregulated by inflammatory factors such as interleukins and tumor necrosis factor-alpha. eight mirnas account for more than 1 % each and collectively represent 70 % of the total number of reads. when looking at the mirna distribution within the different organs, we found the same mirnas to be on the top of each list but also some over-represented mirna are specific to one organ. for instance, mir- accounts for respectively and 10 % of the total reads in testis and ovary, mir- accounts for 42 % in muscles and 12 % in heart. a similar distribution was reported in rainbow trout unfertilized eggs, with slight differences. let- was the most expressed mirna and accounts for 24 % of the total, followed by mir-. in the present study, we confirm that a small subset of mirnas is highly expressed and accounts for approximately 70 % of total mirna counts. this pattern has been observed in salmon fry, but the top expressed mirnas are not the same. ssa-mir- and ssa-mir- account for more than 72 % of the total mirnas in salmon alevin. the two different studies in salmon do not include as many organs as we have in the present study, so that might be the reason why the top expressed mirnas are different. in salmon, by looking at a subset of tissue or alevin, they are probably looking at mirna enrichment in those organs or developmental stages. the strength of our study is that a large number of tissues were sequenced separately. this allows us to perform analysis of mirna expression among the tissues using unsupervised clustering analysis. the heatmap in fig.  shows the tissue distribution of annotated mirnas including all isoforms. a large number of mirnas are clearly overexpressed in a subset of organs/tissues or even only in a single organ/tissue. in mammals, it was demonstrated that mirnas are expressed in a tissue-dependent manner and rainbow trout mirnas are clearly following the same rule. this mirna distribution was validated by rt-qpcr for several selected mirnas. as shown in fig.  sequencing and rt-qpcr data are highly correlated for the mirnas studied. the mirnas exhibit different patterns of expression and different level of expression. hereby, we show that srna-sequencing can be used for characterizing mirna repertoire and that expression profiles are confirmed by rt-qpcr. this indicates that the over-representation of some mirnas does not bias the expression dataset. another set of samples was used for the rt-qpcr analysis in order to exclude any rna-seq library preparation bias and to extend the validation to a larger set of samples. some mirnas have a ubiquitous expression suggesting a role in common biological processes such as cell proliferation, differentiation or apoptosis. in humans, mir- is involved in apoptosis by targeting bcl and mir- inhibits cell proliferation and induces cell cycle arrest. previous studies already reported the expression of those ubiquitous mirnas in rainbow trout. salem et al. and ramachandra et al. identified mirnas from an rna pool of nine tissues; muscle, heart, brain, kidney, liver, spleen, intestine, gill, and skin. most of the ubiquitous mirnas identified in this study are present in their list. in contrast, some mirnas have very specific tissue expression pattern. for example, mir- is only expressed in liver and spleen and was shown to be involved in liver metabolism in rainbow trout, and mir- is only expressed in gonads. the function of mir- is not yet understood but it is involved in sex differentiation in chicken. this tissue-enriched expression is mostly conserved from one species to another. for example, mir- was found to be gonad-specific in frog, atlantic halibut, human, mouse, and rat. thus, organ/tissue-enriched mirnas may have a specific role in the organ/tissue and therefore could be key players of evolutionary conserved biological processes.fig. mirna expression in the tissues. unsupervised average linkage clustering analysis of mirna isoforms in rainbow trout tissue. each row represents a mirna isoform and each column a tissue rna sample. data were median-centered prior to the clustering analysis. for each mirna, the expression level within samples is indicated using a color density scale fig. mirna expression correlation between srna-seq and rt-qpcr. pearson correlations between srna-seq and rt-qpcr data were calculated for mirnas that show diverse expression profiles. rna-seq data are on the y-axis and qpcr are on the x-axis identification of novel rainbow trout mirnas the remaining un-annotated small rnas mapped to the rainbow trout genome were subjected to novel mirna prediction. reads that provided multiple significant alignments against the rainbow trout genome were not considered as they are most likely interspersed repeats or long tandem repeats. a total of loci were predicted as mirna genes without any mirna annotation. from those loci, of them had a prediction score greater than as seen previously in fig.  a prediction score of can be used as a threshold for high confidence prediction. most of the already annoted mirnas have a prediction score above the above-mentioned loci were therefore classified as high confidence loci while the remaining loci are classified as low confidence loci. further analyses were performed for high confidence loci. sequencing data were used to build the novel mirna loci as illustrated in fig.  for one example. information about the predicted locus are shown in fig. 7a: it includes structure and sequence data such as free-energy of the hairpin, hairpin sequence and nucleotides distribution. in fig. 7b, the reads are shown as “stacks” below the precursor sequence and align to both ends of the precursor. the count per base is shown in the top of the b panel. as found in known mirnas, several isoforms in length are found in the sequencing dataset with a frame shift of or nucleotides in both ends. expression profiles were also studied for the novel mirnas. as expected most of them display a tissue-specific or tissue-predominant expression profile. this is the case for instance for testis-specific novel mirnas, ovary-predominant novel mirnas, skin-predominant novel mirnas. the novel rainbow trout mirnas may be found in close related species such as salmon but also in any other fish species with the development of srna sequencing in economical important species.fig. novel mirnas in rainbow trout: prediction and tissue distribution. a the illustration shows sequence and structure statistics of one predicted mirna precursor as an example. b the graph shows, as an example, the 54 nt sequence along with reads that aligned to this sequence. the secondary structure of the precursor is shown with the orange parenthesis. c unsupervised average linkage clustering analysis of new mirna in rainbow trout tissue. each row represents a new mirna locus and each column a tissue rna sample. data were median-centered prior to the clustering analysis. for each mirna, the expression level within sample set is indicated using a color density scale conservation of identified rainbow trout mirnas mirnas are known to be conserved among species to a certain extend and therefore we checked whether the identified rainbow trout mirnas were conserved among fish species. we compared our rainbow trout dataset to fish genomes: atlantic salmon, atlantic cod, zebrafish, medaka, stickleback, sea bass, tetraodon and fugu and looked for mirna sequence conservation. as shown in fig. 8a, 7 % of the rainbow trout mirnas are found in all the other selected species, while 67 % of the trout mirnas are found in only one other species. those 7 % of mirnas found in all fish species are common mirnas also expressed in most vertebrates. one to 4 % of the rainbow trout mirnas are found in either of the selected species. interestingly, 14 % of the rainbow trout mirnas are not found in any of those species, indicating that they might be rainbow trout-specific. it was shown previously in other species that a subset of mirnas can be species-specific. for instance, mirnas identified in zebra finch were found to be avian specific and among them, mirnas were found only in zebra finch. moreover, as shown in fig. 8b, most of the rainbow trout mirnas are found in the atlantic salmon genome while only to 13 % are found in the other species. this could be partially explain by the fact that salmon and rainbow trout are closely related species that share the last round of whole genome duplication while the other species are more distant from rainbow trout and did not undergo the ss4r. but, interestingly, the conservation pattern is very similar among these species indicating that after the third round of whole genome duplication, the mirna repertoire in these fish species is quite stable.fig. mirnas conservation among fish species. conservation analysis of the rainbow trout mirnas repertoire among fish species: atlantic salmon, atlantic cod, zebrafish, medaka, sea bass, stickleback, tetraodon and fugu pie chart representing the percentage of rainbow trout mirnas that are found among the fish genomes stack histograms representing the conservation of rainbow trout mirnas in each fish species  in conclusion, this study represents the first characterization of rainbow trout mirna transcriptome from a wide variety of organs/tissues and describes an extensive repertoire of rainbow trout mirnas. we identified mirna loci in the rainbow trout genome including already known mirnas and putative novel mirnas. we also show that differential expression analysis in different tissues is possible using rna sequencing strategy by confirming the expression data by rt-qpcr with high percentage of correlation. this study provides a starting point for future studies aimed at understanding the roles of mirnas in major physiological process such as growth, reproduction, nutrition, and adaptation. this rainbow trout mirnas repertoire provides a novel resource to advance genomic research in salmonids species. 
micrornas have emerged as important post-transcriptional regulators of gene expression in a wide variety of physiological processes. this study represents the first characterization of rainbow trout mirna transcriptome from a wide variety of tissue and sets an extensive repertoire of rainbow trout mirnas. differential expression analysis was performed in order to identify mirnas exhibiting specific or preferential expression among the analyzed tissues. the aim of the current study was to discover and characterize rainbow trout mirnas in a large number of tissues using next-generation sequencing in order to provide an extensive repertoire of rainbow trout mirnas. a total of different samples corresponding to different tissues or organs were individually sequenced and analyzed independently in order to identify a large number of mirnas with high confidence. these rainbow trout mirnas repertoire provide a novel resource to advance genomic research in salmonid species. they can control both temporal and spatial gene expression and are believed to regulate to 70 % of the genes. this led to the identification of mirna loci in the rainbow trout genome, including already known mirnas. it provides a starting point for future studies aimed at understanding the roles of mirnas in major physiological process such as growth, reproduction or adaptation to stress. the expression data from srna sequencing were confirmed by rt-qpcr. data are however limited for fish species, with only out of the fish species present in mirbase. in addition, novel mirnas are described in rainbow trout that had not been previously reported in other species. micrornarainbow troutrepertoiretissue expressiongene regulationissue-copyright-statement© the author 2016
 micrornas have emerged as important post-transcriptional regulators of gene expression in a wide variety of physiological processes. they can control both temporal and spatial gene expression and are believed to regulate to 70 % of the genes. data are however limited for fish species, with only out of the fish species present in mirbase. the aim of the current study was to discover and characterize rainbow trout mirnas in a large number of tissues using next-generation sequencing in order to provide an extensive repertoire of rainbow trout mirnas.  a total of different samples corresponding to different tissues or organs were individually sequenced and analyzed independently in order to identify a large number of mirnas with high confidence. this led to the identification of mirna loci in the rainbow trout genome, including already known mirnas. differential expression analysis was performed in order to identify mirnas exhibiting specific or preferential expression among the analyzed tissues. in most cases, mirnas exhibit a specific pattern of expression in only a few tissues. the expression data from srna sequencing were confirmed by rt-qpcr. in addition, novel mirnas are described in rainbow trout that had not been previously reported in other species.  this study represents the first characterization of rainbow trout mirna transcriptome from a wide variety of tissue and sets an extensive repertoire of rainbow trout mirnas. it provides a starting point for future studies aimed at understanding the roles of mirnas in major physiological process such as growth, reproduction or adaptation to stress. these rainbow trout mirnas repertoire provide a novel resource to advance genomic research in salmonid species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords micrornarainbow troutrepertoiretissue expressiongene regulationissue-copyright-statement© the author 2016 