BACKGROUND
studies of natural populations lie at the core of understanding the evolution of complex, ecologically relevant phenotypes.
high-throughput approaches to the study of gene functions have accelerated discoveries of the genetic underpinnings of many traits in model organisms, but until recently organisms with well-understood ecology typically lacked sophisticated genomic tools.
model laboratory systems have contributed enormously to the understanding of genetics, gene expression, and the functional interactions of genes; however, the ecological relevance of these findings must also be studied within outbred populations responding to natural environmental challenges.
by combining the power of natural systems with similar sets of genomics tools developed in laboratory systems, a deeper understanding of the molecular basis of adaptive traits and the mechanisms of biodiversity is achieved in nature.
for example, the repeated loss of armor in alaskan stickleback populations is due to a parallel regulatory mutation  <cit> , and the divergence of cichlid visual systems appears to be driven by changes in expression of opsins, rather than structural changes  <cit> .
neither of these findings would have been possible without combining the extensive genomic and natural history information available in these systems.
the introduction of high-throughput sequencing technologies has led to significant declines in the time and cost required to generate genomic tools for functional studies.
for example,  <dig> new genomes were published in  <dig> alone .
however, these genomes are still dominated by invertebrates and prokaryotes.
only  <dig> of the  <dig> sequenced genomes were of vertebrates, reflecting the high cost of sequencing large and complex vertebrate genomes.
in contrast, transcriptome sequencing – the sequence of all transcripts present in a single cell type, tissue type, or entire organism under defined conditions – has emerged as a cost-effective means of rapidly acquiring functional sequence information for non-model systems  <cit> .
comprehensive transcriptomes have recently been characterized for several well-studied natural animal species including the glanville fritillary butterfly  <cit> , staghorn coral  <cit> , horned beetle  <cit> , garter snake  <cit> , great tit  <cit> , and asian tiger mosquito  <cit> .
these transcriptomes provide species-specific genomic information needed to employ genomic approaches in natural systems where the tools were previously lacking, but without the prohibitive costs and time required for sequencing of a full genome.
for example, species-specific microarrays can be developed from transcriptome sequence to assess gene expression in natural populations  <cit> , an approach we employ here.
the acquisition of genomic information regarding expressed sequences is a rapid method for identification of meaningful genetic divergence between species  and the early gene-expression divergence  <cit>  that is thought to play a major role in speciation  <cit> .
here, we seek to add to this growing store of genomic information.
the dark-eyed junco  is a classic avian system that has been extensively studied for more than a century and will be made even more useful with functional genomics.
this seasonally-breeding north american sparrow was the first vertebrate animal in which the effect of photoperiod on seasonality was demonstrated  <cit> , and its behavior, ecology, and physiology have been extensively studied  <cit> , as has its tendency to diverge phenotypically and genetically across its geographic range  <cit> .
one population has been monitored for 30 years and has allowed for assessment of temporal and individual variation in hormone levels, parental behavior, extra-pair mating, and breeding phenology  <cit> .
this population has also emerged as a model for phenotypic engineering  <cit> : over  <dig> studies involving the manipulation of the hormonal phenotypes of free-living individuals have made it possible to relate hormonal variation to variation in phenotype .
the addition of transcriptome information will allow for deeper understanding of the mechanisms behind variation in behavior and physiology as well as how natural selection acts on that variation.
further, the junco offers unique opportunities to study rapid evolutionary divergence at the level of populations.
the dark-eyed junco consists of five morphologically distinct subspecific groups that are thought to have diverged over the past  <dig>  years  <cit> .
in addition, approximately 30 years ago a population of juncos colonized the city of san diego and has undergone rapid phenotypic divergence from its ancestral population in physiology and behavior  <cit> .
previous attempts to reveal the phylogenetic relationships among the groups of juncos have been frustrated by the lack of genetic differentiation owing to recent divergence and a paucity of genetic markers  <cit> .
genomic-level information will open new lines of research in this natural system including allowing gene-expression analysis, targeted re-sequencing, and identification of genes recently under selection, each of which will allow greater insight into the evolution and mechanisms of hormone-mediated phenotypes and natural breeding biology.
we sequenced a transcriptome of this ecologically and evolutionarily well-characterized species, returning substantial sequence diversity and expression information from a relatively shallow sequence coverage depth of long reads.
in this report, we describe our approach and demonstrate the utility of these data in defining genes, identifying potential sequence variants, and confirming expression with a custom microarray.
RESULTS
sampling and sequencing
rna was extracted from  <dig> tissues  from one male and one female adult dark-eyed junco  and prepared into normalized sequencing libraries .
two pools, one from each individual, were sequenced in parallel using gs-flx titanium pyrosequencing , yielding  <dig>  million reads totaling  <dig> million nucleotides with a mean length of  <dig> base pairs  after adaptor trimming.
the quality-filtered  reads have been deposited in the sequence reads archive  database under accession numbers srx <dig>  and srx <dig> .
sampling decisions are a critical question for transcriptome projects as the genes recovered are directly related to the tissues, individuals, and states that are chosen for inclusion.
research in the junco has historically focused on adults and their breeding behavior.
the use of only two adult individuals limits the identification of some classes of genes, such as the developmental genes expressed in growing young; however, the decision also improves our ability to confidently assemble sequences by reducing concerns about integrating allelic variation.
future interest in other conditions, or in the identification of more extensive polymorphic markers for comparing populations, will be able to use this assembly as a reference to improve and focus effort on the condition of interest.
in other systems, the relative importance of these questions have led to some projects sequencing only developing individuals  <cit> , across multiple ages , across multiple eco-types , only specific tissues, or simply more individuals .
in the junco system, this initial approach provides, in our opinion, the most widely valuable research tools for currently anticipated applications and the strongest base from which to launch future projects.
assembly
sequencing reads were assembled using gs de novo assembler , resulting in  <dig>  contigs assembled from  <dig>  reads  covering  <dig>  million bases of sequence with an average length of 884 bp and 12-fold average coverage with  <dig>  singletons remaining unassembled .
the assembled contigs have been deposited in the ncbi transcriptome shotgun assembly  database under accession numbers jv157086-jv <dig>  this assembly is similar to other de novo transcriptome assemblies, which have been characterized by  <dig>  to  <dig>  contigs with 63% to 90% of reads assembled  <cit> .
variation between individuals and alleles can artificially break contigs, as can alternative splicing  <cit> , so further assembly was required to more accurately estimate the number of unique genes in this transcriptome.
sequencing reads were assembled into contigs which were further grouped into isotigs and isogroups .
we further combined contigs into groups based on shared broken reads in the initial assembly following manufacturer's directions .
briefly, many contigs are broken apart by the assembly software due to variability that can be induced by gene duplications, splice variants or even allelic variation  <cit> .
the gs mapper assembly software stores information about these breaks and then pools contigs that shared broken reads into clusters called isogroups; the contigs within a cluster that are joined by broken reads are called isotigs.
in total, the junco assembly yielded  <dig>  isotigs  in  <dig>  isogroups .
each isogroup likely represents a gene, while unique isotigs typically represent alternative splice forms; however, the isotigs may also represent divergent alleles, or members of a gene family  <cit> .
the current depth of sequencing precludes an accurate distinction between these possibilities, but future re-sequencing projects and gene expression studies could potentially distinguish between splice variants and recent duplicates.
the ensembl release  <dig>  <cit>  for the zebra finch , accessed via biomart  <cit>  contains  <dig>  predicted or sequenced genes for the zebra finch, suggesting that our assembly of  <dig>  isogroups may be incomplete  or that there are more expressed regions in bird genomes than predicted by current zebra finch sequencing, annotation, and gene models.
reference assembly
in order to confirm the validity of our sequences and to test the de novo assembly against a reference assembly, we also assembled the junco transcriptome using gmap  <cit>  with standard parameters against the closest available draft genome assembly: the zebra finch the first passerine genome sequencing project  <cit> , and a species that diverged from juncos approximately  <dig> ma ago  <cit> .
of the  <dig> , <dig> reads from the junco transcriptome used for this assembly,  <dig> , <dig>  had a significant alignment to the zebra finch genome identified by the software, with an average of  <dig> % identity.
however, due to the limitation imposed by introns, the lengths of the assembled regions from the reference assembly are substantially shorter , and split into many more groups, than our de novo assembly .
for this reason, we chose to use the de novo assembly for all further analyses.
annotation
the isotigs and singletons were queried against the ncbi non-redundant protein database <cit>  using blastx to identify homologous, annotated genes.
from this search,  <dig>  of the isotigs and  <dig>  of the singletons  returned a significant homolog .
the proportion of assignment for isotigs is slightly higher than that of other de novo transcriptomes , possibly because our isotigs are longer than the contigs used for annotation in other transcriptome annotations   <cit> .
isotigs covered an average of  <dig> % of the protein to which they aligned; singletons covered an average of only  <dig> % of the protein to which they aligned, consistent with their shorter length.
isotigs were, on average,  <dig> % identical across the full length of their alignment, while singletons were  <dig> % identical.
sequences were annotated by sequence similarity to multiple databases, yielding the above number of annotations.
because isogroups represent the full genetic unit, we sought to combine the isotigs into a single annotation for each isogroup, resulting in an annotation for 48% of isogroups .
for nearly all annotated isogroups , only a single annotation was identified for all of its member isotigs.
for a subset of isogroups , multiple annotations were identified but these were confirmed synonymous by literature or alignment searches to confidently assign an isogroup annotation .
some isogroups  could not be reduced to a single annotation and might represent errors in assembly or transcripts with no currently identifiable homology.
our ability to assign a single annotation to most  of annotated isogroups suggests that our assembly accurately characterizes many of the genes of the junco.
of the  <dig>  unique annotations identified among the isogroups reduced to a single annotation,  <dig>   are represented by a single isogroup; the remaining  <dig>  are represented by  <dig>  isogroups .
multiple isogroups assigned to a single annotation suggests that some true genes in our dataset may be incompletely assembled, or represent recently diverged gene duplicates.
for example,  <dig> isogroups are annotated as “nebulin,” which is a conserved, single-copy gene in vertebrates  <cit> , coding for an actin binding protein with multiple isoforms  <cit>  that can complicate transcriptome assembly.
the human version of nebulin has  <dig>  amino acids and contains  <dig> exons at least  <dig> of which are alternatively spliced  <cit> .
additionally, some annotations are not for specific gene products: there are  <dig>  isogroups assigned to nine annotations named some variant of “unnamed” or “hypothetical” proteins.
the presence of multiple isogroups annotated as a single gene suggests that further assembly, or additional sequencing, may be necessary to complete the assembly of some large or complex genes.
functional annotation
gene ontology  is a hierarchical description of gene function that classifies genes based on known or predicted function in model organisms  <cit> .
the use of go terms allows a broader assessment of our annotation and permits the assignment of functional roles to individual genes.
there are limitations to assigning go terms by sequence similarity alone, which can result in over-assignment of go terms to genes that have functionally diverged  <cit> .
consequently, we are not placing high levels of confidence in any particular go assignment, but rather we are investigating the large-scale patterns revealed by these functional annotations.
while there are still some concerns with this approach, preliminary microarray results reveal meaningful and expected go terms , suggesting that our functional annotation is sufficient for broad assessments with the caveat that confidence in any given assignment should be limited.
we assigned go terms, based on sequence similarity, using blast2go  <cit>  and set a strict threshold criterion of e < 10- <dig> against the ncbi non-redundant protein database  <cit> .
this process provided at least one go term for 83% of annotated isogroups  and 5% of annotated singletons  at the “inferred from sequence similarity” level of evidence  <cit> .
this reduced ability to functionally classify singletons is likely due to their shorter length , which reduces the length and quality of sequence alignments that are possible, especially considering the stringent criterion employed.
we identified a total of  <dig>  go term annotations representing  <dig>  unique go terms distributed throughout the go graph .
pathway annotation
we additionally annotated the junco transcriptome sequences by mapping to the kyoto encyclopedia of genes and genomes  pathway annotation  <cit> .
briefly, assembled isotigs and singletons were aligned to protein sequences from the ref-seq databases  <cit>  for zebra finch, chicken, mouse, and human using tblastx, resulting in  <dig>  isotigs  and  <dig>  singletons being assigned across all  <dig> unique pathways available for annotation .
these pathways can, in the future, be targeted for manipulation or sequencing.
in addition, gene expression studies will likely use this approach to analyze changes to whole pathways, an approach which may be more sensitive and interpretable than focusing on single genes  <cit> , to identify small changes in expression level with phenotypic consequences.
orthology group assignment
in addition to our other annotations, we assigned the junco sequences to orthodb  <cit>  orthology groups.
we aligned junco sequences to the ensembl protein database for zebra finch  <cit>  with blastx with a criteria of e < 10- <dig>  and assigned the top alignment  to each isotig and singleton.
we then determined whether all isotigs in an isogroup were assigned to the same protein.
this returned a single annotation for  <dig>  isogroups , and multiple annotations for  <dig> isogroups that we excluded from further portions of this analysis.
in addition,  <dig>  singletons  were assigned to an ensembl protein.
among the isogroups,  <dig>  annotations were assigned to only a single isogroup, and an additional  <dig>  were assigned to multiple isogroups.
including the singletons returned  <dig>  additional unique annotations.
we then used the orthodb database  <cit>  to assign each junco sequence as the orthology group that the corresponding zebra finch protein.
this process resulted in assignment to  <dig>  of the  <dig>  orthology groups previously identified in zebra finch  <cit> .
these data will allow future studies to focus on the divergence of orthologous gene families between junco and closely related species and may aid in the identification of recently duplicated genes in the junco.
completeness
in order to assess the completeness of this transcriptome based on our sequencing efforts, we searched for significant sequence alignments for the assembled isotigs and singletons against the eukaryotic clusters of orthologous group  database  <cit> .
specifically, we searched for a set of  <dig> genes identified to be present as single copies in all bilaterians  studied to date  <cit> .
this reciprocal blast analysis identified homologs for  <dig> core genes  in our dataset .
this high level of coverage is slightly lower  to that found for large-scale genome sequences, which generally identify 99% of these core genes  <cit> .
the best junco alignment for each kog group aligned with a mean of  <dig> % identity and covered an average of  <dig> %  of the aligned protein.
this alignment coverage is likely artificially reduced by the fact that only one isotig  was counted for this length, meaning that any alternative splicing  could result in a low reported coverage.
other transcriptome projects have not, to our knowledge, used this analysis.
these results provide strong support that the junco transcriptome has been sequenced to sufficient depth and from a sufficiently diverse pool of sampled rna to uncover most of the expected genes.
confirmation of expression
to confirm that our putative transcripts are expressed, we constructed a custom 12-plex microarray  and conducted an expression experiment involving four tissues  in male and female juncos.
the full results of this experiment will be described elsewhere; this initial analysis focuses simply on confirming the expression of genes in the tissues of wild-caught individuals.
we confirmed the expression of  <dig>   of the contigs  isogroups) and  <dig>   of the singletons on the array in at least one sex-tissue combination .
the isogroups and singletons that did not show expression in these analyzed tissues may be tissue/condition-specific genes that are unexpressed in our sampled tissues, or could represent spurious sequences that do not accurately represent expressed transcripts.
on the whole, this result suggests that our sequenced genes are largely accurate reflections of expressed genes in the junco.
sequence variants
allelic variants, such as single nucleotide polymorphisms , are powerful tools for population genetic analysis and identification of population structure.
the junco system currently only has nine microsatellite markers, which have been used extensively for paternity analysis in one population  <cit> .
in addition,  <dig> snps were identified across multiple junco species, and this number was sufficient to identify species, but not subspecies, divergence  <cit> .
the addition of more genetic markers will greatly improve the ability to do large population comparisons, including further refining the current junco subspecies phylogeny  <cit> , and identifying variants that may play a role in the current rapid divergence of the genus junco.
to this end, we have conservatively identified  <dig>  unique potential snps in  <dig>  isogroups of this transcriptome  for an average rate of  <dig>  potential variants per  <dig> basepairs of assembled sequence.
while this rate of sequence variant identification is low compared to transcriptomes sequencing a greater number of individuals  <cit> , these potential sequence markers will provide a strong starting point for future studies on population genetics and divergence in the genus junco.
CONCLUSIONS
we have successfully sequenced a transcriptome of an ecological model songbird, the dark-eyed junco, using pyrosequencing and de novo assembly.
through our assembly process, we identified  <dig>  putative genes – half of which have been annotated and three-quarters of which were validated by microarray investigation.
these genes, including over  <dig>  potential sequence variants, will immediately begin to inform the study of the junco and will provide a valuable resource for the study of many songbird species.
based on the identification of putatively conserved genes, we have demonstrated 90% coverage.
the acquisition of these sequence data adds genomic resources to another non-model system.
by combining these data  with the historical study of a natural population, a solid foundation has been laid to advance the study of ecology, evolution, and behavior.
we have already begun to exploit these novel tools in gene expression studies, showing in this approach that the majority of our sequenced genes are expressed under at least some natural conditions.
further research will extend these tools to deeper study of the transcriptomic responses of juncos to environmental stimuli, as well as assist in guiding traditional sequencing projects.

 studies of natural populations lie at the core of understanding the evolution of complex, ecologically relevant phenotypes. high-throughput approaches to the study of gene functions have accelerated discoveries of the genetic underpinnings of many traits in model organisms, but until recently organisms with well-understood ecology typically lacked sophisticated genomic tools. model laboratory systems have contributed enormously to the understanding of genetics, gene expression, and the functional interactions of genes; however, the ecological relevance of these findings must also be studied within outbred populations responding to natural environmental challenges. by combining the power of natural systems with similar sets of genomics tools developed in laboratory systems, a deeper understanding of the molecular basis of adaptive traits and the mechanisms of biodiversity is achieved in nature. for example, the repeated loss of armor in alaskan stickleback populations is due to a parallel regulatory mutation, and the divergence of cichlid visual systems appears to be driven by changes in expression of opsins, rather than structural changes. neither of these findings would have been possible without combining the extensive genomic and natural history information available in these systems. the introduction of high-throughput sequencing technologies has led to significant declines in the time and cost required to generate genomic tools for functional studies. for example, new genomes were published in alone. however, these genomes are still dominated by invertebrates and prokaryotes. only of the sequenced genomes were of vertebrates, reflecting the high cost of sequencing large and complex vertebrate genomes. in contrast, transcriptome sequencing – the sequence of all transcripts present in a single cell type, tissue type, or entire organism under defined conditions – has emerged as a cost-effective means of rapidly acquiring functional sequence information for non-model systems. comprehensive transcriptomes have recently been characterized for several well-studied natural animal species including the glanville fritillary butterfly, staghorn coral, horned beetle, garter snake, great tit, and asian tiger mosquito. these transcriptomes provide species-specific genomic information needed to employ genomic approaches in natural systems where the tools were previously lacking, but without the prohibitive costs and time required for sequencing of a full genome. for example, species-specific microarrays can be developed from transcriptome sequence to assess gene expression in natural populations, an approach we employ here. the acquisition of genomic information regarding expressed sequences is a rapid method for identification of meaningful genetic divergence between species and the early gene-expression divergence that is thought to play a major role in speciation. here, we seek to add to this growing store of genomic information. the dark-eyed junco is a classic avian system that has been extensively studied for more than a century and will be made even more useful with functional genomics. this seasonally-breeding north american sparrow was the first vertebrate animal in which the effect of photoperiod on seasonality was demonstrated, and its behavior, ecology, and physiology have been extensively studied, as has its tendency to diverge phenotypically and genetically across its geographic range. one population has been monitored for 30 years and has allowed for assessment of temporal and individual variation in hormone levels, parental behavior, extra-pair mating, and breeding phenology. this population has also emerged as a model for phenotypic engineering: over studies involving the manipulation of the hormonal phenotypes of free-living individuals have made it possible to relate hormonal variation to variation in phenotype. the addition of transcriptome information will allow for deeper understanding of the mechanisms behind variation in behavior and physiology as well as how natural selection acts on that variation. further, the junco offers unique opportunities to study rapid evolutionary divergence at the level of populations. the dark-eyed junco consists of five morphologically distinct subspecific groups that are thought to have diverged over the past  years. in addition, approximately 30 years ago a population of juncos colonized the city of san diego and has undergone rapid phenotypic divergence from its ancestral population in physiology and behavior. previous attempts to reveal the phylogenetic relationships among the groups of juncos have been frustrated by the lack of genetic differentiation owing to recent divergence and a paucity of genetic markers. genomic-level information will open new lines of research in this natural system including allowing gene-expression analysis, targeted re-sequencing, and identification of genes recently under selection, each of which will allow greater insight into the evolution and mechanisms of hormone-mediated phenotypes and natural breeding biology. we sequenced a transcriptome of this ecologically and evolutionarily well-characterized species, returning substantial sequence diversity and expression information from a relatively shallow sequence coverage depth of long reads. in this report, we describe our approach and demonstrate the utility of these data in defining genes, identifying potential sequence variants, and confirming expression with a custom microarray.  sampling and sequencing rna was extracted from tissues from one male and one female adult dark-eyed junco and prepared into normalized sequencing libraries. two pools, one from each individual, were sequenced in parallel using gs-flx titanium pyrosequencing, yielding million reads totaling million nucleotides with a mean length of base pairs after adaptor trimming. the quality-filtered reads have been deposited in the sequence reads archive database under accession numbers srx and srx. sampling decisions are a critical question for transcriptome projects as the genes recovered are directly related to the tissues, individuals, and states that are chosen for inclusion. research in the junco has historically focused on adults and their breeding behavior. the use of only two adult individuals limits the identification of some classes of genes, such as the developmental genes expressed in growing young; however, the decision also improves our ability to confidently assemble sequences by reducing concerns about integrating allelic variation. future interest in other conditions, or in the identification of more extensive polymorphic markers for comparing populations, will be able to use this assembly as a reference to improve and focus effort on the condition of interest. in other systems, the relative importance of these questions have led to some projects sequencing only developing individuals, across multiple ages, across multiple eco-types, only specific tissues, or simply more individuals. in the junco system, this initial approach provides, in our opinion, the most widely valuable research tools for currently anticipated applications and the strongest base from which to launch future projects. assembly sequencing reads were assembled using gs de novo assembler, resulting in contigs assembled from reads covering million bases of sequence with an average length of 884 bp and 12-fold average coverage with singletons remaining unassembled. the assembled contigs have been deposited in the ncbi transcriptome shotgun assembly database under accession numbers jv157086-jv this assembly is similar to other de novo transcriptome assemblies, which have been characterized by to contigs with 63% to 90% of reads assembled. variation between individuals and alleles can artificially break contigs, as can alternative splicing, so further assembly was required to more accurately estimate the number of unique genes in this transcriptome. sequencing reads were assembled into contigs which were further grouped into isotigs and isogroups. we further combined contigs into groups based on shared broken reads in the initial assembly following manufacturer's directions. briefly, many contigs are broken apart by the assembly software due to variability that can be induced by gene duplications, splice variants or even allelic variation. the gs mapper assembly software stores information about these breaks and then pools contigs that shared broken reads into clusters called isogroups; the contigs within a cluster that are joined by broken reads are called isotigs. in total, the junco assembly yielded isotigs in isogroups. each isogroup likely represents a gene, while unique isotigs typically represent alternative splice forms; however, the isotigs may also represent divergent alleles, or members of a gene family. the current depth of sequencing precludes an accurate distinction between these possibilities, but future re-sequencing projects and gene expression studies could potentially distinguish between splice variants and recent duplicates. the ensembl release for the zebra finch, accessed via biomart contains predicted or sequenced genes for the zebra finch, suggesting that our assembly of isogroups may be incomplete or that there are more expressed regions in bird genomes than predicted by current zebra finch sequencing, annotation, and gene models. reference assembly in order to confirm the validity of our sequences and to test the de novo assembly against a reference assembly, we also assembled the junco transcriptome using gmap with standard parameters against the closest available draft genome assembly: the zebra finch the first passerine genome sequencing project, and a species that diverged from juncos approximately ma ago. of the, reads from the junco transcriptome used for this assembly,, had a significant alignment to the zebra finch genome identified by the software, with an average of % identity. however, due to the limitation imposed by introns, the lengths of the assembled regions from the reference assembly are substantially shorter, and split into many more groups, than our de novo assembly. for this reason, we chose to use the de novo assembly for all further analyses. annotation the isotigs and singletons were queried against the ncbi non-redundant protein database using blastx to identify homologous, annotated genes. from this search, of the isotigs and of the singletons returned a significant homolog. the proportion of assignment for isotigs is slightly higher than that of other de novo transcriptomes, possibly because our isotigs are longer than the contigs used for annotation in other transcriptome annotations. isotigs covered an average of % of the protein to which they aligned; singletons covered an average of only % of the protein to which they aligned, consistent with their shorter length. isotigs were, on average, % identical across the full length of their alignment, while singletons were % identical. sequences were annotated by sequence similarity to multiple databases, yielding the above number of annotations. because isogroups represent the full genetic unit, we sought to combine the isotigs into a single annotation for each isogroup, resulting in an annotation for 48% of isogroups. for nearly all annotated isogroups, only a single annotation was identified for all of its member isotigs. for a subset of isogroups, multiple annotations were identified but these were confirmed synonymous by literature or alignment searches to confidently assign an isogroup annotation. some isogroups could not be reduced to a single annotation and might represent errors in assembly or transcripts with no currently identifiable homology. our ability to assign a single annotation to most of annotated isogroups suggests that our assembly accurately characterizes many of the genes of the junco. of the unique annotations identified among the isogroups reduced to a single annotation, are represented by a single isogroup; the remaining are represented by isogroups. multiple isogroups assigned to a single annotation suggests that some true genes in our dataset may be incompletely assembled, or represent recently diverged gene duplicates. for example, isogroups are annotated as “nebulin,” which is a conserved, single-copy gene in vertebrates, coding for an actin binding protein with multiple isoforms that can complicate transcriptome assembly. the human version of nebulin has amino acids and contains exons at least of which are alternatively spliced. additionally, some annotations are not for specific gene products: there are isogroups assigned to nine annotations named some variant of “unnamed” or “hypothetical” proteins. the presence of multiple isogroups annotated as a single gene suggests that further assembly, or additional sequencing, may be necessary to complete the assembly of some large or complex genes. functional annotation gene ontology is a hierarchical description of gene function that classifies genes based on known or predicted function in model organisms. the use of go terms allows a broader assessment of our annotation and permits the assignment of functional roles to individual genes. there are limitations to assigning go terms by sequence similarity alone, which can result in over-assignment of go terms to genes that have functionally diverged. consequently, we are not placing high levels of confidence in any particular go assignment, but rather we are investigating the large-scale patterns revealed by these functional annotations. while there are still some concerns with this approach, preliminary microarray results reveal meaningful and expected go terms, suggesting that our functional annotation is sufficient for broad assessments with the caveat that confidence in any given assignment should be limited. we assigned go terms, based on sequence similarity, using blast2go and set a strict threshold criterion of e < 10- against the ncbi non-redundant protein database. this process provided at least one go term for 83% of annotated isogroups and 5% of annotated singletons at the “inferred from sequence similarity” level of evidence. this reduced ability to functionally classify singletons is likely due to their shorter length, which reduces the length and quality of sequence alignments that are possible, especially considering the stringent criterion employed. we identified a total of go term annotations representing unique go terms distributed throughout the go graph. pathway annotation we additionally annotated the junco transcriptome sequences by mapping to the kyoto encyclopedia of genes and genomes pathway annotation. briefly, assembled isotigs and singletons were aligned to protein sequences from the ref-seq databases for zebra finch, chicken, mouse, and human using tblastx, resulting in isotigs and singletons being assigned across all unique pathways available for annotation. these pathways can, in the future, be targeted for manipulation or sequencing. in addition, gene expression studies will likely use this approach to analyze changes to whole pathways, an approach which may be more sensitive and interpretable than focusing on single genes, to identify small changes in expression level with phenotypic consequences. orthology group assignment in addition to our other annotations, we assigned the junco sequences to orthodb orthology groups. we aligned junco sequences to the ensembl protein database for zebra finch with blastx with a criteria of e < 10- and assigned the top alignment to each isotig and singleton. we then determined whether all isotigs in an isogroup were assigned to the same protein. this returned a single annotation for isogroups, and multiple annotations for isogroups that we excluded from further portions of this analysis. in addition, singletons were assigned to an ensembl protein. among the isogroups, annotations were assigned to only a single isogroup, and an additional were assigned to multiple isogroups. including the singletons returned additional unique annotations. we then used the orthodb database to assign each junco sequence as the orthology group that the corresponding zebra finch protein. this process resulted in assignment to of the orthology groups previously identified in zebra finch. these data will allow future studies to focus on the divergence of orthologous gene families between junco and closely related species and may aid in the identification of recently duplicated genes in the junco. completeness in order to assess the completeness of this transcriptome based on our sequencing efforts, we searched for significant sequence alignments for the assembled isotigs and singletons against the eukaryotic clusters of orthologous group database. specifically, we searched for a set of genes identified to be present as single copies in all bilaterians studied to date. this reciprocal blast analysis identified homologs for core genes in our dataset. this high level of coverage is slightly lower to that found for large-scale genome sequences, which generally identify 99% of these core genes. the best junco alignment for each kog group aligned with a mean of % identity and covered an average of % of the aligned protein. this alignment coverage is likely artificially reduced by the fact that only one isotig was counted for this length, meaning that any alternative splicing could result in a low reported coverage. other transcriptome projects have not, to our knowledge, used this analysis. these results provide strong support that the junco transcriptome has been sequenced to sufficient depth and from a sufficiently diverse pool of sampled rna to uncover most of the expected genes. confirmation of expression to confirm that our putative transcripts are expressed, we constructed a custom 12-plex microarray and conducted an expression experiment involving four tissues in male and female juncos. the full results of this experiment will be described elsewhere; this initial analysis focuses simply on confirming the expression of genes in the tissues of wild-caught individuals. we confirmed the expression of of the contigs isogroups) and of the singletons on the array in at least one sex-tissue combination. the isogroups and singletons that did not show expression in these analyzed tissues may be tissue/condition-specific genes that are unexpressed in our sampled tissues, or could represent spurious sequences that do not accurately represent expressed transcripts. on the whole, this result suggests that our sequenced genes are largely accurate reflections of expressed genes in the junco. sequence variants allelic variants, such as single nucleotide polymorphisms, are powerful tools for population genetic analysis and identification of population structure. the junco system currently only has nine microsatellite markers, which have been used extensively for paternity analysis in one population. in addition, snps were identified across multiple junco species, and this number was sufficient to identify species, but not subspecies, divergence. the addition of more genetic markers will greatly improve the ability to do large population comparisons, including further refining the current junco subspecies phylogeny, and identifying variants that may play a role in the current rapid divergence of the genus junco. to this end, we have conservatively identified unique potential snps in isogroups of this transcriptome for an average rate of potential variants per basepairs of assembled sequence. while this rate of sequence variant identification is low compared to transcriptomes sequencing a greater number of individuals, these potential sequence markers will provide a strong starting point for future studies on population genetics and divergence in the genus junco.  we have successfully sequenced a transcriptome of an ecological model songbird, the dark-eyed junco, using pyrosequencing and de novo assembly. through our assembly process, we identified putative genes – half of which have been annotated and three-quarters of which were validated by microarray investigation. these genes, including over potential sequence variants, will immediately begin to inform the study of the junco and will provide a valuable resource for the study of many songbird species. based on the identification of putatively conserved genes, we have demonstrated 90% coverage. the acquisition of these sequence data adds genomic resources to another non-model system. by combining these data with the historical study of a natural population, a solid foundation has been laid to advance the study of ecology, evolution, and behavior. we have already begun to exploit these novel tools in gene expression studies, showing in this approach that the majority of our sequenced genes are expressed under at least some natural conditions. further research will extend these tools to deeper study of the transcriptomic responses of juncos to environmental stimuli, as well as assist in guiding traditional sequencing projects. 
it is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. we used gs-flx titanium sequencing of two normalized libraries of pooled rna samples to characterize a transcriptome of the dark-eyed junco, a north american sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior. though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. in contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. annotation of roughly half of the putative genes was accomplished using sequence similarity, and expression was confirmed for the majority with a preliminary microarray analysis.
 though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. in contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. it is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. we used gs-flx titanium sequencing of two normalized libraries of pooled rna samples to characterize a transcriptome of the dark-eyed junco, a north american sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior.  from a broad pool of rna sampled from tissues throughout the body of a male and a female junco, we sequenced a total of million nucleotides from million reads that were assembled de novo into putative transcripts representing gene sets covering million nucleotides with 12-fold average depth of coverage. annotation of roughly half of the putative genes was accomplished using sequence similarity, and expression was confirmed for the majority with a preliminary microarray analysis. of core bilaterian genes, were recovered within our characterized gene set. gene ontology, orthodb orthology groups, and kegg pathway annotation provide further functional information about the sequences, and potential snps were identified.  the extensive sequence information returned by this effort adds to the growing store of genomic data on diverse species. the extent of coverage and annotation achieved and confirmation of expression, show that transcriptome sequencing provides useful information for ecological model systems that have historically lacked genomic tools. the junco-specific microarray developed here is allowing investigations of gene expression responses to environmental and hormonal manipulations – extending the historic work on natural history and hormone-mediated phenotypes in this system. transcriptomeavespyrosequencingmicroarrayjunco titanium cdna sequencingsingle nucleotide polymorphism. 