BACKGROUND
crustaceans are one of the four major groups that make up the phylum arthropoda, the most speciose and morphologically diverse animal group  <cit> .
despite the fact that arthropods as a whole make up the majority of animal species diversity and biomass, until recently the only arthropod represented in the list of nih model organisms http://www.nih.gov/science/models/ was the fruit fly drosophila melanogaster.
the water flea daphnia pulex was recently added to this list, and is the only crustacean to date with a publicly accessible sequenced genome  <cit> .
as crustaceans are now widely recognized as sister group to the hexapods  <cit> , the phylogenetic position of d. pulex suggests that it could serve as a useful outgroup to insects, providing meaningful comparisons with the many insights into developmental and disease biology provided by work on d. melanogaster.
however, there are no genomic resources on a scale comparable to the d. pulex genome, and so it is still not known to what extent the characteristics of the water flea genome are specific to this animal's ecology or shared by other crustaceans  <cit> .
moreover, comparisons of biomedically relevant processes and mechanisms between d. pulex and other model organisms must be informed by robust phylogenetic hypotheses.
at the moment, which specific subgroup of crustaceans is closest to the hexapods  is still a matter of debate  <cit> , but several phylogenetic hypotheses suggest that branchiopods may be more distant from insects than other crustacean groups  <cit> .
crustaceans have long been the subject of ecological and evolutionary study, as well as being lucrative commercial species for human consumption.
even for many of the most intensively studied of these  <cit> , surprisingly few genomic projects have been reported.
most of the crustacean est projects completed to date, notably for the farmed shrimp litopenaeus vannamei   <cit> , the salmon ectoparasite copepod lepeophtheirus salmonis   <cit> , the porcelain crab petrolisthes cinctipes   <cit> , and the lobster homarus americanus   <cit> , have all used sanger sequencing of cdna libraries.
next generation sequencing technologies are increasingly affordable, accessible and robust even for organisms lacking a sequenced genome  <cit> , but have been reported to be applied to a crustacean de novo transcriptome only once, in the antarctic krill euphausia superba  <cit> .
this organism is the subject of ecological and climate change research, but is not a viable laboratory model organism due to its specialized habitat.
while new understanding of developmental and molecular mechanisms in d. pulex are expected to follow from its genome sequence, it is important to note that crustaceans have been the subject of comparative embryology for over a century  <cit> , and in recent decades, of evolutionary developmental biology .
the morphological and molecular mechanistic variations of early embryogenesis, modifications of their body plans and appendage diversifications displayed by crustaceans have all been the subject of studies too numerous to describe here.
however, until recently most comparative analyses of crustaceans have been limited to the study of gene expression or experimental embryology, as crustacean models where functional genetic testing is possible are still few in number.
the limiting factor for comparative functional experiments is often obtaining specific coding sequences of sufficient length.
a large-scale genomic resource for a model crustacean would therefore greatly facilitate development and deployment of transgenic tools.
the amphipod parhyale hawaiensis has emerged as an important laboratory model crustacean species over the last several years  <cit> .
p. hawaiensis was first described in the hawaiian islands  <cit> , but it occupies intertidal marine habitats worldwide.
laboratory husbandry is easy and affordable, and inbred lab cultures produce hundreds of embryos year-round, providing ample material for developmental studies.
fate map and cell lineage analyses of the early embryo show that all three germ layers and the germ line are determined by the eight cell stage  <cit> , and clonal populations show predictable patterns at least up until gastrulation  <cit> .
despite this apparently "mosaic" embryonic development, significant regulative properties have also been described for the embryonic mesoderm and ectoderm  <cit> .
molecular techniques for the study of development, including stable transgenesis  <cit>  and gene knockdown  <cit>  are arguably better established for p. hawaiensis than for any other laboratory crustacean model.
however, the number of p. hawaiensis developmental genes available as genbank accessions is less than  <dig>  progress in understanding the development of p. hawaiensis is thus is limited by the relative paucity of publicly available cloned coding and regulatory regions.
indeed, since the development of germ line transgenesis in p. hawaiensis, its use in developmental studies has been reported only three times  <cit> .
analysis of phenotypes of genomic transgene insertions  <cit>  and case studies of intron sizes  <cit>  are consistent with an extremely large genome size for this amphipod.
accordingly, the genome size of p. hawaiensis is estimated to be  <dig>  gb , very near that of homo sapiens  <cit> .
a genomic bac library for p. hawaiensis has been created and partially sequenced  <cit>  but is not yet available in genbank.
the genome of the closely related amphipod jassa marmorata, with a much smaller genome of  <dig> mbp  has been approved for whole genome, bac end, and est sequencing by the joint genome institute  <cit> , but this work is still ongoing, and laboratory culture of this amphipod is difficult.
a large transcriptome dataset for p. hawaiensis would thus be a highly valuable resource for several scientific communities, and would in addition assist with the annotation of planned amphipod genomic projects.
recent construction of a de novo transcriptome for the milkweed bug oncopeltus fasciatus, which also lacks a sequenced genome  <cit> , suggested that  <dig> pyrosequencing would be a fruitful approach to obtaining a large scale p. hawaiensis transcriptome.
here we present the de novo assembly of a maternal and embryonic transcriptome for p. hawaiensis, sequenced with  <dig> titanium pyrosequencing.
we describe particular features of the p. hawaiensis transcriptome that were revealed during assembly and annotation, including the presence of trans-splicing and enrichment for c2h <dig> zn finger domain-containing transcripts.
we annotate orthologues of genes involved in several major developmental patterning processes, gametogenesis in males and females, and members of major conserved animal signaling pathways.
we observe a high proportion of apparently unique sequences in the transcriptome, and discuss these findings in the light of observations on the d. pulex genome and other existing crustacean genomic resources.
RESULTS
collection and preparation of material for ovarian and embryonic cdna libraries
our goal was to create a transcriptome containing genes relevant to embryonic development, including both maternally provided and zygotically transcribed genes.
all three germ layers and the germ line are determined by the eight cell stage  <cit> , and we wished to capture transcripts from that early stage .
we also wished to sample the intensively studied germ band stage , when major body axes have been patterned and trunk segmentation is ongoing  <cit> , as well as later stages  when organogenesis is predominant.
we therefore collected ovaries  and embryos from all stages of embryogenesis , extracted mrna and prepared cdna for  <dig> pyrosequencing.
because early stage embryos have many fewer cells than later stage embryos, we anticipated that transcripts present in early embryos might suffer low representation if our collection contained equal numbers of embryos of all stages.
we therefore collected greater numbers of earlier stages than of later stages .
sequencing and assembly of the p. hawaiensis transcriptome
we sequenced a total of  <dig> , <dig> reads  with a median read length of  <dig> bp .
for assembly of the de novo transcriptome we used newbler v <dig>  .
newbler's terminology for assembled reads distinguishes between contigs , isotigs , and isogroups .
for continuity, we use this terminology throughout this paper to refer to the products of the newbler assembly.
the default parameters of newbler v <dig>  were used to screen for adapters and eliminate poor quality reads.
the resulting  <dig> , <dig> reads  were then assembled .
<dig> , <dig>  of the screened reads were incorporated into assembled sequences , with  <dig>   singletons remaining.
<dig>  reads  were excluded because they were only partially assembled , from repeat regions , outliers , or too short .
the assembly contained  <dig>  contigs, which grouped into  <dig>  isotigs.
<dig>   of these isotigs were made up of only one contig, and the average number of contigs per isotigs was  <dig> .
the isotig n <dig> length was  <dig>  bp, and the number of isogroups was  <dig>   of these comprised only one isotig, and the mean number of isotigs per isogroup was  <dig> ).
the average coverage per contig was  <dig>  reads/bp .
averaging across all bases in the entire assembly, the average coverage per base pair was  <dig> , meaning that every base pair in the transcriptome was sequenced  <dig>  times on average.
this coverage is high compared to typical numbers for de novo transcriptome assemblies, and should be helpful for distinguishing snps and indels from potential sequencing errors in raw reads  <cit> .
fasta files of all assembly products are freely available from the authors.
blast mapping of non-redundant transcriptome sequences
using an e-value cutoff of 1e- <dig>  we first used blastx to map all non-redundant assembly sequences to nr , and obtained a total of  <dig>  blast hits .
of the  <dig>  isotigs,  <dig>   had at least one hit , and of the  <dig>  singletons,  <dig>   had hits.
the majority of these blast hits were unique: among the isotigs there were  <dig>   unique hits, and among the singletons there were  <dig>   unique hits .
in summary, among all non-redundant assembly sequences , we obtained  <dig>  unique blast hits .
these blast results may mean that the transcriptome contains as many as  <dig>  unique gene transcripts.
however, as for all de novo assemblies, several caveats must be considered.
first, different regions of a single transcript may have different best top blast hits.
our assembly likely contains sequences that belong to the same transcript but are too far apart to be assembled together, and so would be considered "different genes."
as a result, the number of unique blast hits may be an overestimate of unique gene number .
second,  <dig>  isotigs  and  <dig>  singletons  did not yield blast hits that met our e-value cutoff of e- <dig>  these values are comparable to or higher than those obtained in other de novo transcriptome analyses  <cit> .
however, these unmatched sequences may represent transcript fragments whose similarities to known genes is too poor to meet our e-value cutoff, or are non-coding.
it is therefore not formally possible from de novo assembly to know whether the  <dig>  unique blast hit number over- or under-estimates the true number of genes contained in our transcriptome.
<dig>  nr = ncbi non-redundant database
<dig>  because isogroups are collections of isotigs that are hypothesized to originate from the same gene, they do not comprise a single sequence and so cannot be compared by blast to nr.
<dig>  the custom script uniqueblast.pl was applied only to non-redundant assembly sequences .
transcriptome gene ontology  term annotation
we used blast2go  <cit>  to obtain the gene ontology terms associated with the top  <dig> blast hits for each non-redundant assembly sequence.
of the  <dig>  sequences with unique blast hits as per our 1e- <dig> e-value cutoff,  <dig>   of these had go terms associated with them.
to determine whether or not major categories of genes were missing or underrepresented in our transcriptome, we compared the proportions of sequences in selected go term groups to the proportions in these categories observed in the predicted transcript complement of the only crustacean with a sequenced genome, d. pulex .
we did not find significant differences in the proportion of genes in the examined go term categories between the p. hawaiensis transcriptome and the water flea genome, suggesting that our transcriptome does not lack major functional categories of genes.
interestingly, not only were the proportions similar for the two crustaceans, they also closely matched the profiles observed for the sequenced genome of the insect d. melanogaster, and a de novo insect transcriptome from a milkweed bug  <cit>  , suggesting that arthropod species as widely diverged as fruit flies and water fleas share similar proportional gene expression profiles in certain functional genetic categories.
unusual characteristics of the p. hawaiensis transcriptome
p. hawaiensis has been found to employ trans-splicing among its genetic regulatory mechanisms  <cit> .
in this mechanism, sections of transcripts transcribed from independent genomic loci are spliced together post-transcriptionally to form a novel transcript.
such trans-spliced transcripts are recognizable because they contain a diagnostic splice-leader sequence.
we wished to determine to what extent this additional transcript complexity affected our assembly and our ability to assign high-confidence blast annotations.
we therefore processed the trimmed, pre-assembly reads to remove those containing the diagnostic splice-leader sequences , and performed a newbler v <dig>  assembly on the remaining reads.
the results of this assembly were not noticeably different from the complete assembly, nor did the number of unique blast hits increase .
we therefore concluded that the presence of trans-spliced transcripts did not significantly affect our transcriptome assembly or annotation.
when we considered the species identity of the top blast hit for each isotig and singleton , we found that a high proportion  of our assembly sequences most closely matched sequences from other arthropods .
however, an unexpectedly high proportion  was from the lancelet branchiostoma floridae, which is phylogenetically very far removed from p. hawaiensis.
when we examined those sequences with a lancelet sequence as top hit, we noticed that  for many of them, the top  <dig> blast hits were all b. floridae; and  the majority of them seemed to contain c2h <dig> zinc fingers with a specific linker sequence  between c2h <dig> domains.
the genome of at least one other arthropod, the pea aphid acyrthosiphon pisum, has been found to be unusually rich in genes containing the same c2h <dig> zinc finger and diagnostic linker sequence  <cit> .
we reasoned that if both the amphioxus  <cit>  and amphipod genomes happened to contain a high proportion of such genes, this could be responsible for the apparent high similarity of several transcripts in these two species.
to test this, we removed reads containing c2h <dig> zinc finger-encoding sequences, following a previously defined low-stringency definition of a c2h <dig> zinc finger domain  <cit> .
we then assembled the remaining reads with newbler v <dig> , and again scanned for and removed contigs and isotigs containing the motif.
the remaining assembly products were mapped against nr using blastx, and the species identities of their top hits were compared with those obtained from the complete assembly.
we found that the new assembly retained a large proportion  of arthropod hits, but that the number of b. floridae hits had dropped to  <dig> %, and was now comparable to the proportion of hits obtained from other deuterostome phyla .
comparison with other arthropod genomic resources
the blast hit rate of the p. hawaiensis isotigs was  <dig> %.
this is higher than those reported in other de novo transcriptome analyses  <cit> , including that of an arctic crustacean  <cit> , but much lower than the  <dig> % obtained from a previously analyzed de novo insect maternal/embryonic transcriptome  <cit> .
the ~70% of p. hawaiensis isotigs without a high confidence blast hit could be either p. hawaiensis-specific genes, or genes with relatively lower similarity to known genes.
in addition, it is possible that many of our transcripts represent untranslated sequences rather than coding regions , and therefore do not match nr protein sequences.
the relatively low proportion of hits may also reflect the fact that until recently  very few crustacean sequences were included in nr.
the d. pulex genome also contains a high proportion  of apparently daphnia-specific genes, thought to be largely the result of amplification of selected gene families  <cit> .
noting that the e-value cutoff used for the d. pulex analyses  was slightly more relaxed than that used in our initial analysis , we repeated the blastx of our non-redundant p. hawaiensis transcriptome sequences against nr with an e-value cutoff of 1e- <dig>  this yielded  <dig>  unique blast hits .
we therefore found that even adjusting the e-value cutoff to that used for characterization of the d. pulex genome, ~59% of the sequences in this p. hawaiensis transcriptome lack significant similarity to other characterized animal sequences.
these observations suggest that crustaceans may have more species- or clade-specific genes than previously appreciated.
alternatively, these high numbers of apparently lineage-restricted genes may simply reflect the paucity of crustacean genomic resources currently in public databases.
those p. hawaiensis transcriptome sequences which failed to obtain a significant blast hit when compared with nr might share more similarity with other crustaceans.
to test this, we used blast to compare all p. hawaiensis transcriptome sequences that had failed our 1e- <dig> e-value cutoff against nr, with the predicted d. pulex transcriptome  using blastx and a 1e- <dig> e-value cutoff.
we then used blast to compare the obtained sequences with nr to determine their putative identities.
we did not set an e-value limit for this second blast, in order to recover at least some minimal information about the identity of these genes .
we found that  <dig> % of these sequences came from arthropods, only  <dig> % of which were crustaceans.
the low crustacean representation in nr makes it difficult to obtain high confidence blast hits for this group.
we therefore focused on determining what proportion of "nr orphan" p. hawaiensis sequences were highly similar to sequences from d. pulex by comparing the e-values for blast hits against both nr and against the d. pulex predicted transcriptome.
we found that of the  <dig> p. hawaiensis sequences with higher similarity to d. pulex genes than to anything in nr ,  <dig>  of these had e-values at least an order of magnitude higher for d. pulex compared to nr, and  <dig> of these  had e-values greater than one for nr hits, but d. pulex hit e-values of 1e- <dig> or lower.
most of this "daphnia-like" group matched arthropod sequences that were previously annotated as "hypothetical proteins," suggesting that non-insect crustacean sequence annotation could improve future annotation of the existing insect genomes.
the d. pulex genome has been found to contain a high number of genes   <cit> .
without a genome sequence for p. hawaiensis, we cannot accurately estimate gene number in order to perform a rigorous comparison with d. pulex.
however, our transcriptome assembly identified  <dig>  isogroups.
because isogroups are groups of isotigs assembled from the same set of contigs, isogroups may represent putative genes, with each isotig of the isogroup representing a transcript variant, for example a splice variant.
we therefore speculate that p. hawaiensis, with a genome over one order of magnitude larger than that of d. pulex , may also have a high gene number of at least  <dig>  genes.
however, using isogroup number of this de novo transcriptome as a proxy for total gene number has two significant limitations.
the first is the result of our chosen tissue sampling strategy: this transcriptome does not capture postembryonic gene expression.
after hatching, expression of several genes with exclusively juvenile or adult roles is likely, including at minimum additional genes associated with molting, behavior, and gametogenesis.
it is therefore possible that the number of isogroups in our assembly underestimates the true gene number in p. hawaiensis.
the second limitation is the unavoidable result of any de novo assembly, which is that if two sequences from the same transcript do not share significant overlap, they will appear as separate assembly products rather than as a single transcript .
this could result in the number of isogroups being an overestimate of the true number of genes.
a further complication results from newbler v <dig> 's method of handling isogroups made of multiple isotigs.
when we performed our assembly, we limited the number of isotigs in one isogroup to  <dig> .
this has the advantage of avoiding isogroups composed of large numbers of isotigs, as we suspect that in p. hawaiensis, as shown for other animals, the vast majority of genes have fewer than ten splicing isoforms  <cit> .
however, it can also result in problematic isogroup number calculation, because isogroups that exceed the number of isotigs per isogroup threshold are returned to the assembly file as contigs rather than isotigs, thus inflating the gene number estimate.
in summary, in order to determine whether high numbers of species- or clade-specific genes is a general characteristic of crustaceans, and the true extent of species-specific genes for p. hawaiensis, more deep genomic resources will have to be developed for this amphipod and for more crustaceans of diverse classes.
the high gene number of d. pulex is thought to be due to lineage-specific expansions of gene families  <cit> .
these expansions may play adaptive roles in the water flea's ecology  <cit> , or they may be a general feature of crustacean genomes that was previously unappreciated due to the paucity of crustacean genomic resources.
it is therefore of interest to determine the extent of gene family expansion in p. hawaiensis.
in order for our de novo transcriptome to provide a rigorous answer to this question, we would need to distinguish between transcripts of paralogues, and sequences originating from the same transcript that do not overlap enough to belong to the same isotig, or even obtain the same set of top blast hits.
this distinction is not unambiguously possible, given the absence of a reference genome.
however, we performed a preliminary analysis of putative gene expansion in p. hawaiensis, focusing on those gene families found to be expanded in the d. pulex genome.
our analysis conservatively included only those p. hawaiensis isotigs that had a top blast hit against a duplicated d. pulex gene.
we also made sure that these isotigs had the same set of top blast hits but belonged to different isogroups, and were therefore likely to represent paralogues rather than splice variants .
we found that in general, highly expanded d. pulex gene families had more putative paralogues  in p. hawaiensis, relative to less expanded gene families .
it is therefore possible that gene family expansions are a common feature of certain enzymatic gene families in some crustaceans, although genome sequencing will ultimately be needed to provide definitive answers to this question.
assessment of depth and transcript coverage of the transcriptome
although the  <dig>  unique blast hits that we identified may represent unique genes, as discussed extensively above we cannot verify how many transcripts are encoded by the genome in the absence of an annotated genome sequence.
however, we wished to estimate how deeply we had sequenced those transcripts that were present in our oogenesis/embryogenesis cdna sample.
to do this, we assembled progressively larger random subsets of the total reads.
for each subassembly, we used blast to compare our non-redundant transcriptome sequences with nr, and assessed the number of unique blast hits to see to what extent adding sequence data from the same sample improved gene discovery.
even with the maximum number of over  <dig> million reads, we did not observe a plateau in the gene discovery rate .
this suggests that despite the considerable depth of our coverage, sequencing more reads from the same sample could yield even more new gene discovery.
this is in contrast with a recently constructed de novo maternal and embryonic transcriptome for an insect, which was comprised of only  <dig> million reads and yet saturated gene discovery in the cdna sample that was sequenced  <cit> .
the increased complexity of the amphipod transcriptome may reflect its large genome size , high putative gene number , or large predicted intron sizes  <cit> .
total isotig length increased steadily as progressively larger subsets of reads were assembled .
while small numbers of isotigs over  <dig>  bp could be obtained with as few as ~ <dig>  reads, robust recovery of isotigs longer than  <dig>  bp required assembly of at least 60% of our total reads, or ~ <dig>  million reads.
this demonstrates that increased depth of sequencing, in addition to improving gene discovery, has the added benefit of increasing predicted transcript lengths, thereby facilitating their annotation and making them more immediately useful for downstream applications.
in a related analysis, we searched the transcriptome for the presence of the small number of p. hawaiensis developmental genes available as genbank accessions, and found that  <dig> %  were present .
however, only for one of these genes  did our transcriptome add sequence data to the genbank accession .
this may be a reflection of both the relatively rarity of these transcripts, and the fact that those genes identified to date have been the subject of intense developmental studies, and so sequences of considerable length have already been cloned to close to full length  <cit> .
the assembly yielded isotigs as long as  <dig>  bp, with average length  <dig>  bp .
however, we wished to determine what fraction of true full transcript length was likely to be contained by these isotigs.
to do this, we used the methods of o'neil and colleagues  <cit>  in calculating the ortholog hit ratio for isotigs, contigs, and singletons.
we found that  <dig> % of isotigs represented over 50% of putative true full-length transcripts compared with predicted d. pulex transcripts, and  <dig> % of isotigs were over 80% full length .
these ratios were not significantly higher than those obtained by comparing transcriptome sequences to d. melanogaster transcripts , suggesting that p. hawaiensis sequences have comparable similarity to those of the water flea and the fruit fly.
further, comparing transcripts from the fully sequenced genomes of d. pulex and d. melanogaster yields ortholog hit ratio values of  <dig> %  and  <dig> %  respectively, which are similar to the p. hawaiensis/d.
pulex comparison values.
these values are consistent with the increasing support for hypotheses of crustacean paraphyly, which predict large divergences between all of the lineages leading to these arthropod species .
the current state of understanding of crustacean-hexapod phylogenetic relationships  therefore does not allow straightforward predictions of which pair of these three transcriptomes should be most similar to each other.
annotation of signaling pathway genes
future functional genetic studies in p. hawaiensis will likely focus on elucidating the function of highly conserved metazoan genes in this amphipod.
we therefore annotated the transcriptome for the presence of genes belonging to major conserved animal signaling pathways  <cit> .
using the kegg pathways as a guideline  <cit> , we searched for p. hawaiensis homologues of these genes using blast using an e-value cutoff of 1e- <dig>  in most cases, the d. melanogaster homologue of a gene was used as a query, but for some searches, homologues from other organisms were used as queries .
for the notch, tgfβ, wnt, hedgehog, jak/stat and mapk pathways, considering pathway members known from all animals, we identified likely p. hawaiensis homologues of an average of  <dig> %  of pathway genes .
if we consider only those pathway members with known d. melanogaster homologues , this proportion is an average of  <dig> %.
the proportion of genes found for each pathway ranged from  <dig> %  to  <dig> % .
several genes of interest were found among the singletons.
although singletons are sometimes discarded before transcriptome annotation , our data suggest that even these unassembled reads can be a rich source of gene discovery.
the transcriptome sequences for these genes ranged in length from  <dig> bp  to  <dig>  bp , and the majority are at least  <dig> bp long, making them immediately useful for in situ hybridization, rnai-mediated gene knockdown, and race  <cit>  in the case that longer or flanking genomic sequences are required for specific applications.
interestingly, for several signaling pathway members without a d. melanogaster homologue, we found p. hawaiensis homologues , suggesting that in some respects, amphipod signaling pathways may bear greater resemblance to vertebrate pathways than fruit flies.
annotation of developmental and gametogenesis genes
given the tractability of p. hawaiensis as a developmental model, we sought to make this transcriptome of maximal immediate use to the amphipod and broader developmental biology and biomedical research communities.
we therefore undertook manual annotation of over  <dig> genes involved in embryonic patterning, developmental pathways  and gametogenesis in both males and females .
we used lists of genes known to function in these processes in drosophila as a starting point http://www.sdbonline.org/fly/aimain/1aahome.htm, and identified over  <dig> likely p. hawaiensis homologues of these genes.
as we observed for our annotation of signaling pathway genes, the majority of matching transcriptome reads are of sufficient length to allow immediate use in gene expression and function studies.
several of the genes contained multiple hits in our transcriptome, including a large number of singletons.
for some genes, these multiple hits included consecutively numbered isotigs with very similar lengths.
we examined a subset of these genes to determine whether or not these apparently highly similar isotigs were in fact different from each other.
we found that such sequences usually differed from each other at a small number of nucleotide positions, either because of low sequencing quality in one of the component reads, or because of snps or small indels .
although annotation of snps in this transcriptome is beyond the scope of this study, we anticipate that the snps present in this transcriptome could serve as a useful tool for population-level variation studies in the future.
CONCLUSIONS
we have generated a maternal and embryonic transcriptome of the amphipod crustacean parhyale hawaiensis using  <dig> titanium pyrosequencing.
to our knowledge, this represents the second largest crustacean genomic resource, behind the genome of the cladoceran d. pulex, and the largest de novo assembled pyrosequencing-based transcriptome to date.
we confirmed the previously reported presence of trans-splicing in p. hawaiensis  <cit> , but found that the presence of these trans-spliced transcripts did not greatly increase the complexity of the transcriptome or impede assembly.
the p. hawaiensis transcriptome appears to be enriched for a particular class of c2h <dig> zn finger-coding transcripts, which share high similarity with several branchiostoma floridae transcripts, and is also reported to be an enriched sequence class in the genome of the pea aphid acyrthosiphon pisum  <cit> .
we annotated the de novo transcriptome for a large number of developmentally relevant genes, including major conserved metazoan signaling pathways.
we found that even after assembly of over  <dig> million reads, gene discovery continued to increase, suggesting that the extremely large genome size of this amphipod may reflect high gene numbers, high transcript complexity, or both.
these data should both facilitate future developmental and evolutionary studies using this emerging model crustacean species, and contribute to future work in crustacean comparative genomics.
the bulk of existing arthropod genomic resources are for insects, while the sister group to the insects, the crustaceans, remains relatively unexplored.
the large genome size and low relative similarity to existing annotated arthropod genomes may be challenges to potential future efforts to sequence the p. hawaiensis genome.
however, high throughput short read sequencing technologies such as illumina or solid  <cit> , combined with the transcriptome described here, should make such projects tractable.
finally, this dataset should assist phylogenomic approaches to resolution of controversies in crustacean phylogenetic relationships, including the relationship between crustaceans and hexapods.
d. pulex belongs to the branchiopoda, and while some phylogenetic analyses place this group closest to the hexapods  <cit> , others suggest that the malacostraca  are sister to the hexapods  <cit> .
yet others suggest that branchiopoda may be the most derived group within a monophyletic clade containing all crustaceans except for malacostraca, remipedia and cephalocarida  <cit> .
the largest phylogenomic assessment of this problem to date places both branchiopoda and malacostraca in a monophyletic clade that is sister to a  clade  <cit> .
moreover, daphnia species do not branch basally within the branchiopoda, and indeed are placed in the most derived branchiopod clade by most phylogenetic analyses  <cit> .
these competing hypotheses emphasize the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models.
in order to place future comparative, ecological, environmental and biomedical studies using crustacean models in an appropriate phylogenetic context, additional crustacean genomic resources will be necessary.

 crustaceans are one of the four major groups that make up the phylum arthropoda, the most speciose and morphologically diverse animal group. despite the fact that arthropods as a whole make up the majority of animal species diversity and biomass, until recently the only arthropod represented in the list of nih model organisms http://www.nih.gov/science/models/ was the fruit fly drosophila melanogaster. the water flea daphnia pulex was recently added to this list, and is the only crustacean to date with a publicly accessible sequenced genome. as crustaceans are now widely recognized as sister group to the hexapods, the phylogenetic position of d. pulex suggests that it could serve as a useful outgroup to insects, providing meaningful comparisons with the many insights into developmental and disease biology provided by work on d. melanogaster. however, there are no genomic resources on a scale comparable to the d. pulex genome, and so it is still not known to what extent the characteristics of the water flea genome are specific to this animal's ecology or shared by other crustaceans. moreover, comparisons of biomedically relevant processes and mechanisms between d. pulex and other model organisms must be informed by robust phylogenetic hypotheses. at the moment, which specific subgroup of crustaceans is closest to the hexapods is still a matter of debate, but several phylogenetic hypotheses suggest that branchiopods may be more distant from insects than other crustacean groups. crustaceans have long been the subject of ecological and evolutionary study, as well as being lucrative commercial species for human consumption. even for many of the most intensively studied of these, surprisingly few genomic projects have been reported. most of the crustacean est projects completed to date, notably for the farmed shrimp litopenaeus vannamei, the salmon ectoparasite copepod lepeophtheirus salmonis, the porcelain crab petrolisthes cinctipes, and the lobster homarus americanus, have all used sanger sequencing of cdna libraries. next generation sequencing technologies are increasingly affordable, accessible and robust even for organisms lacking a sequenced genome, but have been reported to be applied to a crustacean de novo transcriptome only once, in the antarctic krill euphausia superba. this organism is the subject of ecological and climate change research, but is not a viable laboratory model organism due to its specialized habitat. while new understanding of developmental and molecular mechanisms in d. pulex are expected to follow from its genome sequence, it is important to note that crustaceans have been the subject of comparative embryology for over a century, and in recent decades, of evolutionary developmental biology. the morphological and molecular mechanistic variations of early embryogenesis, modifications of their body plans and appendage diversifications displayed by crustaceans have all been the subject of studies too numerous to describe here. however, until recently most comparative analyses of crustaceans have been limited to the study of gene expression or experimental embryology, as crustacean models where functional genetic testing is possible are still few in number. the limiting factor for comparative functional experiments is often obtaining specific coding sequences of sufficient length. a large-scale genomic resource for a model crustacean would therefore greatly facilitate development and deployment of transgenic tools. the amphipod parhyale hawaiensis has emerged as an important laboratory model crustacean species over the last several years. p. hawaiensis was first described in the hawaiian islands, but it occupies intertidal marine habitats worldwide. laboratory husbandry is easy and affordable, and inbred lab cultures produce hundreds of embryos year-round, providing ample material for developmental studies. fate map and cell lineage analyses of the early embryo show that all three germ layers and the germ line are determined by the eight cell stage, and clonal populations show predictable patterns at least up until gastrulation. despite this apparently "mosaic" embryonic development, significant regulative properties have also been described for the embryonic mesoderm and ectoderm. molecular techniques for the study of development, including stable transgenesis and gene knockdown are arguably better established for p. hawaiensis than for any other laboratory crustacean model. however, the number of p. hawaiensis developmental genes available as genbank accessions is less than progress in understanding the development of p. hawaiensis is thus is limited by the relative paucity of publicly available cloned coding and regulatory regions. indeed, since the development of germ line transgenesis in p. hawaiensis, its use in developmental studies has been reported only three times. analysis of phenotypes of genomic transgene insertions and case studies of intron sizes are consistent with an extremely large genome size for this amphipod. accordingly, the genome size of p. hawaiensis is estimated to be gb, very near that of homo sapiens. a genomic bac library for p. hawaiensis has been created and partially sequenced but is not yet available in genbank. the genome of the closely related amphipod jassa marmorata, with a much smaller genome of mbp has been approved for whole genome, bac end, and est sequencing by the joint genome institute, but this work is still ongoing, and laboratory culture of this amphipod is difficult. a large transcriptome dataset for p. hawaiensis would thus be a highly valuable resource for several scientific communities, and would in addition assist with the annotation of planned amphipod genomic projects. recent construction of a de novo transcriptome for the milkweed bug oncopeltus fasciatus, which also lacks a sequenced genome, suggested that pyrosequencing would be a fruitful approach to obtaining a large scale p. hawaiensis transcriptome. here we present the de novo assembly of a maternal and embryonic transcriptome for p. hawaiensis, sequenced with titanium pyrosequencing. we describe particular features of the p. hawaiensis transcriptome that were revealed during assembly and annotation, including the presence of trans-splicing and enrichment for c2h zn finger domain-containing transcripts. we annotate orthologues of genes involved in several major developmental patterning processes, gametogenesis in males and females, and members of major conserved animal signaling pathways. we observe a high proportion of apparently unique sequences in the transcriptome, and discuss these findings in the light of observations on the d. pulex genome and other existing crustacean genomic resources.  collection and preparation of material for ovarian and embryonic cdna libraries our goal was to create a transcriptome containing genes relevant to embryonic development, including both maternally provided and zygotically transcribed genes. all three germ layers and the germ line are determined by the eight cell stage, and we wished to capture transcripts from that early stage. we also wished to sample the intensively studied germ band stage, when major body axes have been patterned and trunk segmentation is ongoing, as well as later stages when organogenesis is predominant. we therefore collected ovaries and embryos from all stages of embryogenesis, extracted mrna and prepared cdna for pyrosequencing. because early stage embryos have many fewer cells than later stage embryos, we anticipated that transcripts present in early embryos might suffer low representation if our collection contained equal numbers of embryos of all stages. we therefore collected greater numbers of earlier stages than of later stages. sequencing and assembly of the p. hawaiensis transcriptome we sequenced a total of, reads with a median read length of bp. for assembly of the de novo transcriptome we used newbler v. newbler's terminology for assembled reads distinguishes between contigs, isotigs, and isogroups. for continuity, we use this terminology throughout this paper to refer to the products of the newbler assembly. the default parameters of newbler v were used to screen for adapters and eliminate poor quality reads. the resulting, reads were then assembled., of the screened reads were incorporated into assembled sequences, with singletons remaining. reads were excluded because they were only partially assembled, from repeat regions, outliers, or too short. the assembly contained contigs, which grouped into isotigs. of these isotigs were made up of only one contig, and the average number of contigs per isotigs was. the isotig n length was bp, and the number of isogroups was of these comprised only one isotig, and the mean number of isotigs per isogroup was ). the average coverage per contig was reads/bp. averaging across all bases in the entire assembly, the average coverage per base pair was, meaning that every base pair in the transcriptome was sequenced times on average. this coverage is high compared to typical numbers for de novo transcriptome assemblies, and should be helpful for distinguishing snps and indels from potential sequencing errors in raw reads. fasta files of all assembly products are freely available from the authors. blast mapping of non-redundant transcriptome sequences using an e-value cutoff of 1e- we first used blastx to map all non-redundant assembly sequences to nr, and obtained a total of blast hits. of the isotigs, had at least one hit, and of the singletons, had hits. the majority of these blast hits were unique: among the isotigs there were unique hits, and among the singletons there were unique hits. in summary, among all non-redundant assembly sequences, we obtained unique blast hits. these blast results may mean that the transcriptome contains as many as unique gene transcripts. however, as for all de novo assemblies, several caveats must be considered. first, different regions of a single transcript may have different best top blast hits. our assembly likely contains sequences that belong to the same transcript but are too far apart to be assembled together, and so would be considered "different genes." as a result, the number of unique blast hits may be an overestimate of unique gene number. second, isotigs and singletons did not yield blast hits that met our e-value cutoff of e- these values are comparable to or higher than those obtained in other de novo transcriptome analyses. however, these unmatched sequences may represent transcript fragments whose similarities to known genes is too poor to meet our e-value cutoff, or are non-coding. it is therefore not formally possible from de novo assembly to know whether the unique blast hit number over- or under-estimates the true number of genes contained in our transcriptome. nr = ncbi non-redundant database because isogroups are collections of isotigs that are hypothesized to originate from the same gene, they do not comprise a single sequence and so cannot be compared by blast to nr. the custom script uniqueblast.pl was applied only to non-redundant assembly sequences. transcriptome gene ontology term annotation we used blast2go to obtain the gene ontology terms associated with the top blast hits for each non-redundant assembly sequence. of the sequences with unique blast hits as per our 1e- e-value cutoff, of these had go terms associated with them. to determine whether or not major categories of genes were missing or underrepresented in our transcriptome, we compared the proportions of sequences in selected go term groups to the proportions in these categories observed in the predicted transcript complement of the only crustacean with a sequenced genome, d. pulex. we did not find significant differences in the proportion of genes in the examined go term categories between the p. hawaiensis transcriptome and the water flea genome, suggesting that our transcriptome does not lack major functional categories of genes. interestingly, not only were the proportions similar for the two crustaceans, they also closely matched the profiles observed for the sequenced genome of the insect d. melanogaster, and a de novo insect transcriptome from a milkweed bug, suggesting that arthropod species as widely diverged as fruit flies and water fleas share similar proportional gene expression profiles in certain functional genetic categories. unusual characteristics of the p. hawaiensis transcriptome p. hawaiensis has been found to employ trans-splicing among its genetic regulatory mechanisms. in this mechanism, sections of transcripts transcribed from independent genomic loci are spliced together post-transcriptionally to form a novel transcript. such trans-spliced transcripts are recognizable because they contain a diagnostic splice-leader sequence. we wished to determine to what extent this additional transcript complexity affected our assembly and our ability to assign high-confidence blast annotations. we therefore processed the trimmed, pre-assembly reads to remove those containing the diagnostic splice-leader sequences, and performed a newbler v assembly on the remaining reads. the results of this assembly were not noticeably different from the complete assembly, nor did the number of unique blast hits increase. we therefore concluded that the presence of trans-spliced transcripts did not significantly affect our transcriptome assembly or annotation. when we considered the species identity of the top blast hit for each isotig and singleton, we found that a high proportion of our assembly sequences most closely matched sequences from other arthropods. however, an unexpectedly high proportion was from the lancelet branchiostoma floridae, which is phylogenetically very far removed from p. hawaiensis. when we examined those sequences with a lancelet sequence as top hit, we noticed that for many of them, the top blast hits were all b. floridae; and the majority of them seemed to contain c2h zinc fingers with a specific linker sequence between c2h domains. the genome of at least one other arthropod, the pea aphid acyrthosiphon pisum, has been found to be unusually rich in genes containing the same c2h zinc finger and diagnostic linker sequence. we reasoned that if both the amphioxus and amphipod genomes happened to contain a high proportion of such genes, this could be responsible for the apparent high similarity of several transcripts in these two species. to test this, we removed reads containing c2h zinc finger-encoding sequences, following a previously defined low-stringency definition of a c2h zinc finger domain. we then assembled the remaining reads with newbler v, and again scanned for and removed contigs and isotigs containing the motif. the remaining assembly products were mapped against nr using blastx, and the species identities of their top hits were compared with those obtained from the complete assembly. we found that the new assembly retained a large proportion of arthropod hits, but that the number of b. floridae hits had dropped to %, and was now comparable to the proportion of hits obtained from other deuterostome phyla. comparison with other arthropod genomic resources the blast hit rate of the p. hawaiensis isotigs was %. this is higher than those reported in other de novo transcriptome analyses, including that of an arctic crustacean, but much lower than the % obtained from a previously analyzed de novo insect maternal/embryonic transcriptome. the ~70% of p. hawaiensis isotigs without a high confidence blast hit could be either p. hawaiensis-specific genes, or genes with relatively lower similarity to known genes. in addition, it is possible that many of our transcripts represent untranslated sequences rather than coding regions, and therefore do not match nr protein sequences. the relatively low proportion of hits may also reflect the fact that until recently very few crustacean sequences were included in nr. the d. pulex genome also contains a high proportion of apparently daphnia-specific genes, thought to be largely the result of amplification of selected gene families. noting that the e-value cutoff used for the d. pulex analyses was slightly more relaxed than that used in our initial analysis, we repeated the blastx of our non-redundant p. hawaiensis transcriptome sequences against nr with an e-value cutoff of 1e- this yielded unique blast hits. we therefore found that even adjusting the e-value cutoff to that used for characterization of the d. pulex genome, ~59% of the sequences in this p. hawaiensis transcriptome lack significant similarity to other characterized animal sequences. these observations suggest that crustaceans may have more species- or clade-specific genes than previously appreciated. alternatively, these high numbers of apparently lineage-restricted genes may simply reflect the paucity of crustacean genomic resources currently in public databases. those p. hawaiensis transcriptome sequences which failed to obtain a significant blast hit when compared with nr might share more similarity with other crustaceans. to test this, we used blast to compare all p. hawaiensis transcriptome sequences that had failed our 1e- e-value cutoff against nr, with the predicted d. pulex transcriptome using blastx and a 1e- e-value cutoff. we then used blast to compare the obtained sequences with nr to determine their putative identities. we did not set an e-value limit for this second blast, in order to recover at least some minimal information about the identity of these genes. we found that % of these sequences came from arthropods, only % of which were crustaceans. the low crustacean representation in nr makes it difficult to obtain high confidence blast hits for this group. we therefore focused on determining what proportion of "nr orphan" p. hawaiensis sequences were highly similar to sequences from d. pulex by comparing the e-values for blast hits against both nr and against the d. pulex predicted transcriptome. we found that of the p. hawaiensis sequences with higher similarity to d. pulex genes than to anything in nr, of these had e-values at least an order of magnitude higher for d. pulex compared to nr, and of these had e-values greater than one for nr hits, but d. pulex hit e-values of 1e- or lower. most of this "daphnia-like" group matched arthropod sequences that were previously annotated as "hypothetical proteins," suggesting that non-insect crustacean sequence annotation could improve future annotation of the existing insect genomes. the d. pulex genome has been found to contain a high number of genes. without a genome sequence for p. hawaiensis, we cannot accurately estimate gene number in order to perform a rigorous comparison with d. pulex. however, our transcriptome assembly identified isogroups. because isogroups are groups of isotigs assembled from the same set of contigs, isogroups may represent putative genes, with each isotig of the isogroup representing a transcript variant, for example a splice variant. we therefore speculate that p. hawaiensis, with a genome over one order of magnitude larger than that of d. pulex, may also have a high gene number of at least genes. however, using isogroup number of this de novo transcriptome as a proxy for total gene number has two significant limitations. the first is the result of our chosen tissue sampling strategy: this transcriptome does not capture postembryonic gene expression. after hatching, expression of several genes with exclusively juvenile or adult roles is likely, including at minimum additional genes associated with molting, behavior, and gametogenesis. it is therefore possible that the number of isogroups in our assembly underestimates the true gene number in p. hawaiensis. the second limitation is the unavoidable result of any de novo assembly, which is that if two sequences from the same transcript do not share significant overlap, they will appear as separate assembly products rather than as a single transcript. this could result in the number of isogroups being an overestimate of the true number of genes. a further complication results from newbler v's method of handling isogroups made of multiple isotigs. when we performed our assembly, we limited the number of isotigs in one isogroup to. this has the advantage of avoiding isogroups composed of large numbers of isotigs, as we suspect that in p. hawaiensis, as shown for other animals, the vast majority of genes have fewer than ten splicing isoforms. however, it can also result in problematic isogroup number calculation, because isogroups that exceed the number of isotigs per isogroup threshold are returned to the assembly file as contigs rather than isotigs, thus inflating the gene number estimate. in summary, in order to determine whether high numbers of species- or clade-specific genes is a general characteristic of crustaceans, and the true extent of species-specific genes for p. hawaiensis, more deep genomic resources will have to be developed for this amphipod and for more crustaceans of diverse classes. the high gene number of d. pulex is thought to be due to lineage-specific expansions of gene families. these expansions may play adaptive roles in the water flea's ecology, or they may be a general feature of crustacean genomes that was previously unappreciated due to the paucity of crustacean genomic resources. it is therefore of interest to determine the extent of gene family expansion in p. hawaiensis. in order for our de novo transcriptome to provide a rigorous answer to this question, we would need to distinguish between transcripts of paralogues, and sequences originating from the same transcript that do not overlap enough to belong to the same isotig, or even obtain the same set of top blast hits. this distinction is not unambiguously possible, given the absence of a reference genome. however, we performed a preliminary analysis of putative gene expansion in p. hawaiensis, focusing on those gene families found to be expanded in the d. pulex genome. our analysis conservatively included only those p. hawaiensis isotigs that had a top blast hit against a duplicated d. pulex gene. we also made sure that these isotigs had the same set of top blast hits but belonged to different isogroups, and were therefore likely to represent paralogues rather than splice variants. we found that in general, highly expanded d. pulex gene families had more putative paralogues in p. hawaiensis, relative to less expanded gene families. it is therefore possible that gene family expansions are a common feature of certain enzymatic gene families in some crustaceans, although genome sequencing will ultimately be needed to provide definitive answers to this question. assessment of depth and transcript coverage of the transcriptome although the unique blast hits that we identified may represent unique genes, as discussed extensively above we cannot verify how many transcripts are encoded by the genome in the absence of an annotated genome sequence. however, we wished to estimate how deeply we had sequenced those transcripts that were present in our oogenesis/embryogenesis cdna sample. to do this, we assembled progressively larger random subsets of the total reads. for each subassembly, we used blast to compare our non-redundant transcriptome sequences with nr, and assessed the number of unique blast hits to see to what extent adding sequence data from the same sample improved gene discovery. even with the maximum number of over million reads, we did not observe a plateau in the gene discovery rate. this suggests that despite the considerable depth of our coverage, sequencing more reads from the same sample could yield even more new gene discovery. this is in contrast with a recently constructed de novo maternal and embryonic transcriptome for an insect, which was comprised of only million reads and yet saturated gene discovery in the cdna sample that was sequenced. the increased complexity of the amphipod transcriptome may reflect its large genome size, high putative gene number, or large predicted intron sizes. total isotig length increased steadily as progressively larger subsets of reads were assembled. while small numbers of isotigs over bp could be obtained with as few as ~ reads, robust recovery of isotigs longer than bp required assembly of at least 60% of our total reads, or ~ million reads. this demonstrates that increased depth of sequencing, in addition to improving gene discovery, has the added benefit of increasing predicted transcript lengths, thereby facilitating their annotation and making them more immediately useful for downstream applications. in a related analysis, we searched the transcriptome for the presence of the small number of p. hawaiensis developmental genes available as genbank accessions, and found that % were present. however, only for one of these genes did our transcriptome add sequence data to the genbank accession. this may be a reflection of both the relatively rarity of these transcripts, and the fact that those genes identified to date have been the subject of intense developmental studies, and so sequences of considerable length have already been cloned to close to full length. the assembly yielded isotigs as long as bp, with average length bp. however, we wished to determine what fraction of true full transcript length was likely to be contained by these isotigs. to do this, we used the methods of o'neil and colleagues in calculating the ortholog hit ratio for isotigs, contigs, and singletons. we found that % of isotigs represented over 50% of putative true full-length transcripts compared with predicted d. pulex transcripts, and % of isotigs were over 80% full length. these ratios were not significantly higher than those obtained by comparing transcriptome sequences to d. melanogaster transcripts, suggesting that p. hawaiensis sequences have comparable similarity to those of the water flea and the fruit fly. further, comparing transcripts from the fully sequenced genomes of d. pulex and d. melanogaster yields ortholog hit ratio values of % and % respectively, which are similar to the p. hawaiensis/d. pulex comparison values. these values are consistent with the increasing support for hypotheses of crustacean paraphyly, which predict large divergences between all of the lineages leading to these arthropod species. the current state of understanding of crustacean-hexapod phylogenetic relationships therefore does not allow straightforward predictions of which pair of these three transcriptomes should be most similar to each other. annotation of signaling pathway genes future functional genetic studies in p. hawaiensis will likely focus on elucidating the function of highly conserved metazoan genes in this amphipod. we therefore annotated the transcriptome for the presence of genes belonging to major conserved animal signaling pathways. using the kegg pathways as a guideline, we searched for p. hawaiensis homologues of these genes using blast using an e-value cutoff of 1e- in most cases, the d. melanogaster homologue of a gene was used as a query, but for some searches, homologues from other organisms were used as queries. for the notch, tgfβ, wnt, hedgehog, jak/stat and mapk pathways, considering pathway members known from all animals, we identified likely p. hawaiensis homologues of an average of % of pathway genes. if we consider only those pathway members with known d. melanogaster homologues, this proportion is an average of %. the proportion of genes found for each pathway ranged from % to %. several genes of interest were found among the singletons. although singletons are sometimes discarded before transcriptome annotation, our data suggest that even these unassembled reads can be a rich source of gene discovery. the transcriptome sequences for these genes ranged in length from bp to bp, and the majority are at least bp long, making them immediately useful for in situ hybridization, rnai-mediated gene knockdown, and race in the case that longer or flanking genomic sequences are required for specific applications. interestingly, for several signaling pathway members without a d. melanogaster homologue, we found p. hawaiensis homologues, suggesting that in some respects, amphipod signaling pathways may bear greater resemblance to vertebrate pathways than fruit flies. annotation of developmental and gametogenesis genes given the tractability of p. hawaiensis as a developmental model, we sought to make this transcriptome of maximal immediate use to the amphipod and broader developmental biology and biomedical research communities. we therefore undertook manual annotation of over genes involved in embryonic patterning, developmental pathways and gametogenesis in both males and females. we used lists of genes known to function in these processes in drosophila as a starting point http://www.sdbonline.org/fly/aimain/1aahome.htm, and identified over likely p. hawaiensis homologues of these genes. as we observed for our annotation of signaling pathway genes, the majority of matching transcriptome reads are of sufficient length to allow immediate use in gene expression and function studies. several of the genes contained multiple hits in our transcriptome, including a large number of singletons. for some genes, these multiple hits included consecutively numbered isotigs with very similar lengths. we examined a subset of these genes to determine whether or not these apparently highly similar isotigs were in fact different from each other. we found that such sequences usually differed from each other at a small number of nucleotide positions, either because of low sequencing quality in one of the component reads, or because of snps or small indels. although annotation of snps in this transcriptome is beyond the scope of this study, we anticipate that the snps present in this transcriptome could serve as a useful tool for population-level variation studies in the future.  we have generated a maternal and embryonic transcriptome of the amphipod crustacean parhyale hawaiensis using titanium pyrosequencing. to our knowledge, this represents the second largest crustacean genomic resource, behind the genome of the cladoceran d. pulex, and the largest de novo assembled pyrosequencing-based transcriptome to date. we confirmed the previously reported presence of trans-splicing in p. hawaiensis, but found that the presence of these trans-spliced transcripts did not greatly increase the complexity of the transcriptome or impede assembly. the p. hawaiensis transcriptome appears to be enriched for a particular class of c2h zn finger-coding transcripts, which share high similarity with several branchiostoma floridae transcripts, and is also reported to be an enriched sequence class in the genome of the pea aphid acyrthosiphon pisum. we annotated the de novo transcriptome for a large number of developmentally relevant genes, including major conserved metazoan signaling pathways. we found that even after assembly of over million reads, gene discovery continued to increase, suggesting that the extremely large genome size of this amphipod may reflect high gene numbers, high transcript complexity, or both. these data should both facilitate future developmental and evolutionary studies using this emerging model crustacean species, and contribute to future work in crustacean comparative genomics. the bulk of existing arthropod genomic resources are for insects, while the sister group to the insects, the crustaceans, remains relatively unexplored. the large genome size and low relative similarity to existing annotated arthropod genomes may be challenges to potential future efforts to sequence the p. hawaiensis genome. however, high throughput short read sequencing technologies such as illumina or solid, combined with the transcriptome described here, should make such projects tractable. finally, this dataset should assist phylogenomic approaches to resolution of controversies in crustacean phylogenetic relationships, including the relationship between crustaceans and hexapods. d. pulex belongs to the branchiopoda, and while some phylogenetic analyses place this group closest to the hexapods, others suggest that the malacostraca are sister to the hexapods. yet others suggest that branchiopoda may be the most derived group within a monophyletic clade containing all crustaceans except for malacostraca, remipedia and cephalocarida. the largest phylogenomic assessment of this problem to date places both branchiopoda and malacostraca in a monophyletic clade that is sister to a clade. moreover, daphnia species do not branch basally within the branchiopoda, and indeed are placed in the most derived branchiopod clade by most phylogenetic analyses. these competing hypotheses emphasize the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models. in order to place future comparative, ecological, environmental and biomedical studies using crustacean models in an appropriate phylogenetic context, additional crustacean genomic resources will be necessary. 
annotation of several hundred genes revealed p. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways. insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models. we found an unusually high proportion of c2h zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid acyrthosiphon pisum. arthropods are the most diverse animal phylum, but their genomic resources are relatively few. while the genome of the branchiopod daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. in particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod parhyale hawaiensis. we discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as d. pulex and p. hawaiensis, as well as the need for development of further substantial crustacean genomic resources. using pyrosequencing, we sequenced over billion bases of this cdna, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. to generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cdna from ovaries and embryos of p. hawaiensis. the amphipod p. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. our assembly products yielded unique blast hits against nr. these included over predicted transcripts with significant similarity to d. pulex sequences but not to sequences of any other animal.
 arthropods are the most diverse animal phylum, but their genomic resources are relatively few. while the genome of the branchiopod daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. in particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod parhyale hawaiensis. insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models.  to generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cdna from ovaries and embryos of p. hawaiensis. using pyrosequencing, we sequenced over billion bases of this cdna, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. we found an unusually high proportion of c2h zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid acyrthosiphon pisum. consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. our assembly products yielded unique blast hits against nr. these included over predicted transcripts with significant similarity to d. pulex sequences but not to sequences of any other animal. annotation of several hundred genes revealed p. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways.  the amphipod p. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. we discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as d. pulex and p. hawaiensis, as well as the need for development of further substantial crustacean genomic resources. 