BACKGROUND
the flatworm planarian dugesia japonica inhabits fresh water in east asian areas and is the most common planarian in japan  <cit> .
planarians are members of the phylum platyhelminthes, a group of organisms that are thought to have acquired a central nervous system  with a simple brain structure early during evolution  <cit> .
the constitution of the planarian cns and neural network has been visualized in detail by whole mount in situ hybridization and immunofluorescence studies, and fluorescent dye tracing experiments  <cit> : the cns is composed of a brain  in the head region and a pair of ventral nerve cords extending anterior to posterior along the ventral side of the body, with the brain and ventral nerve cords being morphologically distinguishable structures.
neurons from a variety of sensory organs, such as eyes and auricles, project to different portions of the main lobes  <cit> .
these interneurons are involved in information processing of external signals and work to regulate behavior and learning/memory  <cit> .
the planarian brain acts as an information center of the nervous system, and shows fundamental evolutionarily conserved features of the animal brain not only morphologically but also functionally  <cit> .
in addition, planarians are attracting attention as new model organism for regeneration, including brain regeneration, research.
interestingly, planarians can regenerate their well-organized brains from any portion of their bodies utilizing adult somatic pluripotent stem cells   <cit> .
the rna interference technique can be applied in planarians to identify gene functions  <cit> , and single-cell level gene profiling is also possible based on a combination of fluorescence activated cell sorting  and quantitative reverse transcription polymerase chain reaction   <cit> .
in recent years, resources for comparative transcriptome analysis among members of the phylum platyhelminthes have accumulated rapidly.
the planarian schmidtea mediterranea and blood-fluke schistosoma mansoni genome sequences have been analyzed  <cit> , and transcriptome resources and analyses have been reported  <cit> .
unigene build # <dig> for planarian s. mediterranea, which is based on the sanger sequencing method, contains  <dig>  clusters from  <dig>  est sequences, which were obtained from juvenile and adult libraries  <cit> .
some studies with massive numbers of sequencing reads produced from next generation sequencing technologies, include the illumina hiseq, roche  <dig> and life technologies solid, have been reported  <cit> .
however, there is no genomic resource for d. japonica, and only limited transcriptome information is available for this species.
despite the large evolutionary distance between these two planarians  <cit> , they share not just morphological similarity, but also genes, cns features and regeneration ability  <cit> .
the cdna libraries of the schistosome s. mansoni cover its various life stages: egg, miracidium, sporocyst, cercaria, larva and adult, with a total of  <dig>  sequences and  <dig>  clusters   <cit> .
schistosomes are triploblastic animals and members of platyhelminthes, like planarians, with which they share not only body shape but also basic organismal functions.
specifically, they have bilateral symmetry, a functional brain and peripheral nerves, ventral suckers, digestive and excretory organs; and lack a cardiovascular system  <cit> .
moreover, many genes and their amino acid sequences are well conserved between schistosomes and planarians.
however, whereas planarians are free-living flatworms that prey on other organisms, schistosomes, which are major agents of the disease schistosomiasis and parasitize multiple hosts and organs, change their morphology to adapt to their living environments  <cit> .
the life cycles of these two genuses are thus in sharp contrast, requiring brain functions and metabolic processes that are quite different.
to establish a database of genetic information for planarian transcriptome studies, we performed a large-scale est project for the planarian d. japonica using head cdna libraries.
we adopted sanger sequencing in order to decrease the sequence gaps, frame-shift errors, and the misassembly that can occur due to splice variants and to the short reads produced by next generation sequencing.
these factors are important for the identification of long consensus sequences between conserved proteins.
we compared the percentage of amino acid substitutions between d. japonica and its sister species s. mediterranea using the homologue proteins to identify genes whose mutability enables accommodation to different environmental conditions.
for this analysis, we developed a method to extract gene groups that have different rates of evolution in close species that have very-well-conserved proteins.
we have already published a partial analysis of d. japonica transcriptome  <cit> , and have identified several genes that are specifically expressed in the cns  <cit> .
however, those studies were insufficient for the exhaustive comparative analyses between planarians and members of the same family or the same phylum necessary for clarifying the composition and evolution of the cns.
as compared with model organisms, the gene information of platyhelminthes is very limited.
for these reasons we used gene ontology  <cit> , which is based on information across many species, including vertebrates and non-vertebrates, and serves as a common platform to compare and annotate non-model organisms.
in this study, we focused on the cns-development genes, which should give information about the evolutionary position of platyhelminthes.
to examine the genomic evolution and the presence of gene expression, we compared the d. japonica unigenes with not only s. mansoni unigenes but also the predicted protein information from the genome sequence.
the traces we thereby found on the genome suggested the possibility that these genes were derived from the common ancestor of these two genuses, and the divergent gene expression between these genuses provided information about their adaptation to their specific habitats.
RESULTS
est sequencing
a non-normalized cdna library was constructed using poly+ rna isolated from the heads of adult planarians.
two different experimental methods and dna sequencers were used for the cdna template amplification and dna sequencing reaction .
after trimming of vector sequences, about  <dig> % of reads passed the high-quality control for phred base calling  <cit> , and finally a total of  <dig>  5’-end and  <dig>  3’-end reads enabled the assembly analysis to proceed accurately.
for  <dig> clones, the reading gap was closed to get the whole clone sequence by the primer walking method using custom primers based on the est sequence.
all est reads and full-length clone sequences have been submitted to ddbj.
the accession numbers are 5’ ests , 3’ ests  and full-insert sequences .
* ddbj entries registered by previous research.
de novo transcriptome assembly
before de novo assembly, to generate accurate unigenes,  <dig>  paired-assembly contigs were produced  using paired-end sequences of the same clone and cap <dig> assembler software  <cit> .
after the addition of  <dig>  ddbj entries registered from previous research , the complete sequence materials without the original reads that were members of paired-assembly contigs were further assembled into  <dig>  contigs using tgicl software  <cit> .
in addition,  <dig>  sequences remained as singletons, resulting in a total of  <dig>  unique sequences .
the average length of contigs was  <dig>  bp, and the sum of all unique sequences was  <dig>  mbp, including singletons .
figure  <dig> shows the distribution of the number of contigs with a particular length among the unigenes.
the longest contig length was  <dig>  bp.
the histogram of contig depth showed that contigs with fewer than  <dig> copies and singletons accounted for 87% of unique sequences.
in contrast, only  <dig> highly expressed contigs dominated the whole transcriptome sequences .
these profiles were consistent with the results of general non-normalized transcriptome analysis  <cit> .
to estimate the transcriptome coverage for the data set, we assembled  <dig>  replicate random sequences and calculated the non-redundant gene numbers .
the workflow for the assembly construction process is shown in figure  <dig>
summary of the de novo transcriptome assembly using filter-passed and vector trimmed reads, pre-assembly contigs, and full-insert sequences.
the depth of contigs showing less than  <dig> copies of expression consist mostly of the unigene variation.
unigene annotations
the annotations of the d. japonica transcriptome were based on three types of approach: homology searching by blast  <cit> , conserved protein domain detection  <cit> , and gene ontology  <cit>  classification.
the blastx search against the ncbi protein reference sequences database   <cit>  resulted in  <dig>   unigene hits with significant similarity.
the taxonomic distribution per organism using the best hit showed high similarity  with the schistosome, which belongs to the same phylum as planarians .
many planarian genes showed similarity to genes in not only the schistosome but also other organisms, including the hemichordate s. kowalevskii , chordate b. floridae , echinoderm s. purpuratus , and vertebrate d. rerio .
the conserved domain information for the transcriptome was obtained through the pfam database  <cit>  using rps-blast, which scans a set of pre-calculated position-specific scoring matrices with a protein query.
a total  <dig>   conserved protein domains with  <dig>  variations were confirmed in the complete set of unigenes.
protein kinase domains were the most frequent, with  <dig> hits, and the second- and third- most frequent domains were ankyrin repeats  and rna recognition motifs  .
domains with less than  <dig> hits consist mostly of the result .
to address the functional categories of the d. japonica transcriptome, all the unigenes were assigned a gene ontology  classification based on blastx hits against the uniprotkb/swiss-prot database  <cit> , which has reliable information for go terms, and the annotation based on related studies.
by referring to each go term from the uniprot database, the terms associated with the unigenes were consolidated into higher classes using go slim digestion  <cit>  via software  <cit> .
amino acid substitutions between two planarians
the protein blast software identifies the conserved regions and the degrees of similarity between query and subject amino acid sequences.
blast shows not only identical amino acids at a given position in the alignment, but also homologous substitutions, which are determined from the scoring matrix .
a method for calculating the identical match ratio   was applied to find strongly and weakly conserved proteins between the two planarians d. japonica and s. mediterranea.
after comparative analysis of each unigene by tblastx, to obtain sufficient homologous pairs for the extraction, some filter options were applied to the results.
finally, a total of  <dig>  pairs remained as homologous genes, and the top 15%  and the bottom 15%  of them regarding the identical match ratio  were sorted into functional categories by rps-blast analysis using the eukaryotic clusters of orthologous groups  database  <cit> .
of these,  <dig>  genes were classified into  <dig> kog categories with  <dig> kog functions.
no genes were classified as ‘cell motility’ kog function.
extraction of the planarian cns-development genes
to obtain insight into the evolutionary origin of a functional brain, central nervous system -development genes were extracted from the d. japonica unigenes using go annotations as a common knowledge base.
the category “cns-development” was chosen here because we reasoned that the strong evolutionary/structural conservation of the planarian and higher animal brains implies conservation of many brain developmental pathways.
based on the results of blastx analysis against uniprotkb, each primary hit was defined as a putative planarian protein and function.
the go term ‘central nervous system development’  has  <dig> subcategories, each of which has a considerable number of subfamilies and terms.
because a protein which had multifunctions was mapped to each respective term separately, the total count of hits classed to cns-development genes was increased by such duplications.
the classification analysis produced a total of  <dig> matches and  <dig> cns-development genes, with  <dig> subcategories .
the most highly represented subcategory was ‘brain development’ , with  <dig> matches, and the second- and third-most frequent were ‘spinal cord development’  and ‘central nervous system neuron differentiation’ , with  <dig> and  <dig> matches, respectively.
in addition,  <dig> matches were found for the subcategory ‘ventral cord development’.
+ indicate the presence of gene which homologous with d. japonica cns-development gene in s. mediterranea and the schistosoma.
comparative analysis of cns-development genes among platyhelminths
two types of schistosome genome dataset, the primary genome sequence and predicted protein sequences based on the genome sequence, and the primary genome sequence of s. mediterranea, were used for exploring the platyhelminthes genes that are homologous with d. japonica cns-development genes.
the primary genome sequence provides evidence useful for distinguishing between genes with low expression and deleted genes.
the predicted protein sequences were directly dependent on the computational process, and were not tested regarding whether they were supported by biological evidence of the gene expression, but contributed to the detection of genes that have small exons split by long intron regions.
in the case of direct searching against primary genome sequences, the d. japonica unigenes were mapped by blat software  <cit>  using the parameters of the six translation frames to compare the protein-protein sequences.
in addition, the predicted protein sequences were analyzed using the tblastn program.
the use of translated nucleotide databases and translated nucleotide query enables absorption of the third codon position mutations, and therefore it is useful for comparison of evolutionarily distant genes.
in these searches, if either one of the two scores exceeded the threshold, the matched gene was defined as conserved.
a transcriptome-transcriptome analysis among platyhelminths was performed using tblastx rather than blastn for the same reason as described above.
the summary showed that the d. japonica cns-development genes were highly conserved among platyhelminths .
in the case of comparison with s. mediterranea, all of the  <dig> genes were detected in the genome sequence, and almost all of the genes  were found in the transcriptome data set obtained by next generation sequencing.
in total, 75/ <dig>  d. japonica cns-development genes were conserved in the schistosome genome.
most of these genes were in subcategories other than brain development, suggesting that the cns-development genes were well conserved during evolution.
in contrast to the high genome concordance, the results of transcriptome-transcriptome analysis showed a low concordance rate of 54/ <dig> , excluding genes in the categories ‘astrocyte differentiation’ and ‘oligodendrocyte differentiation’.
discussion
d. japonica transcriptome
we performed large scale d. japonica transcriptome analysis using non-normalized cdna libraries constructed from head tissue to construct a transcriptome database for comparative genomics and studies of brain evolution.
compared with normalized cdna libraries, in which the proportion of high-copy housekeeping genes is decreased, non-normalized cdna libraries have many redundant transcripts, but they keep the information of the original expression levels and are expected to provide raw expression profiles  <cit> .
next generation sequencing is a powerful tool that has provided massive datasets for genome and transcriptome analysis, including some datasets for the s. mediterranea transcriptome  <cit> .
however, for comparative analysis between two planarians, it is important to use long and gapless consensus sequences of conserved proteins.
to obtain such sequences, next-generation sequencing is not always the best tool.
for example, roche  <dig> cannot correctly read homo-polymer sequences like aaaa or tttt , and this limitation may cause many frame-shift errors in a gene sequence.
also, although illumina hiseq generates a large volume of reads, they are rather short, and this makes it difficult to obtain long and correct contig sequences without high quality reference information.
the paired-end application is a useful way to link separated contigs, but long gaps often arise when there are no consensus sequences.
in contrast, sanger sequencing provides long, high-quality mrna reads, which are important for comparative analysis using consensus sequences of conserved protein domains.
furthermore, long reads can help to distinguish between paralogues and alternative splicing variants, and thus are useful for transcriptome research of non-model organisms for which high-quality genomic information is lacking.
in this study, we obtained a total of  <dig>  unigenes with  <dig>  bp average length as a result of de novo assembly using  <dig>  newly acquired long sanger sequences.
two types of pre-assembled reads, gap-closed sequences obtained by a primer walking method, and overlap-joined contigs from the same clone, contributed to extending the contig length.
additionally, many singletons that were unassembled ests also had sufficient length to identify the function.
the histogram of assembly statistics  and functional annotations of the assembled unigenes, based on conserved domain search by rps-blast , indicated that we had built an encompassing and low-bias gene profile that provided enough information for comparative transcriptome analysis among two planarians and a schistosome.
regarding approaches to non-model organism transcriptomes, the algorithms applied and dataset selection are of the same importance as de novo assembling for finding new evolutionary events or pathways.
here, we report two different approaches to finding such events, not simply the acquisition of basic knowledge about the d. japonica transcriptome.
comparative analysis between two planarians
the acquisitions of gene mutations and corresponding amino acid substitutions are not random events, but rather are affected by the pressure of natural selection.
it is generally accepted that similar proteins with homologous substitutions share basic molecular functions in organisms  <cit> .
the biotic features  of the two planarians studied here, d. japonica and s. mediterranea, are quite similar, and large numbers of their genes and their encoded amino acid sequences are well conserved.
however, the nucleic acid sequences of many homologous genes have low similarity between these two planarians, and there are many genes containing homologous substitutions for amino acids in conserved regions, although the profiles and details remain unknown.
for example, piwi <dig> alignment shows 94% identical matches of amino acids over the entire sequence .
but in the case of ggt <dig>  only 70% of amino acids are the same, although the percentage rises to 88% if homologous substitutions are included .
based on the hypothesis that proteins with many substitutable amino acids could be candidates for members of gene groups that are able to undergo changes to accommodate to different environments, we developed a new method for detecting the fraction of substitutable amino acids within individual genes.
to view the trends in functional groups of proteins rather than in each gene, we classified the results calculated by this method into functions and categories based on kog annotation.
the log <dig> ratio between the number of proteins with homologous substitutions and the number with identical matches revealed distinctive features of function and category .
although a very long time has passed since their divergence from their common ancestor, amino acids in proteins involved in basal mechanisms such as functions of the ‘information storage and processing’ category and cellular structure-related functions ‘cytoskeleton’ and ‘extracellular structures’ have not undergone replacement even by amino acids that have similar biochemical properties, presumably as a result of selection pressure.
by contrast, in the function ‘defense mechanisms’, which is directly linked to an organism’s environment, every gene mapped to this function displayed a high rate of substitution.
among the proteins in the category ‘metabolism’, the energy metabolism functions ‘carbohydrate transport and metabolism’ and ‘energy production and conversion’ showed low rates of substitution, whereas genes related to digestion processes  had high rates of substitution, perhaps reflecting responsiveness to their feeding habitat.
it is interesting that many accumulations of amino-acid substitutions were observed in proteins that were well conserved not only between the two planarians examined here, but among many species, and were expected to maintain their biological function .
previous studies of schistosomes suggested that these animals are incapable of de novo synthesis of sterols or free fatty acids and must use precursors of fatty acids from the host  <cit> .
it could be considered that the effects of selection pressure on metabolism-related genes are decreased in parasitic life, because an appropriate nutritional environment is provided by the host.
these previously reported results support our hypothesis that, in addition to selective pressure from the environment, the potential of genes to accommodate substitutions is also an important factor in evolution.
evolutionary conservation of cns-development genes
as mentioned above, platyhelminths are considered to be primitive animals that possess a functional cns closely related to the evolutionary origin of the cns.
the d. japonica cns is composed of an anterior cephalic ganglion and ventral nerve cords, which are morphologically distinguishable structures.
planarians can sense light and chemical signals transmitted through their eyes and chemosensors, respectively, to their brain, and show different behaviors according to the type of signal.
in addition the partial results of previous transcriptome analysis, we previously reported several genes specifically expressed in the cns, such as synaptotagmin, prohormone convertase  <dig>  and netrin  <cit> .
but, as compared with model organisms, the gene information for platyhelminths is very limited, and this makes it difficult to perform whole transcriptome comparison.
for this reason we used gene ontology and the category “cns-development”, which is based on information across many species, including vertebrates and non-vertebrates.
we think this annotation can serve well as a common platform for comparing and annotating among platyhelminths.
both the functionality and complexity of the planarian cns are supported by our identification of  <dig> d. japonica genes related to cns development, based on the gene ontology and uniprotkb knowledge base.
homology search with s. mediterranea showed that the genes are quite highly conserved between these two planarian species.
all of the  <dig> genes were found in the s. mediterranea genome sequence and almost all of these genes  were found in the s. mediterranea transcriptome data set derived by next generation sequencing.
this number of cns-development-related genes is not much lower than that in model organisms known to have highly functional brains, namely, d. melanogaster , d. rerio , g. gallus  and m. musculus   <cit> .
in addition, these numbers are clearly larger than the number of cns-development-related genes in c. elegans , which may have lost a complex cns, and suggest the possibility that the planarian has a functional brain at not only the developmental level but also the genomic level.
the cns of s. mansoni consists of a central ganglion, six pairs of main nerve trunks, and longitudinal nerve cords, which extend along the length of the body  <cit> .
additionally, six different types of sensory papillae are known, and are considered to be valuable structures for penetration and navigation through the vasculature in the body of the host.
using the predicted protein sequences of the schistosome and performing an exhaustive search of all possible cns-development genes,  <dig> homologs of the  <dig> genes in d. japonica were successfully detected.
this result indicates that the planarian cns-development genes already existed before the divergence of planarians and schistosomes from their last common ancestor.
interestingly, expression of about one-third of the cns genes was not observed in the schistosome unigene database, which was built using transcript sequences.
because the schistosome unigenes were constructed from  <dig> different developmental stages,  <dig>  libraries, and  <dig>  sequences, their completeness is considered to be extremely high, and therefore these cns-development genes not observed in this schistosome database can be concluded to have extremely low or no expression.
it is possible that, in contrast to the free-living lifestyle of planarians, the parasitic life of schistosomes resulted in selective degeneration of unnecessary genes in schistosomes.
the features of the brain are determined to a large degree by the differential regulation of the transcriptome during an animal’s life cycle.
schistosomes not only inhabit various tissues of their intermediate and definitive hosts, but also traverse freshwater environments to change hosts.
viewed in this light, it might have been expected that the schistosome would have had more gene components related to the cns, but instead it showed a reduced number of such genes.
this might be because planarians face more complex circumstances than schistosomes regarding enemies or climate throughout their lifetime, or because schistosomes construct complicated components using more limited gene sets.
further studies will be needed to clarify the relation between gene diversification and external factors in organisms’ environments.
to validate possible scenarios of planarian cns evolution, this database will be valuable for the identification of relevant genes.
more extensive analyses of genes and genomic resources will be needed to better understand the functions and evolution of the cns.
CONCLUSIONS
we have produced  <dig>  high-quality est reads from a head library of the planarian d. japonica, and de novo assembly analysis of these ests produced  <dig>  unigene sequences.
similarity search against public databases and conserved domain analysis of predicted protein sequences indicated that this dataset is a useful resource for comparative transcriptome studies.
comparison of homologous genes between two planarian species led us to hypothesize that not only the pressure of natural selection but also the potential of genes is important for an acceleration of the accumulation of amino-acid substitutions, a hypothesis that is supported by previous studies showing that schistosomes are defective in lipid metabolism.
a total of  <dig> planarian cns-development genes were extracted using gene ontology annotation, and this result suggested the possibility that the planarian has a functional brain both developmentally and genetically.
mapping of these genes onto the schistosome genome showed that the 91% of the planarian cns-development genes were conserved within the schistosome genome.
however, approximately one-third of the planarian cns genes were not expressed in the schistosome.
these analyses suggest that the establishment of the planarian cns occurred before the divergence of planarians from their common ancestor with schistosomes, but that these two genuses subsequently diversified to adapt to their differing circumstances regarding the complexity needed for a free versus a parasitic life.
this database of the d. japonica transcriptome constructed here provides an important resource not only for planarian research, but also for comparative analyses of the cns.

 the flatworm planarian dugesia japonica inhabits fresh water in east asian areas and is the most common planarian in japan. planarians are members of the phylum platyhelminthes, a group of organisms that are thought to have acquired a central nervous system with a simple brain structure early during evolution. the constitution of the planarian cns and neural network has been visualized in detail by whole mount in situ hybridization and immunofluorescence studies, and fluorescent dye tracing experiments: the cns is composed of a brain in the head region and a pair of ventral nerve cords extending anterior to posterior along the ventral side of the body, with the brain and ventral nerve cords being morphologically distinguishable structures. neurons from a variety of sensory organs, such as eyes and auricles, project to different portions of the main lobes. these interneurons are involved in information processing of external signals and work to regulate behavior and learning/memory. the planarian brain acts as an information center of the nervous system, and shows fundamental evolutionarily conserved features of the animal brain not only morphologically but also functionally. in addition, planarians are attracting attention as new model organism for regeneration, including brain regeneration, research. interestingly, planarians can regenerate their well-organized brains from any portion of their bodies utilizing adult somatic pluripotent stem cells. the rna interference technique can be applied in planarians to identify gene functions, and single-cell level gene profiling is also possible based on a combination of fluorescence activated cell sorting and quantitative reverse transcription polymerase chain reaction. in recent years, resources for comparative transcriptome analysis among members of the phylum platyhelminthes have accumulated rapidly. the planarian schmidtea mediterranea and blood-fluke schistosoma mansoni genome sequences have been analyzed, and transcriptome resources and analyses have been reported. unigene build # for planarian s. mediterranea, which is based on the sanger sequencing method, contains clusters from est sequences, which were obtained from juvenile and adult libraries. some studies with massive numbers of sequencing reads produced from next generation sequencing technologies, include the illumina hiseq, roche and life technologies solid, have been reported. however, there is no genomic resource for d. japonica, and only limited transcriptome information is available for this species. despite the large evolutionary distance between these two planarians, they share not just morphological similarity, but also genes, cns features and regeneration ability. the cdna libraries of the schistosome s. mansoni cover its various life stages: egg, miracidium, sporocyst, cercaria, larva and adult, with a total of sequences and clusters. schistosomes are triploblastic animals and members of platyhelminthes, like planarians, with which they share not only body shape but also basic organismal functions. specifically, they have bilateral symmetry, a functional brain and peripheral nerves, ventral suckers, digestive and excretory organs; and lack a cardiovascular system. moreover, many genes and their amino acid sequences are well conserved between schistosomes and planarians. however, whereas planarians are free-living flatworms that prey on other organisms, schistosomes, which are major agents of the disease schistosomiasis and parasitize multiple hosts and organs, change their morphology to adapt to their living environments. the life cycles of these two genuses are thus in sharp contrast, requiring brain functions and metabolic processes that are quite different. to establish a database of genetic information for planarian transcriptome studies, we performed a large-scale est project for the planarian d. japonica using head cdna libraries. we adopted sanger sequencing in order to decrease the sequence gaps, frame-shift errors, and the misassembly that can occur due to splice variants and to the short reads produced by next generation sequencing. these factors are important for the identification of long consensus sequences between conserved proteins. we compared the percentage of amino acid substitutions between d. japonica and its sister species s. mediterranea using the homologue proteins to identify genes whose mutability enables accommodation to different environmental conditions. for this analysis, we developed a method to extract gene groups that have different rates of evolution in close species that have very-well-conserved proteins. we have already published a partial analysis of d. japonica transcriptome, and have identified several genes that are specifically expressed in the cns. however, those studies were insufficient for the exhaustive comparative analyses between planarians and members of the same family or the same phylum necessary for clarifying the composition and evolution of the cns. as compared with model organisms, the gene information of platyhelminthes is very limited. for these reasons we used gene ontology, which is based on information across many species, including vertebrates and non-vertebrates, and serves as a common platform to compare and annotate non-model organisms. in this study, we focused on the cns-development genes, which should give information about the evolutionary position of platyhelminthes. to examine the genomic evolution and the presence of gene expression, we compared the d. japonica unigenes with not only s. mansoni unigenes but also the predicted protein information from the genome sequence. the traces we thereby found on the genome suggested the possibility that these genes were derived from the common ancestor of these two genuses, and the divergent gene expression between these genuses provided information about their adaptation to their specific habitats.  est sequencing a non-normalized cdna library was constructed using poly+ rna isolated from the heads of adult planarians. two different experimental methods and dna sequencers were used for the cdna template amplification and dna sequencing reaction. after trimming of vector sequences, about % of reads passed the high-quality control for phred base calling, and finally a total of 5’-end and 3’-end reads enabled the assembly analysis to proceed accurately. for clones, the reading gap was closed to get the whole clone sequence by the primer walking method using custom primers based on the est sequence. all est reads and full-length clone sequences have been submitted to ddbj. the accession numbers are 5’ ests, 3’ ests and full-insert sequences. * ddbj entries registered by previous research. de novo transcriptome assembly before de novo assembly, to generate accurate unigenes, paired-assembly contigs were produced using paired-end sequences of the same clone and cap assembler software. after the addition of ddbj entries registered from previous research, the complete sequence materials without the original reads that were members of paired-assembly contigs were further assembled into contigs using tgicl software. in addition, sequences remained as singletons, resulting in a total of unique sequences. the average length of contigs was bp, and the sum of all unique sequences was mbp, including singletons. figure shows the distribution of the number of contigs with a particular length among the unigenes. the longest contig length was bp. the histogram of contig depth showed that contigs with fewer than copies and singletons accounted for 87% of unique sequences. in contrast, only highly expressed contigs dominated the whole transcriptome sequences. these profiles were consistent with the results of general non-normalized transcriptome analysis. to estimate the transcriptome coverage for the data set, we assembled replicate random sequences and calculated the non-redundant gene numbers. the workflow for the assembly construction process is shown in figure summary of the de novo transcriptome assembly using filter-passed and vector trimmed reads, pre-assembly contigs, and full-insert sequences. the depth of contigs showing less than copies of expression consist mostly of the unigene variation. unigene annotations the annotations of the d. japonica transcriptome were based on three types of approach: homology searching by blast, conserved protein domain detection, and gene ontology classification. the blastx search against the ncbi protein reference sequences database resulted in unigene hits with significant similarity. the taxonomic distribution per organism using the best hit showed high similarity with the schistosome, which belongs to the same phylum as planarians. many planarian genes showed similarity to genes in not only the schistosome but also other organisms, including the hemichordate s. kowalevskii, chordate b. floridae, echinoderm s. purpuratus, and vertebrate d. rerio. the conserved domain information for the transcriptome was obtained through the pfam database using rps-blast, which scans a set of pre-calculated position-specific scoring matrices with a protein query. a total conserved protein domains with variations were confirmed in the complete set of unigenes. protein kinase domains were the most frequent, with hits, and the second- and third- most frequent domains were ankyrin repeats and rna recognition motifs. domains with less than hits consist mostly of the result. to address the functional categories of the d. japonica transcriptome, all the unigenes were assigned a gene ontology classification based on blastx hits against the uniprotkb/swiss-prot database, which has reliable information for go terms, and the annotation based on related studies. by referring to each go term from the uniprot database, the terms associated with the unigenes were consolidated into higher classes using go slim digestion via software. amino acid substitutions between two planarians the protein blast software identifies the conserved regions and the degrees of similarity between query and subject amino acid sequences. blast shows not only identical amino acids at a given position in the alignment, but also homologous substitutions, which are determined from the scoring matrix. a method for calculating the identical match ratio was applied to find strongly and weakly conserved proteins between the two planarians d. japonica and s. mediterranea. after comparative analysis of each unigene by tblastx, to obtain sufficient homologous pairs for the extraction, some filter options were applied to the results. finally, a total of pairs remained as homologous genes, and the top 15% and the bottom 15% of them regarding the identical match ratio were sorted into functional categories by rps-blast analysis using the eukaryotic clusters of orthologous groups database. of these, genes were classified into kog categories with kog functions. no genes were classified as ‘cell motility’ kog function. extraction of the planarian cns-development genes to obtain insight into the evolutionary origin of a functional brain, central nervous system -development genes were extracted from the d. japonica unigenes using go annotations as a common knowledge base. the category “cns-development” was chosen here because we reasoned that the strong evolutionary/structural conservation of the planarian and higher animal brains implies conservation of many brain developmental pathways. based on the results of blastx analysis against uniprotkb, each primary hit was defined as a putative planarian protein and function. the go term ‘central nervous system development’ has subcategories, each of which has a considerable number of subfamilies and terms. because a protein which had multifunctions was mapped to each respective term separately, the total count of hits classed to cns-development genes was increased by such duplications. the classification analysis produced a total of matches and cns-development genes, with subcategories. the most highly represented subcategory was ‘brain development’, with matches, and the second- and third-most frequent were ‘spinal cord development’ and ‘central nervous system neuron differentiation’, with and matches, respectively. in addition, matches were found for the subcategory ‘ventral cord development’. + indicate the presence of gene which homologous with d. japonica cns-development gene in s. mediterranea and the schistosoma. comparative analysis of cns-development genes among platyhelminths two types of schistosome genome dataset, the primary genome sequence and predicted protein sequences based on the genome sequence, and the primary genome sequence of s. mediterranea, were used for exploring the platyhelminthes genes that are homologous with d. japonica cns-development genes. the primary genome sequence provides evidence useful for distinguishing between genes with low expression and deleted genes. the predicted protein sequences were directly dependent on the computational process, and were not tested regarding whether they were supported by biological evidence of the gene expression, but contributed to the detection of genes that have small exons split by long intron regions. in the case of direct searching against primary genome sequences, the d. japonica unigenes were mapped by blat software using the parameters of the six translation frames to compare the protein-protein sequences. in addition, the predicted protein sequences were analyzed using the tblastn program. the use of translated nucleotide databases and translated nucleotide query enables absorption of the third codon position mutations, and therefore it is useful for comparison of evolutionarily distant genes. in these searches, if either one of the two scores exceeded the threshold, the matched gene was defined as conserved. a transcriptome-transcriptome analysis among platyhelminths was performed using tblastx rather than blastn for the same reason as described above. the summary showed that the d. japonica cns-development genes were highly conserved among platyhelminths. in the case of comparison with s. mediterranea, all of the genes were detected in the genome sequence, and almost all of the genes were found in the transcriptome data set obtained by next generation sequencing. in total, 75/ d. japonica cns-development genes were conserved in the schistosome genome. most of these genes were in subcategories other than brain development, suggesting that the cns-development genes were well conserved during evolution. in contrast to the high genome concordance, the results of transcriptome-transcriptome analysis showed a low concordance rate of 54/, excluding genes in the categories ‘astrocyte differentiation’ and ‘oligodendrocyte differentiation’. discussion d. japonica transcriptome we performed large scale d. japonica transcriptome analysis using non-normalized cdna libraries constructed from head tissue to construct a transcriptome database for comparative genomics and studies of brain evolution. compared with normalized cdna libraries, in which the proportion of high-copy housekeeping genes is decreased, non-normalized cdna libraries have many redundant transcripts, but they keep the information of the original expression levels and are expected to provide raw expression profiles. next generation sequencing is a powerful tool that has provided massive datasets for genome and transcriptome analysis, including some datasets for the s. mediterranea transcriptome. however, for comparative analysis between two planarians, it is important to use long and gapless consensus sequences of conserved proteins. to obtain such sequences, next-generation sequencing is not always the best tool. for example, roche cannot correctly read homo-polymer sequences like aaaa or tttt, and this limitation may cause many frame-shift errors in a gene sequence. also, although illumina hiseq generates a large volume of reads, they are rather short, and this makes it difficult to obtain long and correct contig sequences without high quality reference information. the paired-end application is a useful way to link separated contigs, but long gaps often arise when there are no consensus sequences. in contrast, sanger sequencing provides long, high-quality mrna reads, which are important for comparative analysis using consensus sequences of conserved protein domains. furthermore, long reads can help to distinguish between paralogues and alternative splicing variants, and thus are useful for transcriptome research of non-model organisms for which high-quality genomic information is lacking. in this study, we obtained a total of unigenes with bp average length as a result of de novo assembly using newly acquired long sanger sequences. two types of pre-assembled reads, gap-closed sequences obtained by a primer walking method, and overlap-joined contigs from the same clone, contributed to extending the contig length. additionally, many singletons that were unassembled ests also had sufficient length to identify the function. the histogram of assembly statistics and functional annotations of the assembled unigenes, based on conserved domain search by rps-blast, indicated that we had built an encompassing and low-bias gene profile that provided enough information for comparative transcriptome analysis among two planarians and a schistosome. regarding approaches to non-model organism transcriptomes, the algorithms applied and dataset selection are of the same importance as de novo assembling for finding new evolutionary events or pathways. here, we report two different approaches to finding such events, not simply the acquisition of basic knowledge about the d. japonica transcriptome. comparative analysis between two planarians the acquisitions of gene mutations and corresponding amino acid substitutions are not random events, but rather are affected by the pressure of natural selection. it is generally accepted that similar proteins with homologous substitutions share basic molecular functions in organisms. the biotic features of the two planarians studied here, d. japonica and s. mediterranea, are quite similar, and large numbers of their genes and their encoded amino acid sequences are well conserved. however, the nucleic acid sequences of many homologous genes have low similarity between these two planarians, and there are many genes containing homologous substitutions for amino acids in conserved regions, although the profiles and details remain unknown. for example, piwi alignment shows 94% identical matches of amino acids over the entire sequence. but in the case of ggt only 70% of amino acids are the same, although the percentage rises to 88% if homologous substitutions are included. based on the hypothesis that proteins with many substitutable amino acids could be candidates for members of gene groups that are able to undergo changes to accommodate to different environments, we developed a new method for detecting the fraction of substitutable amino acids within individual genes. to view the trends in functional groups of proteins rather than in each gene, we classified the results calculated by this method into functions and categories based on kog annotation. the log ratio between the number of proteins with homologous substitutions and the number with identical matches revealed distinctive features of function and category. although a very long time has passed since their divergence from their common ancestor, amino acids in proteins involved in basal mechanisms such as functions of the ‘information storage and processing’ category and cellular structure-related functions ‘cytoskeleton’ and ‘extracellular structures’ have not undergone replacement even by amino acids that have similar biochemical properties, presumably as a result of selection pressure. by contrast, in the function ‘defense mechanisms’, which is directly linked to an organism’s environment, every gene mapped to this function displayed a high rate of substitution. among the proteins in the category ‘metabolism’, the energy metabolism functions ‘carbohydrate transport and metabolism’ and ‘energy production and conversion’ showed low rates of substitution, whereas genes related to digestion processes had high rates of substitution, perhaps reflecting responsiveness to their feeding habitat. it is interesting that many accumulations of amino-acid substitutions were observed in proteins that were well conserved not only between the two planarians examined here, but among many species, and were expected to maintain their biological function. previous studies of schistosomes suggested that these animals are incapable of de novo synthesis of sterols or free fatty acids and must use precursors of fatty acids from the host. it could be considered that the effects of selection pressure on metabolism-related genes are decreased in parasitic life, because an appropriate nutritional environment is provided by the host. these previously reported results support our hypothesis that, in addition to selective pressure from the environment, the potential of genes to accommodate substitutions is also an important factor in evolution. evolutionary conservation of cns-development genes as mentioned above, platyhelminths are considered to be primitive animals that possess a functional cns closely related to the evolutionary origin of the cns. the d. japonica cns is composed of an anterior cephalic ganglion and ventral nerve cords, which are morphologically distinguishable structures. planarians can sense light and chemical signals transmitted through their eyes and chemosensors, respectively, to their brain, and show different behaviors according to the type of signal. in addition the partial results of previous transcriptome analysis, we previously reported several genes specifically expressed in the cns, such as synaptotagmin, prohormone convertase and netrin. but, as compared with model organisms, the gene information for platyhelminths is very limited, and this makes it difficult to perform whole transcriptome comparison. for this reason we used gene ontology and the category “cns-development”, which is based on information across many species, including vertebrates and non-vertebrates. we think this annotation can serve well as a common platform for comparing and annotating among platyhelminths. both the functionality and complexity of the planarian cns are supported by our identification of d. japonica genes related to cns development, based on the gene ontology and uniprotkb knowledge base. homology search with s. mediterranea showed that the genes are quite highly conserved between these two planarian species. all of the genes were found in the s. mediterranea genome sequence and almost all of these genes were found in the s. mediterranea transcriptome data set derived by next generation sequencing. this number of cns-development-related genes is not much lower than that in model organisms known to have highly functional brains, namely, d. melanogaster, d. rerio, g. gallus and m. musculus. in addition, these numbers are clearly larger than the number of cns-development-related genes in c. elegans, which may have lost a complex cns, and suggest the possibility that the planarian has a functional brain at not only the developmental level but also the genomic level. the cns of s. mansoni consists of a central ganglion, six pairs of main nerve trunks, and longitudinal nerve cords, which extend along the length of the body. additionally, six different types of sensory papillae are known, and are considered to be valuable structures for penetration and navigation through the vasculature in the body of the host. using the predicted protein sequences of the schistosome and performing an exhaustive search of all possible cns-development genes, homologs of the genes in d. japonica were successfully detected. this result indicates that the planarian cns-development genes already existed before the divergence of planarians and schistosomes from their last common ancestor. interestingly, expression of about one-third of the cns genes was not observed in the schistosome unigene database, which was built using transcript sequences. because the schistosome unigenes were constructed from different developmental stages, libraries, and sequences, their completeness is considered to be extremely high, and therefore these cns-development genes not observed in this schistosome database can be concluded to have extremely low or no expression. it is possible that, in contrast to the free-living lifestyle of planarians, the parasitic life of schistosomes resulted in selective degeneration of unnecessary genes in schistosomes. the features of the brain are determined to a large degree by the differential regulation of the transcriptome during an animal’s life cycle. schistosomes not only inhabit various tissues of their intermediate and definitive hosts, but also traverse freshwater environments to change hosts. viewed in this light, it might have been expected that the schistosome would have had more gene components related to the cns, but instead it showed a reduced number of such genes. this might be because planarians face more complex circumstances than schistosomes regarding enemies or climate throughout their lifetime, or because schistosomes construct complicated components using more limited gene sets. further studies will be needed to clarify the relation between gene diversification and external factors in organisms’ environments. to validate possible scenarios of planarian cns evolution, this database will be valuable for the identification of relevant genes. more extensive analyses of genes and genomic resources will be needed to better understand the functions and evolution of the cns.  we have produced high-quality est reads from a head library of the planarian d. japonica, and de novo assembly analysis of these ests produced unigene sequences. similarity search against public databases and conserved domain analysis of predicted protein sequences indicated that this dataset is a useful resource for comparative transcriptome studies. comparison of homologous genes between two planarian species led us to hypothesize that not only the pressure of natural selection but also the potential of genes is important for an acceleration of the accumulation of amino-acid substitutions, a hypothesis that is supported by previous studies showing that schistosomes are defective in lipid metabolism. a total of planarian cns-development genes were extracted using gene ontology annotation, and this result suggested the possibility that the planarian has a functional brain both developmentally and genetically. mapping of these genes onto the schistosome genome showed that the 91% of the planarian cns-development genes were conserved within the schistosome genome. however, approximately one-third of the planarian cns genes were not expressed in the schistosome. these analyses suggest that the establishment of the planarian cns occurred before the divergence of planarians from their common ancestor with schistosomes, but that these two genuses subsequently diversified to adapt to their differing circumstances regarding the complexity needed for a free versus a parasitic life. this database of the d. japonica transcriptome constructed here provides an important resource not only for planarian research, but also for comparative analyses of the cns. 
a new method devised here revealed that proteins related to metabolism and defense mechanisms have high flexibility of amino-acid substitutions within the planarian family. planarians have a bilobed brain with nine lateral branches from which a variety of external signals are projected into different portions of the main lobes. in addition, our results indicate that the planarian cns-development genes already existed before the divergence of planarians and schistosomes from their common ancestor. comparative analysis revealed that 91% of the planarian cns-development genes could be mapped onto the schistosome genome, but one-third of these shared genes were not expressed in the schistosome. here we conducted large-scale est analysis of the head region of the planarian dugesia japonica to construct a database of the head-region transcriptome, and then performed comparative analyses among related species. analysis comparing homologous genes between two planarian species showed that the potential of genes is important for accumulation of amino-acid substitutions. eight-two cns-development genes were found in the planarian. the presence of many cns-development genes in our database supports the notion that the planarian has a fundamental brain with regard to evolution and development at not only the morphological/functional, but also the genomic, level. a total of high-quality est reads were obtained from a head library of the planarian dugesia japonica, and unigene sequences were produced by de novo assembly. planarians are considered to be among the extant animals close to one of the earliest groups of organisms that acquired a central nervous system during evolution. furthermore, planarians have robust regenerative ability and are attracting attention as a new model organism for the study of regeneration. we constructed a database that is a useful resource for comparative planarian transcriptome studies.
 planarians are considered to be among the extant animals close to one of the earliest groups of organisms that acquired a central nervous system during evolution. planarians have a bilobed brain with nine lateral branches from which a variety of external signals are projected into different portions of the main lobes. various interneurons process different signals to regulate behavior and learning/memory. furthermore, planarians have robust regenerative ability and are attracting attention as a new model organism for the study of regeneration. here we conducted large-scale est analysis of the head region of the planarian dugesia japonica to construct a database of the head-region transcriptome, and then performed comparative analyses among related species.  a total of high-quality est reads were obtained from a head library of the planarian dugesia japonica, and unigene sequences were produced by de novo assembly. a new method devised here revealed that proteins related to metabolism and defense mechanisms have high flexibility of amino-acid substitutions within the planarian family. eight-two cns-development genes were found in the planarian. comparative analysis revealed that 91% of the planarian cns-development genes could be mapped onto the schistosome genome, but one-third of these shared genes were not expressed in the schistosome.  we constructed a database that is a useful resource for comparative planarian transcriptome studies. analysis comparing homologous genes between two planarian species showed that the potential of genes is important for accumulation of amino-acid substitutions. the presence of many cns-development genes in our database supports the notion that the planarian has a fundamental brain with regard to evolution and development at not only the morphological/functional, but also the genomic, level. in addition, our results indicate that the planarian cns-development genes already existed before the divergence of planarians and schistosomes from their common ancestor. 