BACKGROUND
the genome and transcriptome of oats  are one of the least explored among cereal grain crops.
while the complexity associated with its large and repetitive genome  is an impediment, it is also clear that fewer efforts have been devoted to oat genome research.
for instance, as of november  <dig> there were only  <dig>  oat nucleotide sequences in genbank  <cit>  which, assuming no sequence duplication, only represents approximately  <dig> % of the estimated  <dig> gb oat genome.
this dearth of genome information is an obstacle to applying modern genetic and genomic methods for oat improvement, such as modifying the content and composition of various nutritional and health promoting compounds.
of particular interest are avenanthramides, tocols , and digestive fiber .
the potential health benefits of avenanthramides in humans are largely based on their function as antioxidants  <cit> .
tocols, including vitamin e, prevent lipid oxidative damage  <cit> .
a diet rich in the cell wall polysaccharide β-glucan is associated with a reduced risk of heart disease and reduced incidence of type ii diabetes  <cit> .
despite interest in the health-promoting properties of oat, our understanding of the genetics and molecular properties of avenanthramide, tocol, and β-glucan content and composition is still in its infancy, in part due to the complexity and large size of the polyploid oat genome.
further, because oat has less global economic importance than other cool season cereal grains such as wheat and barley, less funding has been directed toward oat research, including the development of genomic resources.
in the absence of a genome sequence and related genomic information, oat genetics and genomics can leverage genome information from related species.
for instance, the genome sequence of the model grass brachypodium distachyon   <cit>  has shown potential for assisting oat genomics research because their genomes share large blocks of synteny despite differences in genome size and ploidy  <cit> .
however, an ensemble of genomic resources for oat itself would be even more useful.
recent advances in sequencing technologies, collectively known as next generation sequencing , have transformed genomic research.
ngs has made possible high-throughput transcriptome sequencing , giving rise to a multitude of transcriptomes and transcript profiling studies in many organisms, including numerous plant species.
for instance, rna-seq has provided evidence for protein-coding gene prediction and annotation  <cit> , within-gene marker discovery  <cit> , and accurate and sensitive gene expression measurement  <cit> .
for species with a sequenced genome, rna-seq can assist in delimiting intron-exon boundaries and differential splicing to refine gene models  <cit> .
in oat, cdna sequencing has been employed for single nucleotide polymorphism  identification and marker development  <cit> .
the authors outlined a rapid and effective high-throughput pipeline for snp discovery and genotyping that could be used in other species with poorly-characterized genomes.
some of the snps developed were used to construct the first complete tetraploid oat linkage map  <cit> .
currently the most challenging aspect of rna-seq is the post-processing analysis of reads.
a number of algorithms have been developed to accommodate analysis of massive amounts of rna-seq data.
typically, each algorithm is more appropriate for a specific type of sequencing technology, target sequence, organism, and experimental condition.
two of the software packages specifically developed for the assembly of transcriptomes are velvet/oases  <cit> , collectively referred as oases hereafter, and trinity  <cit> .
both are able to assemble short reads without a reference genome by analyzing collections of de bruijn graphs constructed based on series of overlapping k-mers.
velvet was initially designed for genomic dna assembly, with oases later added to address particulars of transcriptome assembly such as alternative splicing and high variability in gene expression that impact read coverage.
trinity was designed specifically for transcriptome assembly.
both packages are able to differentiate slightly dissimilar versions of a particular gene.
in this study, we employed high-throughput paired-end illumina technology to generate  <dig>  gb of read sequence to explore the oat seed transcriptome.
the specific objectives were i) to reconstruct a comprehensive transcriptome encompassing four stages of seed development; ii) to create, characterize, and annotate a gene expression atlas of the developing oat seed; and iii) to employ this atlas to examine gene expression associated with the synthesis of health-promoting compounds.
this assembly constitutes the first comprehensive transcriptome study in oat seeds, and provides a valuable new resource for the oat community to assist efforts aimed at enhancing oat seed nutritional quality and other traits.
in addition, it constitutes the first direct comparison between two of the most widely used assembly programs in a polyploid organism, and thus provides guidelines for future assemblies.
RESULTS
illumina sequencing and read assembly
a flowchart overview of the steps followed in the assembly process is outlined in figure  <dig>  to obtain a broad sample of the oat seed transcriptome, four independent cdna libraries were constructed from de-hulled oat seeds sampled at four developmental stages:  <dig>   <dig>   <dig>  and 28 days after anthesis .
libraries were sequenced using illumina hiseq  <dig> technology, with nearly  <dig> million 100-base paired-end raw reads generated across the four stages.
after removal of primer adaptor sequences and filtering low quality reads,  <dig> million high-quality reads remained.
erroneous base calls are more prone to happen near the 3′-end of the cdna fragment.
thus, to increase the assembly accuracy, reads were further trimmed according to their individual base-call quality-score  .
the  <dig> million reads were assembled with oases and trinity.
because trinity demands considerably more computational resources, each of the four libraries had to be independently assembled when this package was used.
in contrast the four libraries were combined within a single oases assembly to increase the number of starting reads and the expected coverage.
additionally, while longer k-mers lead to more specificity , they also lower coverage and sensitivity and thus perform poorly on genes expressed at low levels  <cit> .
in assemblers where this parameter can be modified a balance must be reached during analysis.
in its current version, trinity does not allow k-mer value modification; rather it is fixed to a 25-mer.
however, with oases several k-mer sizes  were tested.
we could not test 25-mer on oases with the four libraries combined dataset due to memory limitations, since with shorter k-mers more k-mer-size fragments have to be allocated.
here, we use the term ‘transcript isoform’ or ‘transcript’ to refer to each individual sequence in the assembly.
the terms ‘locus’ or ‘loci’ are used to group together similar transcript isoforms.
this assignment of isoforms to loci is performed by the trinity and oases assemblers based on sequence variations detected as the assembly process progress.
thus, separate transcript isoforms within a locus might represent splice variants or other highly similar sequences such as homeoalleles .
a two-step evaluation was applied on all resultant trinity and oases assemblies to benchmark their quality.
in the first step, several broadly used assembly-quality parameters were assessed.
these included the number of assembled reads, the number of transcripts assembled, average transcript length, and the n <dig> value.
each parameter was calculated for the different k-mer oases assemblies and for each of the four independently assembled libraries in trinity.
a summary of all quality parameters measured is shown in additional file  <dig>  an informative assay of assembly quality in the absence of a reference genome is to compare the assemblies against well-validated databases.
thus, in the second step both the uniprot-plants and uniref <dig> databases, as well as the predicted complete set of translated coding sequences of brachypodium, were selected to examine the quality of the assemblies .
for each de novo assembly tested, the following three types of transcript sequences representative of every locus were compared with the sequences in the databases: i) the longest transcript isoform of each locus; ii) the isoform with the highest confidence; and iii) non-redundant transcripts identified by clustering of all de novo transcripts.
this clustering procedure reduces the size of the assembly by combining together highly similar isoforms .
based on the quality-parameters assessed, all trinity assemblies were less accurate, had shorter transcripts, and contained fewer putative unique protein coding sequences than the assemblies constructed with oases.
of the eleven k-mers tested in oases, a good balance between transcript length, specificity, and diversity  was found for the 67-kmer assembly.
this assembly, which contains  <dig>  sequences, was termed de novo oat seed transcriptome assembly  and used as the reference oat seed transcriptome assembly for further analysis .
transcripts were further filtered to reduce presence of sequences with many undetermined calls , or shorter than  <dig> nt.
the remaining  <dig>  transcript sequences have been deposited at ddbj/embl/genbank under the accession gaje <dig>  the version described in this paper is the first version, gaje <dig>  to rule out the possibility that the superiority of oases assemblies may be a consequence of the higher number of initial reads used, each library was also independently assembled with oases.
as expected, fewer transcripts were assembled in the individual libraries using the same 67-mer:  <dig> ,  <dig> ,  <dig> , and  <dig>  for  <dig>   <dig>   <dig>  and  <dig> daa libraries, respectively, as compared to dnost .
also, the average transcript length , and n <dig>  was shorter.
however, these individual library oases assemblies were superior to their counterpart library trinity assemblies.
in addition, the same 25-mer that is fixed for trinity was used with oases on the individual libraries and produced  <dig>   <dig> ,  <dig> , and  <dig>  transcripts of  <dig> ,  <dig> ,  <dig> , and  <dig>  average length, and a n <dig> of  <dig> ,  <dig> ,  <dig> , and  <dig> , for  <dig>   <dig>   <dig>  and  <dig> daa libraries, respectively.
all three parameters are higher than the ones obtained for trinity assemblies.
analysis and annotation of dnost
the dnost yielded over 55 mb of assembled sequence, with an average transcript length of  <dig>  nt and average sequencing depth of  <dig> ×.
transcript length distribution is shown in additional file  <dig>  the dnost assembly contains  <dig>  transcript isoforms, which represent a total of  <dig>  distinct assembled loci .
as noted earlier, each locus may include several highly similar transcript isoforms, whose sequence differences could reflect splice variants, homeologs and paralogs, and sequencing errors.
for instance, when the longest transcript isoform per locus was blasted  against the uniprot-plants database, there were  <dig>  hits representing  <dig> % of the  <dig>  loci .
however, only  <dig>   of them corresponded to unique uniprot-plants entries.
because only one isoform  per locus was considered, the degree of redundancy may be attributable mostly to the inherent duplication of the hexaploid oat genome, and suggests that homeologous genes may be assembled within the same locus, as well as in different loci, depending on how divergent the homeologs are.
in addition, to assist the identification of putative homeologous genes, regardless of the locus into they were assembled, a search was conducted with the dnost assembly against itself .
although families of close paralogs are most certainly present, due to the stricter parameters used, this search is more likely to retrieve homeologous relationships by grouping together highly similar transcript isoforms.
we found  <dig>  relationships and denoted the results the homeologous set file .
within the hsf, the longest transcript was chosen as the representative of the locus .
nt: nucleotides.
read depth may have a significant impact on the ability to discriminate between homeologs in polyploid genomes.
to examine this in detail, reads were mapped back to a particular chosen locus  and piled up to illustrate read depth .
this locus was chosen as an example because it is homologous to an enzyme in the tocol pathway, and was assembled into three isoforms, consistent with three oat homeologs.
in effect, in areas with low-medium read-depth, differentiation between snps is problematic.
for instance, the two zoomed-in putative snps in the low read-covered region of the assembled gene  were overlooked by the assembly software and called as a single base for all three isoforms.
however, the presence of a polymorphism is suggested by the number of reads bearing a different base.
the expected high degree of homozygosity of the oat genotype used for rna-seq suggests that the single nucleotide variations observed in the assembled transcripts are most likely due to true homeoalleles and not to genetic heterozygosity.
while, the latter cannot be excluded as source of snps, it is presumed to be less frequent than homeoallele sequence variation.
in areas with more read depth , snp discrimination occurred.
generally, we observed effective discrimination by the assembler above a read depth of 75– <dig>
all de novo assembled dnost transcripts were annotated  against genbank’s non-redundant  protein database .
predicted proteins, including many with putative functions assigned, could be retrieved for  <dig>   of the  <dig>  dnost transcript isoforms, which is a percentage similar or above previous studies involving plant species without sequenced genomes  <cit> .
the redundancy in dnost is indicated by the fact that just  <dig>  of the annotated dnost transcripts corresponded to unique nr peptides.
similarly,  <dig>  brachypodium predicted peptides were identified  that were similar to one or more dnost transcript isoforms, with  <dig> % of dnost transcripts having two or more hits.
therefore, the transcripts identified over four oat seed developmental stages share homology to nearly 40% of the predicted  <dig>  brachypodium protein-coding genes  <cit> .
the dnost transcript sequences were uniquely anchored to the single best hit in the brachypodium genome , and were found to be homogenously distributed in the brachypodium genome and correlated  with the brachypodium coding sequence  density, indicating that a broad diversity of genes is represented in dnost.
functional classification of dnost
functional classification of dnost sequences was performed through a gene ontology  categorization .
the original  <dig>  dnost transcript isoforms were first clustered within and across loci  to  <dig>  representative sequences, to reduce the redundancy of the original assembly.
these clusters were queried  against uniprot-plants database, and subsequently annotated using the go-uniprot association file.
an ontology annotation was found for  <dig>  of the sequences , of which  <dig>  corresponded to unique proteins.
the inferred go terms were distributed in the three main go domains as follows: biological process , cellular component , and molecular function .
biological process was mainly represented by cellular and metabolic processes , representing more than 55% of the annotations, followed by response to stimulus  and biological regulation .
when the sequences were categorized according to the cellular component main term ,  <dig> % of them corresponded to cell or cell part categories, and  <dig> % to organelle or organelle part.
a hypergeometric statistical test was employed to identify over-represented  go categories and genes present in dnost more often than expected by chance, as compared to uniprot-plants database .
among over-represented cellular components were cell wall and other categories associated to developing tissues.
overrepresented molecular functions were: transferase activity, catalytic activity, nucleotide binding, atp binding, kinase activity, phosphotransferase activity, and protein binding.
overrepresented biological processes were glycolysis and several other metabolic processes.
transcripts associated with the synthesis of important health-promoting compounds appear to be well-represented.
for instance, the cell wall subcategory contained  <dig> unique tran-scripts, including peptidoglycan-based, cellulose- and pectin-containing, and chitin- and β-glucan-containing cell wall.
the most represented go subcategories within molecular function main term were binding , catalytic activity , transporter activity , and transcription regulation activity  .
all four molecular functions are involved in biosynthetic processes, reflecting the developing nature of the tissues analyzed.
in addition, all dnost transcript isoforms were classified according to their major mapman envisaged metabolic routes  and their normalized raw digital expression counts  .
results reinforce previous observations that the genes represented in dnost are diverse in nature and cover many key cell processes.
in agreement with go classification, the mapman metabolism overview functional classification also shows high numbers of transcripts involved in synthetic processes as compared to degradation processes, consistent with a developing tissue.
microsatellite markers in dnost
microsatellite  markers are broadly used for marker-assisted selection in crop breeding due to the ease of their implementation and co-dominant nature.
we scanned the dnost for gene-derived ssr markers with the potential to be used in oat breeding programs.
in total,  <dig>  ssrs were found within  <dig>  different transcripts.
a summary of the putative ssrs is shown in additional file  <dig>  primers targeting the ssrs were designed when possible .
the most abundant ssrs were the tri-repeats .
the rest were distributed as follows: mono-repeats , di , tetra , penta , and hexa .
excluding mono-repeats, the percentage of di, tri, tetra, penta and hexa was  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig> %, respectively.
the ssr-bearing transcripts were aligned  against the brachypodium genome sequence to pinpoint the syntenic location in which polymorphisms occur .
their distribution along the brachypodium genome is uniform and consistent with gene density.
construction of oat seed gene indices oatseedref <dig> and oatseedref90
to develop a comprehensive compendium of available oat seed expressed sequences, we combined the dnost assembly with oat sequences published by other sources.
the sequence information retrieved from the genbank  consisted of  <dig>  oat seed est sequences totaling  <dig> , <dig> nt with an average length of  <dig>  nt .
these were concatenated with the  <dig>  dnost transcripts to build an index of  <dig>  oat seed expressed sequences.
we named this reference index oatseedref <dig> v <dig>  .
additionally, to reduce redundancy in oatseedref <dig>  oatseedref <dig> was created  in a manner similar to the uniprot reference clusters databases uniref <dig> and uniref <dig> .
clustering reduces the presence of redundant sequences and base miscall errors, but can also eliminate highly similar homeoalleles.
the oatseedref <dig> non-redundant index is composed of  <dig>  sequences, of which  <dig>  are tentative consensus  and  <dig>  singletons, for a total of  <dig> , <dig> nt of sequence information.
of the  <dig>  sequences,  <dig>   were unique to the dnost assembly,  <dig>   were unique to the genbank ests, and  <dig>   resulted from merging of at least one genbank sequence and one of our de novo transcript assemblies.
sequence lengths in oatseedref <dig> range between  <dig> and  <dig>  nt, with an average of  <dig>  nt.
since the minimum contig length in dnost was set to  <dig> nt, all of the shortest sequences  derive from ests retrieved from genbank.
clustering revealed that more than 63% of the oat ests in genbank are overlapping or overlapped by sequences present in dnost.
the oatseedref <dig> sequences were compared  with the brachypodium and uniprot-plants protein databases and  <dig>   and  <dig>   hits were retrieved, respectively.
only  <dig> % of the unique brachypodium proteins corresponded to the set of oat ests in genbank, which indicates that the vast majority of the unique-protein targets come from our de novo assembly.
this comparison further demonstrates that our rna-seq assemblies are an important source of novel oat cdna sequences.
oat health-promoting compounds as a practical demonstration of dnost utility
to demonstrate the utility of dnost, we studied biosynthetic genes for three important health-promoting compounds in oats: avenanthramides, tocols, and β-glucans.
sequences of genes in the respective pathways from close relatives were downloaded from genbank and other sources  and the dnost assembly was searched  for homologous sequences.
homologous transcripts were found for all genes investigated , which supports both the accuracy and completeness of the dnost assembly.
the pivotal enzyme in the biosynthesis of avenanthramides is hht, which catalyzes the final condensation reaction.
searches  against dnost retrieved multiple homologous transcripts to the four reported oat hht isoforms  .
homologies were also found for genes coding two other key enzymes in avenanthramide synthesis, ccoaomt and ccoa3h.
for ccoaomt, eight homologs were found in dnost.
also, the complete ccoaomt cds was obtained by expanding a previously existing genbank sequence.
the second enzyme, ccoa3h, is required for the synthesis of several of the most predominant avenanthramides.
to our knowledge, no ccoa3h gene sequence has been reported for oats.
we found four transcripts homologous to brachypodium  and barley  ccoa3h genes.
the expression of all avenanthramide synthesis homologs was quantified and is shown in heat maps for the main enzymatic steps in the pathway .
homologous transcripts in the heat maps may represent homeoalleles or other highly similar sequences including paralogs.
the results indicate that there are significant differences in the expression of homologous transcripts in many biosynthetic steps, suggesting that homeoalleles and/or paralogs may be differentially expressed.
tocol accumulation profiles in developing oat seeds have only been studied recently, and distinctly different temporal patterns of accumulation for both tocotrienols and tocopherols were found  <cit> .
sequences of the genes in the tocol biosynthetic pathway were sought within the dnost, and transcripts were found for all .
for instance, five transcripts exhibited homology to barley, brachypodium, and wheat hppd .
ggr catalyzes the reduction of ggdp to pdp, and six ggr homologues were assembled in dnost which nearly cover the predicted complete cds.
the committed step in the biosynthesis of tocopherols is condensation of hga and pdp, catalyzed by hpt , for which one oat transcript was found.
similarly, the committed step in tocotrienol biosynthesis is the condensation of hga with ggdp, by hggt.
five highly similar hggt homologue transcripts were found in dnost.
surprisingly, no hggt homologue was found in the brachypodium genome.
seeds of brachypodium accumulate tocotrienols , thus it is likely that the hggt homologue lies in an unsequenced region of the brachypodium genome.
the last three enzymes, vte <dig>  vte <dig>  and vte <dig>  are common for the synthesis of both tocopherols and tocotrienols, and homologues were found for all three genes.
thus, dnost holds promise for linking gene expression to metabolic aspects of tocol accumulation in oat seeds.
β-glucans were the last health-promoting compounds evaluated.
cellulose-synthase  and cellulose-synthase-like  sequences were downloaded from  and compared to dnost assembly.
the retrieved matches included  <dig> ces and  <dig> csl unique transcripts .
the average length of the transcript isoforms assembled for csl and ces was  <dig>  and  <dig> , respectively.
as an example, alignments of barley cls-f <dig>  and the oat homologue  with dnost revealed homology to five isoforms .
comparatively to barley, two insertions and one deletion at the 3′-end appears to produce an early stop codon.
similarly, ten assembled isoforms had 84-85% and 76-78% identity with their barley and arabidopsis ces-a homologues, respectively.
discussion
oat transcripts were de novo assembled from illumina reads derived from four oat seed developmental stages.
assembly was performed with both oases and trinity, and the results were compared.
strict values were chosen for some parameters to assure a more precise assembly.
to our knowledge this is the first comparison between oases and trinity in a polyploid organism, which makes our study a useful guide for future studies on transcriptome analysis in plants, where polyploidy is common.
several quality tests were performed to determine the robustness of each assembly; the length and number of assembled transcripts, and how precisely they match annotated databases are particularly valuable in this regard.
in our assemblies we prioritized sequence accuracy over other criteria, such as total number of transcripts and length of total assembled sequence.
this approach may risk losing rare transcripts, but overall it improves the quality of the assembly.
despite the fact that trinity was specifically developed for transcript assemblies, our quality benchmarks indicated higher quality scores for oases assemblies.
it is likely that the reduced number of putative unique protein coding sequences from the trinity assemblies is due in part to the shorter transcripts produced.
trinity developers initially tested its algorithms on diverse organisms such as fission yeast, mouse, and whitefly  <cit> , but according to our results trinity appears not to be as suitable for complex polyploid transcriptomes such as that of hexaploid oat.
one of the reasons for this result may be that k-mer length is fixed at  <dig> nt; such a small number may not be sufficient to discriminate among highly similar homeologous or even paralogous sequences.
further, the short k-mer length causes trinity to run considerably more slowly than oases run with 67-mer, since more words of size k per read have to be constructed and tested for alignment.
if the multi-thread option of trinity is not selected, oases running with the fixed 25-mer of trinity finished 15– <dig> times faster than trinity.
oases was shown to reconstruct a higher number of gene transcripts than trinity on human and mouse datasets, although the accuracy of the assemblies was comparable  <cit> .
similarly, in the assembly of the tea plant  transcriptome, oases performed better than trinity in most of the parameters studied  <cit> .
however, trinity produced better results for highly expressed genes.
the authors also reported that trinity run  <dig> times slower than oases when the same k-mer value of  <dig> was used.
based on our benchmark analysis, the oases 67-mer-assembly  was selected as the representative for the seed oat transcriptome.
the proportion of dnost loci with at least one homologue in other databases  ranged between  <dig>  and  <dig> % in these comparisons, and was highest for the uniprot-plants database.
these percentages increased up to  <dig> %  when all dnost transcript isoforms were considered.
significantly, nearly 72% of the loci  in dnost had a homologue within the collection of predicted brachypodium peptides, which reinforces a previous study suggesting that brachypodium can be an effective resource to assist oat genetics and genomics research  <cit> .
assembly errors and other sequence variation could produce fusion longer transcripts during the assembly.
we do not believe that this occurred at a high level, as seen in the transcripts analyzed .
although fusion proteins may not be completely discarded, that  <dig> % of dnost transcripts retrieved two or more brachypodium predicted cds is more likely attributable to the presence of transcripts with more than one large conserved domain, or other regions with highly similar sequence .
indeed, the brachypodium genome shows six major chromosomal duplications covering  <dig> % of the genome, which represent ancestral whole-genome duplications, and so detection of paralogs is likely to be a major factor in detecting more than one gene from blast searches  <cit> .
for non-sequenced organisms, large-scale transcriptome assemblies from illumina reads appear to be more robust than from roche/ <dig>  presumably due to the higher coverage attained.
in an assembly of chickpea transcripts obtained from roche/ <dig> rna-seq, only about 58% had similarity  to the uniref <dig> database  <cit> , vs. the nearly 70% in our assembly.
in another study  <cit> , oliver et al.
reported between  <dig>  and  <dig>  transcripts using high-throughput roche/ <dig> sequencing technology on four different oat genotypes, with average transcript lengths ranging between  <dig> and  <dig> nt, vs. the  <dig>  transcripts averaging  <dig>  nt in dnost.
our transcriptome assembly appears to be accurate, as reflected by the results of blast searches against both plant and multi-organism databases.
thus dnost greatly expands the current collection of oat expressed sequences.
for instance, transcription factors are typically expressed at low levels and are most likely to be under-represented in est databases; however, functional classification  analysis of dnost revealed a large group of putative transcription factor sequences.
we estimated that a 75– <dig> read depth is required for homeolog discrimination.
to our knowledge this is the first study to provide such an estimate for a polyploid genome.
in a de novo assembly of three individual human genomes, best false negative allele calls were obtained with considerably lower  read coverage  <cit> .
also, a 20× sequencing depth was required in the de novo assembly of an individual human genome with 75-nt read length to achieve a maximum contig size after which no further increase in length was observed  <cit> .
our assembly also suggests that homeologous genes in oat are both highly conserved and may remain functional, although confirming this would require extensive proteomic and enzymatic analyses.
moreover, the question of whether these sequences represent true homeologs, allelic variants, or artifacts is not empirically addressed in this study.
thus, caution may be advised when using dnost for certain purposes.
for instance, the assembly process may have merged multiple homeologs into a single sequence, shuffling snps among homeologs.
this could be especially relevant where low sequence divergence prevented the assembler from resolving homeologs.
the homeologous forms in oat show similar or even a higher percentage of identity among them than was reported for wheat, with 90-99% identity at the nucleotide level and often identical at the amino acid level  <cit> .
polyploid species pose a challenge because of the presence of homeoalleles that may be difficult to deconvolute at the sequence level.
to assemble highly similar homeologs separately requires a high number of reads and imposing strict parameters for assembly, such as larger k-mers, no mismatches allowed in k-mer alignments, and high minimum read coverage.
clearly, assembling with longer k-mers first requires longer reads.
currently, paired-end read sequencing using illumina technology is limited to  <dig> nt, which still may be not sufficient to precisely discriminate highly similar homeologs in polyploid genomes.
conversely, stricter assembling parameters will reduce the number of transcripts.
while assembling dnost a balance between transcript redundancy and number was pursued, since obtaining a large number of transcripts was also a goal to make an oat library as complete and diverse as possible.
sequence redundancy is intrinsic to polyploid organisms, and this was proven to be the case in dnost.
despite the continuous improvements in ngs technologies and assembly algorithms, it is still extremely challenging assembling highly homologous genes into separate isoforms.
there are numerous opportunities for practical use of dnost.
for example, the number of sequence polymorphisms that have been described in oat suitable to be used as molecular markers is very limited.
for instance, only  <dig> ssr markers from oat were listed on the graingenes database as of november  <dig> .
the more than  <dig>  potential new genic ssr markers identified greatly expand the current ssr repository.
while only a fraction of these may be found to be polymorphic between oat cultivars, validated polymorphic ssrs will provide a new resource for marker-assisted selection.
as we found in oat, tri-nucleotide repeats are the most abundant  in other plant species such as rice, corn, peanut, alfalfa, and arabidopsis  <cit> , indicative of the fact that they are probably preferentially present within transcripts to prevent frame shifts.
the first physically anchored hexaploid oat map has been recently completed  <cit> .
a goldengate assay was employed for genotyping  <dig>  snps as part of the mapping effort.
to examine for the presence of those snps in the dnost transcripts, searches  were conducted using the dna sequence surrounding the snp.
this revealed that  <dig>   of the snps in the goldengate assay were present in at least one transcript .
because goldengate snps were first discovered using cdna from different tissues, these results suggest dnost to be a fairly comprehensive source sequences, despite its seed origin.
as a last example, transcripts for genes associated with the synthesis of health-promoting compounds are present in dnost and thus novel information is available to examine molecular aspects of their synthesis.
for instance, avenanthramides are unique to oat among cereal grains, but the genes involved in their synthesis have not been fully characterized.
a key enzyme in avenanthramide pathway is hht, from which there appear to be multiple isoforms that accept a wide range of substrates with different affinities  <cit> .
complete sequences for three oat hhts  have been reported  <cit> , as well as a partial sequence for ashht <dig> .
we found twelve homologous transcripts to ashht <dig>  eleven to ashht <dig>  thirteen to ashht <dig>  and thirteen ashht <dig>  for a total of sixteen different transcripts.
locus_17720_transcript_1/ <dig>  had high sequence identity to ashht <dig> .
a second transcript isoform  was the most similar  to ashht <dig> .
these two loci shared 96% identity with the differences located in the 3′utr, suggesting that ashht <dig> and ashht <dig> are in fact homeologs.
in another example, five dnost transcripts had at least 95% homology in their predicted cds to ashht4: locus_ <dig> , loci_15223_/ <dig> , locus_ <dig> , and locus_ <dig> .
by aligning all transcripts with the previously known partial ashtt <dig> cdna we were able to extend the ashtt <dig>  <dig> bases towards the 5′ end to complete the cds.
thus, dnost is useful not only to identify oat homologues for genes of interest, but also to obtain complete sequences of partially cloned oat genes.
CONCLUSIONS
there are inherent challenges in developing an accurate oat gene transcript set because of its level of genome duplication, with post-processing of sequences often required to differentiate true homeologues from assembled artifacts.
we have shown that our de novo transcript assembly of developing oat seeds obtained with oases is able to differentiate highly similar genes to a significant extent.
this indicates that the nearly  <dig> fold average coverage we obtained is deep enough to discern homoeoalleles and paralogs to some degree.
nevertheless, post-processing of sequences may still be required to establish whether these sequences represent true homeologs, particularly in transcripts with low coverage.
our study provides an optimized analytical pipeline for other researchers attempting to assemble transcriptome data from polyploid plant species.
we validate that dnost is an excellent source of diverse oat transcripts such as those associated with the synthesis of several oat seed compounds possessing health-promoting properties, and also served as a resource to identify several thousand new potential molecular markers.
thus the oat transcript assembly developed in this study will be useful for a variety of avenues of oat improvement.

 the genome and transcriptome of oats are one of the least explored among cereal grain crops. while the complexity associated with its large and repetitive genome is an impediment, it is also clear that fewer efforts have been devoted to oat genome research. for instance, as of november there were only oat nucleotide sequences in genbank which, assuming no sequence duplication, only represents approximately % of the estimated gb oat genome. this dearth of genome information is an obstacle to applying modern genetic and genomic methods for oat improvement, such as modifying the content and composition of various nutritional and health promoting compounds. of particular interest are avenanthramides, tocols, and digestive fiber. the potential health benefits of avenanthramides in humans are largely based on their function as antioxidants. tocols, including vitamin e, prevent lipid oxidative damage. a diet rich in the cell wall polysaccharide β-glucan is associated with a reduced risk of heart disease and reduced incidence of type ii diabetes. despite interest in the health-promoting properties of oat, our understanding of the genetics and molecular properties of avenanthramide, tocol, and β-glucan content and composition is still in its infancy, in part due to the complexity and large size of the polyploid oat genome. further, because oat has less global economic importance than other cool season cereal grains such as wheat and barley, less funding has been directed toward oat research, including the development of genomic resources. in the absence of a genome sequence and related genomic information, oat genetics and genomics can leverage genome information from related species. for instance, the genome sequence of the model grass brachypodium distachyon has shown potential for assisting oat genomics research because their genomes share large blocks of synteny despite differences in genome size and ploidy. however, an ensemble of genomic resources for oat itself would be even more useful. recent advances in sequencing technologies, collectively known as next generation sequencing, have transformed genomic research. ngs has made possible high-throughput transcriptome sequencing, giving rise to a multitude of transcriptomes and transcript profiling studies in many organisms, including numerous plant species. for instance, rna-seq has provided evidence for protein-coding gene prediction and annotation, within-gene marker discovery, and accurate and sensitive gene expression measurement. for species with a sequenced genome, rna-seq can assist in delimiting intron-exon boundaries and differential splicing to refine gene models. in oat, cdna sequencing has been employed for single nucleotide polymorphism identification and marker development. the authors outlined a rapid and effective high-throughput pipeline for snp discovery and genotyping that could be used in other species with poorly-characterized genomes. some of the snps developed were used to construct the first complete tetraploid oat linkage map. currently the most challenging aspect of rna-seq is the post-processing analysis of reads. a number of algorithms have been developed to accommodate analysis of massive amounts of rna-seq data. typically, each algorithm is more appropriate for a specific type of sequencing technology, target sequence, organism, and experimental condition. two of the software packages specifically developed for the assembly of transcriptomes are velvet/oases, collectively referred as oases hereafter, and trinity. both are able to assemble short reads without a reference genome by analyzing collections of de bruijn graphs constructed based on series of overlapping k-mers. velvet was initially designed for genomic dna assembly, with oases later added to address particulars of transcriptome assembly such as alternative splicing and high variability in gene expression that impact read coverage. trinity was designed specifically for transcriptome assembly. both packages are able to differentiate slightly dissimilar versions of a particular gene. in this study, we employed high-throughput paired-end illumina technology to generate gb of read sequence to explore the oat seed transcriptome. the specific objectives were i) to reconstruct a comprehensive transcriptome encompassing four stages of seed development; ii) to create, characterize, and annotate a gene expression atlas of the developing oat seed; and iii) to employ this atlas to examine gene expression associated with the synthesis of health-promoting compounds. this assembly constitutes the first comprehensive transcriptome study in oat seeds, and provides a valuable new resource for the oat community to assist efforts aimed at enhancing oat seed nutritional quality and other traits. in addition, it constitutes the first direct comparison between two of the most widely used assembly programs in a polyploid organism, and thus provides guidelines for future assemblies.  illumina sequencing and read assembly a flowchart overview of the steps followed in the assembly process is outlined in figure  to obtain a broad sample of the oat seed transcriptome, four independent cdna libraries were constructed from de-hulled oat seeds sampled at four developmental stages: and 28 days after anthesis. libraries were sequenced using illumina hiseq technology, with nearly million 100-base paired-end raw reads generated across the four stages. after removal of primer adaptor sequences and filtering low quality reads, million high-quality reads remained. erroneous base calls are more prone to happen near the 3′-end of the cdna fragment. thus, to increase the assembly accuracy, reads were further trimmed according to their individual base-call quality-score. the million reads were assembled with oases and trinity. because trinity demands considerably more computational resources, each of the four libraries had to be independently assembled when this package was used. in contrast the four libraries were combined within a single oases assembly to increase the number of starting reads and the expected coverage. additionally, while longer k-mers lead to more specificity, they also lower coverage and sensitivity and thus perform poorly on genes expressed at low levels. in assemblers where this parameter can be modified a balance must be reached during analysis. in its current version, trinity does not allow k-mer value modification; rather it is fixed to a 25-mer. however, with oases several k-mer sizes were tested. we could not test 25-mer on oases with the four libraries combined dataset due to memory limitations, since with shorter k-mers more k-mer-size fragments have to be allocated. here, we use the term ‘transcript isoform’ or ‘transcript’ to refer to each individual sequence in the assembly. the terms ‘locus’ or ‘loci’ are used to group together similar transcript isoforms. this assignment of isoforms to loci is performed by the trinity and oases assemblers based on sequence variations detected as the assembly process progress. thus, separate transcript isoforms within a locus might represent splice variants or other highly similar sequences such as homeoalleles. a two-step evaluation was applied on all resultant trinity and oases assemblies to benchmark their quality. in the first step, several broadly used assembly-quality parameters were assessed. these included the number of assembled reads, the number of transcripts assembled, average transcript length, and the n value. each parameter was calculated for the different k-mer oases assemblies and for each of the four independently assembled libraries in trinity. a summary of all quality parameters measured is shown in additional file an informative assay of assembly quality in the absence of a reference genome is to compare the assemblies against well-validated databases. thus, in the second step both the uniprot-plants and uniref databases, as well as the predicted complete set of translated coding sequences of brachypodium, were selected to examine the quality of the assemblies. for each de novo assembly tested, the following three types of transcript sequences representative of every locus were compared with the sequences in the databases: i) the longest transcript isoform of each locus; ii) the isoform with the highest confidence; and iii) non-redundant transcripts identified by clustering of all de novo transcripts. this clustering procedure reduces the size of the assembly by combining together highly similar isoforms. based on the quality-parameters assessed, all trinity assemblies were less accurate, had shorter transcripts, and contained fewer putative unique protein coding sequences than the assemblies constructed with oases. of the eleven k-mers tested in oases, a good balance between transcript length, specificity, and diversity was found for the 67-kmer assembly. this assembly, which contains sequences, was termed de novo oat seed transcriptome assembly and used as the reference oat seed transcriptome assembly for further analysis. transcripts were further filtered to reduce presence of sequences with many undetermined calls, or shorter than nt. the remaining transcript sequences have been deposited at ddbj/embl/genbank under the accession gaje the version described in this paper is the first version, gaje to rule out the possibility that the superiority of oases assemblies may be a consequence of the higher number of initial reads used, each library was also independently assembled with oases. as expected, fewer transcripts were assembled in the individual libraries using the same 67-mer:,,, and for and daa libraries, respectively, as compared to dnost. also, the average transcript length, and n was shorter. however, these individual library oases assemblies were superior to their counterpart library trinity assemblies. in addition, the same 25-mer that is fixed for trinity was used with oases on the individual libraries and produced,, and transcripts of,,, and average length, and a n of,,, and, for and daa libraries, respectively. all three parameters are higher than the ones obtained for trinity assemblies. analysis and annotation of dnost the dnost yielded over 55 mb of assembled sequence, with an average transcript length of nt and average sequencing depth of ×. transcript length distribution is shown in additional file the dnost assembly contains transcript isoforms, which represent a total of distinct assembled loci. as noted earlier, each locus may include several highly similar transcript isoforms, whose sequence differences could reflect splice variants, homeologs and paralogs, and sequencing errors. for instance, when the longest transcript isoform per locus was blasted against the uniprot-plants database, there were hits representing % of the loci. however, only of them corresponded to unique uniprot-plants entries. because only one isoform per locus was considered, the degree of redundancy may be attributable mostly to the inherent duplication of the hexaploid oat genome, and suggests that homeologous genes may be assembled within the same locus, as well as in different loci, depending on how divergent the homeologs are. in addition, to assist the identification of putative homeologous genes, regardless of the locus into they were assembled, a search was conducted with the dnost assembly against itself. although families of close paralogs are most certainly present, due to the stricter parameters used, this search is more likely to retrieve homeologous relationships by grouping together highly similar transcript isoforms. we found relationships and denoted the results the homeologous set file. within the hsf, the longest transcript was chosen as the representative of the locus. nt: nucleotides. read depth may have a significant impact on the ability to discriminate between homeologs in polyploid genomes. to examine this in detail, reads were mapped back to a particular chosen locus and piled up to illustrate read depth. this locus was chosen as an example because it is homologous to an enzyme in the tocol pathway, and was assembled into three isoforms, consistent with three oat homeologs. in effect, in areas with low-medium read-depth, differentiation between snps is problematic. for instance, the two zoomed-in putative snps in the low read-covered region of the assembled gene were overlooked by the assembly software and called as a single base for all three isoforms. however, the presence of a polymorphism is suggested by the number of reads bearing a different base. the expected high degree of homozygosity of the oat genotype used for rna-seq suggests that the single nucleotide variations observed in the assembled transcripts are most likely due to true homeoalleles and not to genetic heterozygosity. while, the latter cannot be excluded as source of snps, it is presumed to be less frequent than homeoallele sequence variation. in areas with more read depth, snp discrimination occurred. generally, we observed effective discrimination by the assembler above a read depth of 75– all de novo assembled dnost transcripts were annotated against genbank’s non-redundant protein database. predicted proteins, including many with putative functions assigned, could be retrieved for of the dnost transcript isoforms, which is a percentage similar or above previous studies involving plant species without sequenced genomes. the redundancy in dnost is indicated by the fact that just of the annotated dnost transcripts corresponded to unique nr peptides. similarly, brachypodium predicted peptides were identified that were similar to one or more dnost transcript isoforms, with % of dnost transcripts having two or more hits. therefore, the transcripts identified over four oat seed developmental stages share homology to nearly 40% of the predicted brachypodium protein-coding genes. the dnost transcript sequences were uniquely anchored to the single best hit in the brachypodium genome, and were found to be homogenously distributed in the brachypodium genome and correlated with the brachypodium coding sequence density, indicating that a broad diversity of genes is represented in dnost. functional classification of dnost functional classification of dnost sequences was performed through a gene ontology categorization. the original dnost transcript isoforms were first clustered within and across loci to representative sequences, to reduce the redundancy of the original assembly. these clusters were queried against uniprot-plants database, and subsequently annotated using the go-uniprot association file. an ontology annotation was found for of the sequences, of which corresponded to unique proteins. the inferred go terms were distributed in the three main go domains as follows: biological process, cellular component, and molecular function. biological process was mainly represented by cellular and metabolic processes, representing more than 55% of the annotations, followed by response to stimulus and biological regulation. when the sequences were categorized according to the cellular component main term, % of them corresponded to cell or cell part categories, and % to organelle or organelle part. a hypergeometric statistical test was employed to identify over-represented go categories and genes present in dnost more often than expected by chance, as compared to uniprot-plants database. among over-represented cellular components were cell wall and other categories associated to developing tissues. overrepresented molecular functions were: transferase activity, catalytic activity, nucleotide binding, atp binding, kinase activity, phosphotransferase activity, and protein binding. overrepresented biological processes were glycolysis and several other metabolic processes. transcripts associated with the synthesis of important health-promoting compounds appear to be well-represented. for instance, the cell wall subcategory contained unique tran-scripts, including peptidoglycan-based, cellulose- and pectin-containing, and chitin- and β-glucan-containing cell wall. the most represented go subcategories within molecular function main term were binding, catalytic activity, transporter activity, and transcription regulation activity. all four molecular functions are involved in biosynthetic processes, reflecting the developing nature of the tissues analyzed. in addition, all dnost transcript isoforms were classified according to their major mapman envisaged metabolic routes and their normalized raw digital expression counts. results reinforce previous observations that the genes represented in dnost are diverse in nature and cover many key cell processes. in agreement with go classification, the mapman metabolism overview functional classification also shows high numbers of transcripts involved in synthetic processes as compared to degradation processes, consistent with a developing tissue. microsatellite markers in dnost microsatellite markers are broadly used for marker-assisted selection in crop breeding due to the ease of their implementation and co-dominant nature. we scanned the dnost for gene-derived ssr markers with the potential to be used in oat breeding programs. in total, ssrs were found within different transcripts. a summary of the putative ssrs is shown in additional file primers targeting the ssrs were designed when possible. the most abundant ssrs were the tri-repeats. the rest were distributed as follows: mono-repeats, di, tetra, penta, and hexa. excluding mono-repeats, the percentage of di, tri, tetra, penta and hexa was,,, and %, respectively. the ssr-bearing transcripts were aligned against the brachypodium genome sequence to pinpoint the syntenic location in which polymorphisms occur. their distribution along the brachypodium genome is uniform and consistent with gene density. construction of oat seed gene indices oatseedref and oatseedref90 to develop a comprehensive compendium of available oat seed expressed sequences, we combined the dnost assembly with oat sequences published by other sources. the sequence information retrieved from the genbank consisted of oat seed est sequences totaling, nt with an average length of nt. these were concatenated with the dnost transcripts to build an index of oat seed expressed sequences. we named this reference index oatseedref v. additionally, to reduce redundancy in oatseedref oatseedref was created in a manner similar to the uniprot reference clusters databases uniref and uniref. clustering reduces the presence of redundant sequences and base miscall errors, but can also eliminate highly similar homeoalleles. the oatseedref non-redundant index is composed of sequences, of which are tentative consensus and singletons, for a total of, nt of sequence information. of the sequences, were unique to the dnost assembly, were unique to the genbank ests, and resulted from merging of at least one genbank sequence and one of our de novo transcript assemblies. sequence lengths in oatseedref range between and nt, with an average of nt. since the minimum contig length in dnost was set to nt, all of the shortest sequences derive from ests retrieved from genbank. clustering revealed that more than 63% of the oat ests in genbank are overlapping or overlapped by sequences present in dnost. the oatseedref sequences were compared with the brachypodium and uniprot-plants protein databases and and hits were retrieved, respectively. only % of the unique brachypodium proteins corresponded to the set of oat ests in genbank, which indicates that the vast majority of the unique-protein targets come from our de novo assembly. this comparison further demonstrates that our rna-seq assemblies are an important source of novel oat cdna sequences. oat health-promoting compounds as a practical demonstration of dnost utility to demonstrate the utility of dnost, we studied biosynthetic genes for three important health-promoting compounds in oats: avenanthramides, tocols, and β-glucans. sequences of genes in the respective pathways from close relatives were downloaded from genbank and other sources and the dnost assembly was searched for homologous sequences. homologous transcripts were found for all genes investigated, which supports both the accuracy and completeness of the dnost assembly. the pivotal enzyme in the biosynthesis of avenanthramides is hht, which catalyzes the final condensation reaction. searches against dnost retrieved multiple homologous transcripts to the four reported oat hht isoforms. homologies were also found for genes coding two other key enzymes in avenanthramide synthesis, ccoaomt and ccoa3h. for ccoaomt, eight homologs were found in dnost. also, the complete ccoaomt cds was obtained by expanding a previously existing genbank sequence. the second enzyme, ccoa3h, is required for the synthesis of several of the most predominant avenanthramides. to our knowledge, no ccoa3h gene sequence has been reported for oats. we found four transcripts homologous to brachypodium and barley ccoa3h genes. the expression of all avenanthramide synthesis homologs was quantified and is shown in heat maps for the main enzymatic steps in the pathway. homologous transcripts in the heat maps may represent homeoalleles or other highly similar sequences including paralogs. the results indicate that there are significant differences in the expression of homologous transcripts in many biosynthetic steps, suggesting that homeoalleles and/or paralogs may be differentially expressed. tocol accumulation profiles in developing oat seeds have only been studied recently, and distinctly different temporal patterns of accumulation for both tocotrienols and tocopherols were found. sequences of the genes in the tocol biosynthetic pathway were sought within the dnost, and transcripts were found for all. for instance, five transcripts exhibited homology to barley, brachypodium, and wheat hppd. ggr catalyzes the reduction of ggdp to pdp, and six ggr homologues were assembled in dnost which nearly cover the predicted complete cds. the committed step in the biosynthesis of tocopherols is condensation of hga and pdp, catalyzed by hpt, for which one oat transcript was found. similarly, the committed step in tocotrienol biosynthesis is the condensation of hga with ggdp, by hggt. five highly similar hggt homologue transcripts were found in dnost. surprisingly, no hggt homologue was found in the brachypodium genome. seeds of brachypodium accumulate tocotrienols, thus it is likely that the hggt homologue lies in an unsequenced region of the brachypodium genome. the last three enzymes, vte vte and vte are common for the synthesis of both tocopherols and tocotrienols, and homologues were found for all three genes. thus, dnost holds promise for linking gene expression to metabolic aspects of tocol accumulation in oat seeds. β-glucans were the last health-promoting compounds evaluated. cellulose-synthase and cellulose-synthase-like sequences were downloaded from and compared to dnost assembly. the retrieved matches included ces and csl unique transcripts. the average length of the transcript isoforms assembled for csl and ces was and, respectively. as an example, alignments of barley cls-f and the oat homologue with dnost revealed homology to five isoforms. comparatively to barley, two insertions and one deletion at the 3′-end appears to produce an early stop codon. similarly, ten assembled isoforms had 84-85% and 76-78% identity with their barley and arabidopsis ces-a homologues, respectively. discussion oat transcripts were de novo assembled from illumina reads derived from four oat seed developmental stages. assembly was performed with both oases and trinity, and the results were compared. strict values were chosen for some parameters to assure a more precise assembly. to our knowledge this is the first comparison between oases and trinity in a polyploid organism, which makes our study a useful guide for future studies on transcriptome analysis in plants, where polyploidy is common. several quality tests were performed to determine the robustness of each assembly; the length and number of assembled transcripts, and how precisely they match annotated databases are particularly valuable in this regard. in our assemblies we prioritized sequence accuracy over other criteria, such as total number of transcripts and length of total assembled sequence. this approach may risk losing rare transcripts, but overall it improves the quality of the assembly. despite the fact that trinity was specifically developed for transcript assemblies, our quality benchmarks indicated higher quality scores for oases assemblies. it is likely that the reduced number of putative unique protein coding sequences from the trinity assemblies is due in part to the shorter transcripts produced. trinity developers initially tested its algorithms on diverse organisms such as fission yeast, mouse, and whitefly, but according to our results trinity appears not to be as suitable for complex polyploid transcriptomes such as that of hexaploid oat. one of the reasons for this result may be that k-mer length is fixed at nt; such a small number may not be sufficient to discriminate among highly similar homeologous or even paralogous sequences. further, the short k-mer length causes trinity to run considerably more slowly than oases run with 67-mer, since more words of size k per read have to be constructed and tested for alignment. if the multi-thread option of trinity is not selected, oases running with the fixed 25-mer of trinity finished 15– times faster than trinity. oases was shown to reconstruct a higher number of gene transcripts than trinity on human and mouse datasets, although the accuracy of the assemblies was comparable. similarly, in the assembly of the tea plant transcriptome, oases performed better than trinity in most of the parameters studied. however, trinity produced better results for highly expressed genes. the authors also reported that trinity run times slower than oases when the same k-mer value of was used. based on our benchmark analysis, the oases 67-mer-assembly was selected as the representative for the seed oat transcriptome. the proportion of dnost loci with at least one homologue in other databases ranged between and % in these comparisons, and was highest for the uniprot-plants database. these percentages increased up to % when all dnost transcript isoforms were considered. significantly, nearly 72% of the loci in dnost had a homologue within the collection of predicted brachypodium peptides, which reinforces a previous study suggesting that brachypodium can be an effective resource to assist oat genetics and genomics research. assembly errors and other sequence variation could produce fusion longer transcripts during the assembly. we do not believe that this occurred at a high level, as seen in the transcripts analyzed. although fusion proteins may not be completely discarded, that % of dnost transcripts retrieved two or more brachypodium predicted cds is more likely attributable to the presence of transcripts with more than one large conserved domain, or other regions with highly similar sequence. indeed, the brachypodium genome shows six major chromosomal duplications covering % of the genome, which represent ancestral whole-genome duplications, and so detection of paralogs is likely to be a major factor in detecting more than one gene from blast searches. for non-sequenced organisms, large-scale transcriptome assemblies from illumina reads appear to be more robust than from roche/ presumably due to the higher coverage attained. in an assembly of chickpea transcripts obtained from roche/ rna-seq, only about 58% had similarity to the uniref database, vs. the nearly 70% in our assembly. in another study, oliver et al. reported between and transcripts using high-throughput roche/ sequencing technology on four different oat genotypes, with average transcript lengths ranging between and nt, vs. the transcripts averaging nt in dnost. our transcriptome assembly appears to be accurate, as reflected by the results of blast searches against both plant and multi-organism databases. thus dnost greatly expands the current collection of oat expressed sequences. for instance, transcription factors are typically expressed at low levels and are most likely to be under-represented in est databases; however, functional classification analysis of dnost revealed a large group of putative transcription factor sequences. we estimated that a 75– read depth is required for homeolog discrimination. to our knowledge this is the first study to provide such an estimate for a polyploid genome. in a de novo assembly of three individual human genomes, best false negative allele calls were obtained with considerably lower read coverage. also, a 20× sequencing depth was required in the de novo assembly of an individual human genome with 75-nt read length to achieve a maximum contig size after which no further increase in length was observed. our assembly also suggests that homeologous genes in oat are both highly conserved and may remain functional, although confirming this would require extensive proteomic and enzymatic analyses. moreover, the question of whether these sequences represent true homeologs, allelic variants, or artifacts is not empirically addressed in this study. thus, caution may be advised when using dnost for certain purposes. for instance, the assembly process may have merged multiple homeologs into a single sequence, shuffling snps among homeologs. this could be especially relevant where low sequence divergence prevented the assembler from resolving homeologs. the homeologous forms in oat show similar or even a higher percentage of identity among them than was reported for wheat, with 90-99% identity at the nucleotide level and often identical at the amino acid level. polyploid species pose a challenge because of the presence of homeoalleles that may be difficult to deconvolute at the sequence level. to assemble highly similar homeologs separately requires a high number of reads and imposing strict parameters for assembly, such as larger k-mers, no mismatches allowed in k-mer alignments, and high minimum read coverage. clearly, assembling with longer k-mers first requires longer reads. currently, paired-end read sequencing using illumina technology is limited to nt, which still may be not sufficient to precisely discriminate highly similar homeologs in polyploid genomes. conversely, stricter assembling parameters will reduce the number of transcripts. while assembling dnost a balance between transcript redundancy and number was pursued, since obtaining a large number of transcripts was also a goal to make an oat library as complete and diverse as possible. sequence redundancy is intrinsic to polyploid organisms, and this was proven to be the case in dnost. despite the continuous improvements in ngs technologies and assembly algorithms, it is still extremely challenging assembling highly homologous genes into separate isoforms. there are numerous opportunities for practical use of dnost. for example, the number of sequence polymorphisms that have been described in oat suitable to be used as molecular markers is very limited. for instance, only ssr markers from oat were listed on the graingenes database as of november. the more than potential new genic ssr markers identified greatly expand the current ssr repository. while only a fraction of these may be found to be polymorphic between oat cultivars, validated polymorphic ssrs will provide a new resource for marker-assisted selection. as we found in oat, tri-nucleotide repeats are the most abundant in other plant species such as rice, corn, peanut, alfalfa, and arabidopsis, indicative of the fact that they are probably preferentially present within transcripts to prevent frame shifts. the first physically anchored hexaploid oat map has been recently completed. a goldengate assay was employed for genotyping snps as part of the mapping effort. to examine for the presence of those snps in the dnost transcripts, searches were conducted using the dna sequence surrounding the snp. this revealed that of the snps in the goldengate assay were present in at least one transcript. because goldengate snps were first discovered using cdna from different tissues, these results suggest dnost to be a fairly comprehensive source sequences, despite its seed origin. as a last example, transcripts for genes associated with the synthesis of health-promoting compounds are present in dnost and thus novel information is available to examine molecular aspects of their synthesis. for instance, avenanthramides are unique to oat among cereal grains, but the genes involved in their synthesis have not been fully characterized. a key enzyme in avenanthramide pathway is hht, from which there appear to be multiple isoforms that accept a wide range of substrates with different affinities. complete sequences for three oat hhts have been reported, as well as a partial sequence for ashht. we found twelve homologous transcripts to ashht eleven to ashht thirteen to ashht and thirteen ashht for a total of sixteen different transcripts. locus_17720_transcript_1/ had high sequence identity to ashht. a second transcript isoform was the most similar to ashht. these two loci shared 96% identity with the differences located in the 3′utr, suggesting that ashht and ashht are in fact homeologs. in another example, five dnost transcripts had at least 95% homology in their predicted cds to ashht4: locus_, loci_15223_/, locus_, and locus_. by aligning all transcripts with the previously known partial ashtt cdna we were able to extend the ashtt bases towards the 5′ end to complete the cds. thus, dnost is useful not only to identify oat homologues for genes of interest, but also to obtain complete sequences of partially cloned oat genes.  there are inherent challenges in developing an accurate oat gene transcript set because of its level of genome duplication, with post-processing of sequences often required to differentiate true homeologues from assembled artifacts. we have shown that our de novo transcript assembly of developing oat seeds obtained with oases is able to differentiate highly similar genes to a significant extent. this indicates that the nearly fold average coverage we obtained is deep enough to discern homoeoalleles and paralogs to some degree. nevertheless, post-processing of sequences may still be required to establish whether these sequences represent true homeologs, particularly in transcripts with low coverage. our study provides an optimized analytical pipeline for other researchers attempting to assemble transcriptome data from polyploid plant species. we validate that dnost is an excellent source of diverse oat transcripts such as those associated with the synthesis of several oat seed compounds possessing health-promoting properties, and also served as a resource to identify several thousand new potential molecular markers. thus the oat transcript assembly developed in this study will be useful for a variety of avenues of oat improvement. 
however, challenges remain for accurate differentiation of homoeoalleles and paralogs, particularly in polyploid organisms with no supporting genome sequence. to our knowledge, this study provides the first direct performance comparison between two major assemblers in a polyploid organism. it expands the number of oat ests 3-fold, and constitutes the first comprehensive transcriptome study in oat. in this study, rna-seq was employed to generate and characterize the first gene expression atlas for hexaploid oat. the software packages trinity and oases were used to produce a transcript assembly from nearly million 100-bp paired-end reads from developing oat seeds. to assess the robustness of dnost, we successfully identified gene transcripts associated with the biosynthetic pathways of three compounds with health-promoting properties, and quantified their expression. the oases 67-kmer assembly, denoted dnost, is over million nucleotides in length and the average transcript length is nucleotides. based on the quality-parameters employed, oases assemblies were superior.
 next generation sequencing provides new opportunities to explore transcriptomes. however, challenges remain for accurate differentiation of homoeoalleles and paralogs, particularly in polyploid organisms with no supporting genome sequence. in this study, rna-seq was employed to generate and characterize the first gene expression atlas for hexaploid oat.  the software packages trinity and oases were used to produce a transcript assembly from nearly million 100-bp paired-end reads from developing oat seeds. based on the quality-parameters employed, oases assemblies were superior. the oases 67-kmer assembly, denoted dnost, is over million nucleotides in length and the average transcript length is nucleotides. the × sequencing depth was adequate to differentiate a large proportion of putative homoeoalleles and paralogs. to assess the robustness of dnost, we successfully identified gene transcripts associated with the biosynthetic pathways of three compounds with health-promoting properties, and quantified their expression.  to our knowledge, this study provides the first direct performance comparison between two major assemblers in a polyploid organism. the workflow we developed provides a useful guide for comparable analyses in other organisms. the transcript assembly developed here is a major advance. it expands the number of oat ests 3-fold, and constitutes the first comprehensive transcriptome study in oat. this resource will be a useful new tool both for analysis of genes relevant to nutritional enhancement of oat, and for improvement of this crop in general. transcriptome assemblyoatrna-seqtocolvitamin eavenanthramideβ-glucantrinityoasesavena 