BACKGROUND
eukaryotic primary mrnas consist of protein-coding regions  and intervening non-coding regions .
the mature mrna transcript, which acts as substrate for translation into protein, is produced by removing introns in a process called splicing.
splicing can be either constitutive, always producing the same mrna, or alternative, via variable inclusion of parts of the primary transcript.
alternative splicing  is thus a mechanism that enables multiple transcripts and proteins to be encoded by the same gene, thereby promoting transcript and protein diversity  <cit> .
furthermore, events of as can provide an additional level of post-transcriptional gene regulation, e.g.
by the production of mrna isoforms with truncated open reading frames that are subject to degradation by the nonsense mediated decay pathway  <cit> .
as is particularly widespread in higher eukaryotes, especially in mammals - it has been estimated that up to 94% of all multi-exonic h. sapiens genes are alternatively spliced  <cit> .
large-scale detection of as usually involves expressed sequence tags , microarray, or rna-seq analysis.
however, not all as events can be detected by these methods.
moreover, nowadays genomic sequence data is being churned out at a much faster rate than transcript data, that is, many genomes have low transcript coverage.
thus, there is a need for independent methods of detecting as.
it has been shown that as involving alternative donors/acceptors separated by 2- <dig> nt, also called "subtle alternative splicing" , is an evolutionarily widespread class of as among animals, and among these, nagnag as, involving acceptors separated by  <dig> nt, is the most common  <cit> .
the terminology "nagnag" refers to events of as that involve two acceptors  which are preceded by any of the four possible nucleotides .
hence, the generic pattern for such tandem splice sites is "nagnag".
nagnag as can result in one of three possibilities  - constitutive use of the first acceptor , constitutive use of the second acceptor , or use of both acceptors, that is, alternative splicing   <cit> .
nagnags contribute 45% of all conserved alternative acceptors in h. sapiens and m. musculus  <cit> .
since the difference between the two isoforms is three nucleotides, no frameshift is induced, and the usual impact of a nagnag as event is the insertion or deletion of one amino acid.
in a recent study, we predicted the splicing outcome at nagnag acceptors in seven animal genomes  with a high degree of accuracy, and 83% of the experimentally validated cases agreed with the predictions  <cit> .
in agreement with previous studies  <cit>  this indicated that the mechanism behind nagnag as seems to be simple, stochastic and conserved in vertebrates and beyond.
while there have been numerous experimental as well as computational studies of as in animals, the study of as in plants is still in its early stages  <cit> .
although as is commonly observed in plants, the overall abundance of as seems to be lower than in animals.
several studies have estimated that between 20%-30% of plant genes undergo as  <cit> , while the current estimate based on deep sequencing of the arabidopsis thaliana transcriptome is 42%-56% of intron-containing genes  <cit> .
est-based detection of as in plants revealed that intron retention appears to be the most common kind of as event in plants  <cit> .
exon-skipping, which is the most common event in animals  <cit> , is much less frequent in plants  <cit> .
the two prevalent models for spliceosome assembly are intron-definition, which applies to short introns  and involves the intron as the initial unit of recognition during spliceosome assembly; and exon-definition, which applies to long introns introns , and involves recognition of the exon as the initial unit for splicing  <cit> .
thus, one would expect inaccurate splicing to result in intron-retention under the intron-definition model, and exon-skipping under the exon-definition model  <cit> .
hence the results showing that intron-retention is the most common as event in plants and exon-skipping in animals are consistent with these models of splicing.
however, alternative acceptors and donors seem to occur at a comparable frequency  <cit> .
in particular, short distance or subtle as events, seem to be just as common, and nagnag acceptors are widespread and abundant; a study on as found  <dig> alternative nagnags in rice and  <dig> in a. thaliana  <cit> .
initial analyses of the model plant p. patens, the first sequenced bryophyte, indicated a distribution of as events similar to other plants studied so far  <cit> .
consequently, we here aimed to characterize and predict the extent of nagnag as in p. patens.
analysis of the available transcript data indicates that nagnag as is just as common in the moss p. patens as in seed plants.
we achieved a high level of performance in silico, and 64% of the cases of well-supported as using independently generated  <dig> data could be correctly predicted.
in agreement with a recent study comparing a. thaliana and o. sativa with mammals  <cit> , our results suggest that the mechanism of nagnag as is similar in plants and animals.
RESULTS
identification of alternative nagnags using sanger ests
since the extent of nagnag occurence in p. patens had not been reported, we sought to identify genomic nagnags.
to find all genomic nagnags , we looked for all annotated intron-exon boundaries which had an ag at three positions upstream or downstream of the annotated acceptor.
this yielded  <dig>  nagnag motifs, of which  <dig>  were covered by sanger ests .
cases where the est evidence supported only one of the nag acceptors were called constitutive, whereas cases with est support for both acceptors were called alternative .
<dig>  of the detected  <dig>  nagnags with sanger est coverage were alternatively spliced , while  <dig>   were exclusively spliced at the first  acceptor  and  <dig>   were spliced only at the second  acceptor .
thus, nagnag as is common in p. patens.
sequence logos for all nagnag splice sites as well as for ei, e and i sites are visualized in figure  <dig>
gene ontology enrichment analysis
to assess whether genes with nagnag as in p. patens are enriched for specific functional categories and whether there is any similarity with a. thaliana and o. sativa in that sense, we analyzed gene ontology  term annotations with gossip  <cit> .
go terms with a fdr corrected p-value  less than  <dig>  were considered significantly different.
we found that  <dig> genes with the term plastid  are statistically enriched  in the set of p. patens genes with est support for an alternative nagnag acceptor .
this could be confirmed by the gossip analysis for the p. patens alternative nagnag genes supported by sanger and  <dig> reads .
in addition, the terms organelle and mitochondrion  were found to be enriched among the nagnag genes in p. patens.
"dna binding"  which is reported for a. thaliana and o. sativa to be enriched in alternative nagnag genes  <cit> , could not be observed for p. patens.
to further examine this inconsistency, the supported alternative nagnag genes from a. thaliana  were subjected to go enrichment analysis as well .
this analysis confirms the term "dna binding" as overrepresented among the a. thaliana nagnag genes , thus consistent with the analyses for a. thaliana and o. sativa  <cit>  as well as for mouse  <cit> .
however, "dna binding" was not found to be enriched in p. patens nagnag genes.
in contrast to the analyses for the seed plants and mouse, fisher's exact test with false discovery rate corrected p-values was used here, instead of a chi-square test.
however, if the parent term of dna binding, nucleotide binding  was also subjected to a chi-square test for p. patens, it was found to be enriched .
the fact that "dna binding" was not found to be enriched in p. patens nagnags might be due to the current status of the p. patens annotation  - e.g.
in many cases the gene models lack 5' and 3' regions and therefore do not cover the whole protein sequence.
on the other hand, mosses and vascular plants diverged more than  <dig> million years ago and thus p. patens alternative nagnag acceptor genes might be different.
nevertheless, the go enrichment analysis in terms of the category "cellular component" reveals that a. thaliana as well as p. patens nagnag genes share a bias towards the term "intracellular organelle", which includes "nucleus" and "plastid" .
in addition to the enriched molecular function "dna binding", our analysis confirmed the functions "rna binding", "transcription factor activity" and "transcription regulator activity" to be also slightly enriched in a. thaliana alternative nagnag acceptor genes , which is coherent with reports for m. musculus  <cit> .
evolutionary conservation of nagnag splicing among plants?
seven clusters of homologous genes with as at nagnag acceptors in the same intron were reported to be conserved between a. thaliana and o. sativa  <cit> .
in order to check whether as nagnag events are conserved between a. thaliana and p. patens, a blast based single linkage clustering was performed, using all transcripts with a sanger-supported nagnag.
altogether,  <dig>  clusters containing a. thaliana and p. patens genes were identified, of which five clusters contained genes with a nagnag motif at the orthologous  intron.
five out of the seven p. patens genes in these clusters were selected for experimental validation.
in all cases only one of the two isoforms could be detected, which is consistent with the support by sanger ests, which in all cases supported only one of the two isoforms.
in addition, in all cases the nagnag motif itself is not conserved between a. thaliana and p. patens .
in a. thaliana, only two of the nagnag motifs contains a gag, whereas four of five in p. patens contain a gag, and are therefore unlikely to represent alternative nagnags .
given the assumption that we are looking at orthologous or at least homologous positions and our transcript evidence is sufficient, this observation can be explained by two possible evolutionary scenarios.
in the first scenario the alternative nagnag sites are ancestral and have diverged in the lineages leading to a. thaliana and p. patens.
while they might have been inactivated by the introduction of a gag in the moss p. patens, they have been retained functional in a. thaliana.
in the second scenario these alternative nagnag acceptors in a. thaliana arose after the divergence of mosses and seed plants.
given the current scarce data, both scenarios appear equally parsimonious.
in order to decide which scenario is true, additional taxa would have to be included into the analysis.
given the current data and analyzes there is evidence for conserved nagnag as events between o. sativa and a. thaliana, but not between p. patens and a. thaliana.
thus, it appears as if nagnag as is not conserved across several hundreds of millions of years  <cit>  or arose secondarily.
prediction of nagnag as in p. patens
the most crucial prerequisite for good prediction performance is a reliable training dataset.
it is critical that the samples are correctly labelled as far as possible.
in terms of datasets of alternative and constitutive exons, this means that we should use the available transcript evidence judiciously, in order to minimise mislabelling.
in other words, we want to avoid the contamination of the set of constitutive exons by alternative exons which currently lack transcript support for being alternative, as well of alternative exons by potentially erroneously labelled exons.
thus, we used filters on the transcript support to improve the reliability of the labels - as in our previous work on nagnag as prediction in animals  <cit> , a training set was constructed based on the following criteria:
constitutive: ≥  <dig> ests supporting either e or i variant,  <dig> for the other;
alternative: ≥  <dig> ests supporting each variant, ≥ 10% of ests supporting minor variant.
this yielded a training dataset of  <dig> nagnags -  <dig> constitutive  and  <dig> ei, or alternative cases.
the classifiers were trained using this dataset.
the remaining  <dig>  nagnags  were used as a test set.
it is noteworthy that the average coverage per constitutive nagnag in this set is only three ests , indicating that there are potentially many undiscovered alternative nagnags in p. patens.
the training data was used with a classifier  in a cross-validation setting.
briefly, the classifier uses part of the training data to learn a model based on the sample labels and the features, and then uses this model to assign posterior probabilities , p and p according to the three possible classed) to each sample.
the predicted nagnag class is the one which receives the maximum score or posterior probability from the classifier.
we computed the receiver operating characteristics , which is a plot of the true positive rate versus the false positive rate, and measured the area under the roc curve , which is a standard measure of the quality of a classifier  <cit> .
an ideal classifier, which makes no errors, would achieve an auc of  <dig> .
we used  <dig> features, and achieved an in silico performance of auc =  <dig> ,  <dig>  and  <dig>  for the ei, e and i forms, respectively .
this performance was obtained under various cross-validation settings /n of the dataset is used to learn, and the remaining 1/n for prediction - this is repeated n times, and the average performance is reported).
generation of additional transcript evidence
as mentioned above, average transcript support was found to be low.
in order to generate more evidence for alternative acceptors, next generation sequencing was carried out.
for this purpose, adult gametophores carrying gametangia  were grown, as this tissue was not well represented in the pre-existing ~ <dig>  sanger reads.
in addition, the cdna was normalized in order to equalize transcript abundance and thus avoid redundancy.
while the ~ <dig>  sanger reads map to  <dig>  gene models, the ~ <dig>   <dig> reads map to  <dig>  gene models.
the  <dig> reads map to a total of  <dig>  gene models that were not covered previously, and identified  <dig> additional alternative nagnags.
even though the  <dig> data cover only 75%  of the nagnags evidenced by sanger ests, they enabled detection of  <dig> alternative nagnags -  <dig> % of the covered nagnags, as compared to  <dig> % using sanger ests.
of these  <dig>   <dig> were previously identified by sanger ests.
there are  <dig> nagnags which have support for only one acceptor in the sanger data, and for only the other acceptor in  <dig> data.
combining the results from sanger and  <dig> data, p. patens has  <dig> alternative nagnags.
again these results show that nagnag as is as widespread in the moss p. patens as it is in the seed plants a. thaliana and o. sativa.
an overview of the nagnag sites covered by the different transcript evidence pools is presented in table  <dig>
experimental confirmation of the nagnag as
experiments were performed on  <dig> candidate nagnags,  <dig> as controls  to see whether the splicing outcomes according to sanger and  <dig> reads could be confirmed by a pcr based approach, and five on the basis of an orthologous alternative nagnag intron in a. thaliana .
of the seven candidates with support for as from sanger or  <dig> datasets, three were predicted to be alternative spliced with p values >  <dig>  .
using sanger sequencing of cdna based pcr products, all three candidates were indeed verified as being alternatively spliced in p. patens protonema and gametophore tissue, respectively.
eight candidate genes were used as potential negative controls, as their p predictions were  <dig>  and lower.
all candidates showed support for the single predicted isoform by means of available transcript evidence and consequently only this single isoform could be detected during experimental validation .
having support for both variants from either the sanger or the  <dig> datasets, but a p <  <dig> , four more candidates were chosen to be validated.
nagnag as could be confirmed for the gene product phypa_ <dig> by sanger sequencing of cdna pcr products, although it has a low p of  <dig>  .
the experimental validation is supported by the sanger dataset, where  <dig> "e" variants as well as  <dig> "i" variants could be identified.
this is the only case where prediction from the naïve bayes classifier does not agree with the experimental results.
in case of phypa_ <dig> and phypa_ <dig>  only one of the two isoforms could be detected, reflecting the low p values.
the sensitivity of sanger sequencing allows detection of as for ratios of the two isoforms of about 3: <dig> or lower, meaning that cases in which the minor isoform abundance is < 25%, as may go undetected even if present.
therefore, validation using fluorescence labeled forward primers and fragment length detection on a capillary sequencer was used to detect the minor isoform abundance for two examples.
in case of phypa_ <dig>  the received data determined by sanger sequencing of pcr products could be confirmed by the more sensitive detection using the fluorescence labeled forward primers.
the two isoforms with three nucleotides difference in length were detected using capillary separation and had a relative abundance of approximately 3: <dig>  .
in case of phypa_ <dig>  only one of the two isoforms could be detected by sanger sequencing as well as in the more sensitive validation using fluorescence labeled primers .
thus, a low p prediction for this candidate seems to be correct as is the case for phypa_ <dig> and phypa_ <dig>  for which only one of the two isoforms could be identified as predicted.
detection of both isoforms either in sanger datasets  or in the  <dig> datasets  could be explained by the higher sensitivity of sequencing as compared to the pcr-based approach or by the fact that adult gametophores were used to generate the  <dig> data, while the validation was carried out in the two principal tissues of the juvenile stage.
thus it cannot not be excluded that these candidates are indeed alternatively spliced.
gag acceptors
twelve of the  <dig> candidate genes possess a gag in the nagnag motif .
using the above described methods, all of them are shown to be not alternatively spliced.
therefore, gag seems not to be used as an alternative acceptor for as in p. patens in most cases, which is in line with the sequence logos .
exceptions could be phypa_ <dig> and phypa_ <dig>  which possess both isoforms regarding sanger and  <dig> datasets.
these two candidates may indeed use gag as acceptors for as, but this remains to be proven.
rare usage of gags as acceptors in p. patens is in agreement with previous work which shows that functional acceptors are only very rarely gags - the order of preference for the nucleotide preceding the ag in functional acceptors is c > t > a > g, which has been shown both by experimental work  <cit>  as well as by in silico analyses of nagnag splicing  <cit> .
when we consider the est and  <dig> evidence in p. patens, only  <dig> %  of gag-containing nagnags are alternative - filtering by transcript support to use only well-supported cases  further reduces this to  <dig> % .
taken together, this strongly suggests that gags function only very rarely as functional acceptors in p. patens .
using  <dig> data for independent validation of predictions
the classifier was trained based on previously existing sanger evidence, the additional  <dig> evidence was used for independent validation.
combining the  <dig> and sanger datasets resulted in  <dig> additional nagnag as events being detected - of these,  <dig> had strong support for as in terms of satisfying the criteria used to define the training dataset .
62%  of these were predicted to be alternative by the naïve bayes classifier.
if we require ≥  <dig> reads per variant while keeping the threshold of minor variant abundance at ≥ 10%, the correct predictions rise to 75% .
when considering as according to  <dig> reads alone, 64%  of the well-supported cases of as are predicted correctly, which increases to 79%  if we require ≥  <dig> reads per variant while keeping the threshold of minor variant abundance at ≥ 10%.
on the other hand, if we look at cases which are constitutive with a support of ≥  <dig> transcripts, according to the combined transcript dataset, only 1/ <dig> e cases and 0/ <dig> i cases are predicted to be alternative.
the naïve bayes classifier predicts  <dig> further cases of as  in p. patens - the high specificity shown by nearly no predicted as in strongly supported constitutive nagnags combined with the sensitivity of 62% in detecting newly discovered strongly supported cases of as shows that there are potentially several hundred as yet undiscovered cases of nagnag as in p. patens.
prediction of nagnag as in p. patens by a classifier trained on h. sapiens data
we had earlier shown that a classifier trained on only h. sapiens nagnag data could predict nagnag splicing outcomes with near-identical accuracy on other vertebrate genomes , and with a slight drop in the case of d. melanogaster and caenorhabditis elegans  <cit> .
therefore, we also tried to predict nagnag as in p. patens using a naive bayes classifier trained on h. sapiens data and achieved an auc of  <dig> ,  <dig>  and  <dig>  for the ei, e and i forms, respectively.
this was achieved using five features  and is similar to that achieved on d. melanogaster earlier  <cit> , reinforcing the notion that nagnag splicing in plants is similar to that in animals.
CONCLUSIONS
here we describe the first computational prediction of alternative splicing  in a non-seed plant and find that nagnag as in p. patens, a moss, can be predicted with high accuracy.
since the extent of nagnags in p. patens had not yet been reported, this work involved both characterization as well prediction of nagnag splicing in p. patens.
using ests, we found that nagnag as is as widespread in the bryophyte p. patens as it is in the seed plants a. thaliana and o. sativa.
thus, nagnag as is likely to be a common feature of as in all land plants, just as it is in animals.
although we detected homologs with nagnag events among the two land plants p. patens and a. thaliana, nagnag splicing seems not to be conserved at the intron level.
using carefully constructed training and test datasets, an in silico performance of auc =  <dig> ,  <dig>  and  <dig>  was achieved for the ei, e and i forms, respectively.
the most informative features  were the nucleotides in the nagnag and its immediate vicinity, and even a relatively simple classifier like the naïve bayes classifier could match the more sophisticated bayesian network and support vector machine.
the performance achieved by a naïve bayes classifier trained on h. sapiens data  was similar to that achieved on d. melanogaster earlier  <cit> .
this indicates that, as in animals, the mechanism behind nagnag as in plants is simple in nature and mostly dependent on the splice site neighborhood.
independent validation of the predictions of the classifier  using  <dig> data showed that 64%  of the well-supported cases of nagnag as could be predicted correctly.
in total, seven candidates were chosen for independent experimental confirmation of the sanger and  <dig> evidence of nagnag splicing.
the experimental confirmation depends on detection of isoforms using sequence electropherograms and is less sensitive than size polymorphism detection using fluorescence-labeled primers.
the latter method was used on two of the seven examples and confirmed the results of the previous method.
while there is transcript support for alternative use of gag acceptors this could not be proven in our experimental validation.
in addition, a further  <dig> experiments were performed - six as negative controls, all of which agreed with the predictions, and five to check for possible conserved nagnag as with a. thaliana, which could not be detected.
when additional  <dig> transcript evidence was used to supplement the sanger est data, a total of  <dig> alternative nagnags were found in p. patens.
since the average coverage per constitutive nagnag was still only approximately ten ests, this number shall likely continue to rise with deeper coverage of the transcriptome.
nevertheless, the results provide the first evidence that nagnag as is widespread in p. patens.
our findings are in agreement with a recent study which showed that nagnag as shares common properties in a. thaliana and o. sativa and animals  <cit> .
this indicates that the mechanism behind nagnag as in land plants is similar to that in animals.
the pervasiveness of nagnag as suggests that it may be a general feature of splicing in animals and plants, and possibly in all eukaryotes.

 eukaryotic primary mrnas consist of protein-coding regions and intervening non-coding regions. the mature mrna transcript, which acts as substrate for translation into protein, is produced by removing introns in a process called splicing. splicing can be either constitutive, always producing the same mrna, or alternative, via variable inclusion of parts of the primary transcript. alternative splicing is thus a mechanism that enables multiple transcripts and proteins to be encoded by the same gene, thereby promoting transcript and protein diversity. furthermore, events of as can provide an additional level of post-transcriptional gene regulation, e.g. by the production of mrna isoforms with truncated open reading frames that are subject to degradation by the nonsense mediated decay pathway. as is particularly widespread in higher eukaryotes, especially in mammals - it has been estimated that up to 94% of all multi-exonic h. sapiens genes are alternatively spliced. large-scale detection of as usually involves expressed sequence tags, microarray, or rna-seq analysis. however, not all as events can be detected by these methods. moreover, nowadays genomic sequence data is being churned out at a much faster rate than transcript data, that is, many genomes have low transcript coverage. thus, there is a need for independent methods of detecting as. it has been shown that as involving alternative donors/acceptors separated by 2- nt, also called "subtle alternative splicing", is an evolutionarily widespread class of as among animals, and among these, nagnag as, involving acceptors separated by nt, is the most common. the terminology "nagnag" refers to events of as that involve two acceptors which are preceded by any of the four possible nucleotides. hence, the generic pattern for such tandem splice sites is "nagnag". nagnag as can result in one of three possibilities - constitutive use of the first acceptor, constitutive use of the second acceptor, or use of both acceptors, that is, alternative splicing. nagnags contribute 45% of all conserved alternative acceptors in h. sapiens and m. musculus. since the difference between the two isoforms is three nucleotides, no frameshift is induced, and the usual impact of a nagnag as event is the insertion or deletion of one amino acid. in a recent study, we predicted the splicing outcome at nagnag acceptors in seven animal genomes with a high degree of accuracy, and 83% of the experimentally validated cases agreed with the predictions. in agreement with previous studies this indicated that the mechanism behind nagnag as seems to be simple, stochastic and conserved in vertebrates and beyond. while there have been numerous experimental as well as computational studies of as in animals, the study of as in plants is still in its early stages. although as is commonly observed in plants, the overall abundance of as seems to be lower than in animals. several studies have estimated that between 20%-30% of plant genes undergo as, while the current estimate based on deep sequencing of the arabidopsis thaliana transcriptome is 42%-56% of intron-containing genes. est-based detection of as in plants revealed that intron retention appears to be the most common kind of as event in plants. exon-skipping, which is the most common event in animals, is much less frequent in plants. the two prevalent models for spliceosome assembly are intron-definition, which applies to short introns and involves the intron as the initial unit of recognition during spliceosome assembly; and exon-definition, which applies to long introns introns, and involves recognition of the exon as the initial unit for splicing. thus, one would expect inaccurate splicing to result in intron-retention under the intron-definition model, and exon-skipping under the exon-definition model. hence the results showing that intron-retention is the most common as event in plants and exon-skipping in animals are consistent with these models of splicing. however, alternative acceptors and donors seem to occur at a comparable frequency. in particular, short distance or subtle as events, seem to be just as common, and nagnag acceptors are widespread and abundant; a study on as found alternative nagnags in rice and in a. thaliana. initial analyses of the model plant p. patens, the first sequenced bryophyte, indicated a distribution of as events similar to other plants studied so far. consequently, we here aimed to characterize and predict the extent of nagnag as in p. patens. analysis of the available transcript data indicates that nagnag as is just as common in the moss p. patens as in seed plants. we achieved a high level of performance in silico, and 64% of the cases of well-supported as using independently generated data could be correctly predicted. in agreement with a recent study comparing a. thaliana and o. sativa with mammals, our results suggest that the mechanism of nagnag as is similar in plants and animals.  identification of alternative nagnags using sanger ests since the extent of nagnag occurence in p. patens had not been reported, we sought to identify genomic nagnags. to find all genomic nagnags, we looked for all annotated intron-exon boundaries which had an ag at three positions upstream or downstream of the annotated acceptor. this yielded nagnag motifs, of which were covered by sanger ests. cases where the est evidence supported only one of the nag acceptors were called constitutive, whereas cases with est support for both acceptors were called alternative. of the detected nagnags with sanger est coverage were alternatively spliced, while were exclusively spliced at the first acceptor and were spliced only at the second acceptor. thus, nagnag as is common in p. patens. sequence logos for all nagnag splice sites as well as for ei, e and i sites are visualized in figure gene ontology enrichment analysis to assess whether genes with nagnag as in p. patens are enriched for specific functional categories and whether there is any similarity with a. thaliana and o. sativa in that sense, we analyzed gene ontology term annotations with gossip. go terms with a fdr corrected p-value less than were considered significantly different. we found that genes with the term plastid are statistically enriched in the set of p. patens genes with est support for an alternative nagnag acceptor. this could be confirmed by the gossip analysis for the p. patens alternative nagnag genes supported by sanger and reads. in addition, the terms organelle and mitochondrion were found to be enriched among the nagnag genes in p. patens. "dna binding" which is reported for a. thaliana and o. sativa to be enriched in alternative nagnag genes, could not be observed for p. patens. to further examine this inconsistency, the supported alternative nagnag genes from a. thaliana were subjected to go enrichment analysis as well. this analysis confirms the term "dna binding" as overrepresented among the a. thaliana nagnag genes, thus consistent with the analyses for a. thaliana and o. sativa as well as for mouse. however, "dna binding" was not found to be enriched in p. patens nagnag genes. in contrast to the analyses for the seed plants and mouse, fisher's exact test with false discovery rate corrected p-values was used here, instead of a chi-square test. however, if the parent term of dna binding, nucleotide binding was also subjected to a chi-square test for p. patens, it was found to be enriched. the fact that "dna binding" was not found to be enriched in p. patens nagnags might be due to the current status of the p. patens annotation - e.g. in many cases the gene models lack 5' and 3' regions and therefore do not cover the whole protein sequence. on the other hand, mosses and vascular plants diverged more than million years ago and thus p. patens alternative nagnag acceptor genes might be different. nevertheless, the go enrichment analysis in terms of the category "cellular component" reveals that a. thaliana as well as p. patens nagnag genes share a bias towards the term "intracellular organelle", which includes "nucleus" and "plastid". in addition to the enriched molecular function "dna binding", our analysis confirmed the functions "rna binding", "transcription factor activity" and "transcription regulator activity" to be also slightly enriched in a. thaliana alternative nagnag acceptor genes, which is coherent with reports for m. musculus. evolutionary conservation of nagnag splicing among plants? seven clusters of homologous genes with as at nagnag acceptors in the same intron were reported to be conserved between a. thaliana and o. sativa. in order to check whether as nagnag events are conserved between a. thaliana and p. patens, a blast based single linkage clustering was performed, using all transcripts with a sanger-supported nagnag. altogether, clusters containing a. thaliana and p. patens genes were identified, of which five clusters contained genes with a nagnag motif at the orthologous intron. five out of the seven p. patens genes in these clusters were selected for experimental validation. in all cases only one of the two isoforms could be detected, which is consistent with the support by sanger ests, which in all cases supported only one of the two isoforms. in addition, in all cases the nagnag motif itself is not conserved between a. thaliana and p. patens. in a. thaliana, only two of the nagnag motifs contains a gag, whereas four of five in p. patens contain a gag, and are therefore unlikely to represent alternative nagnags. given the assumption that we are looking at orthologous or at least homologous positions and our transcript evidence is sufficient, this observation can be explained by two possible evolutionary scenarios. in the first scenario the alternative nagnag sites are ancestral and have diverged in the lineages leading to a. thaliana and p. patens. while they might have been inactivated by the introduction of a gag in the moss p. patens, they have been retained functional in a. thaliana. in the second scenario these alternative nagnag acceptors in a. thaliana arose after the divergence of mosses and seed plants. given the current scarce data, both scenarios appear equally parsimonious. in order to decide which scenario is true, additional taxa would have to be included into the analysis. given the current data and analyzes there is evidence for conserved nagnag as events between o. sativa and a. thaliana, but not between p. patens and a. thaliana. thus, it appears as if nagnag as is not conserved across several hundreds of millions of years or arose secondarily. prediction of nagnag as in p. patens the most crucial prerequisite for good prediction performance is a reliable training dataset. it is critical that the samples are correctly labelled as far as possible. in terms of datasets of alternative and constitutive exons, this means that we should use the available transcript evidence judiciously, in order to minimise mislabelling. in other words, we want to avoid the contamination of the set of constitutive exons by alternative exons which currently lack transcript support for being alternative, as well of alternative exons by potentially erroneously labelled exons. thus, we used filters on the transcript support to improve the reliability of the labels - as in our previous work on nagnag as prediction in animals, a training set was constructed based on the following criteria: constitutive: ≥ ests supporting either e or i variant, for the other; alternative: ≥ ests supporting each variant, ≥ 10% of ests supporting minor variant. this yielded a training dataset of nagnags - constitutive and ei, or alternative cases. the classifiers were trained using this dataset. the remaining nagnags were used as a test set. it is noteworthy that the average coverage per constitutive nagnag in this set is only three ests, indicating that there are potentially many undiscovered alternative nagnags in p. patens. the training data was used with a classifier in a cross-validation setting. briefly, the classifier uses part of the training data to learn a model based on the sample labels and the features, and then uses this model to assign posterior probabilities, p and p according to the three possible classed) to each sample. the predicted nagnag class is the one which receives the maximum score or posterior probability from the classifier. we computed the receiver operating characteristics, which is a plot of the true positive rate versus the false positive rate, and measured the area under the roc curve, which is a standard measure of the quality of a classifier. an ideal classifier, which makes no errors, would achieve an auc of. we used features, and achieved an in silico performance of auc =, and for the ei, e and i forms, respectively. this performance was obtained under various cross-validation settings /n of the dataset is used to learn, and the remaining 1/n for prediction - this is repeated n times, and the average performance is reported). generation of additional transcript evidence as mentioned above, average transcript support was found to be low. in order to generate more evidence for alternative acceptors, next generation sequencing was carried out. for this purpose, adult gametophores carrying gametangia were grown, as this tissue was not well represented in the pre-existing ~ sanger reads. in addition, the cdna was normalized in order to equalize transcript abundance and thus avoid redundancy. while the ~ sanger reads map to gene models, the ~ reads map to gene models. the reads map to a total of gene models that were not covered previously, and identified additional alternative nagnags. even though the data cover only 75% of the nagnags evidenced by sanger ests, they enabled detection of alternative nagnags - % of the covered nagnags, as compared to % using sanger ests. of these were previously identified by sanger ests. there are nagnags which have support for only one acceptor in the sanger data, and for only the other acceptor in data. combining the results from sanger and data, p. patens has alternative nagnags. again these results show that nagnag as is as widespread in the moss p. patens as it is in the seed plants a. thaliana and o. sativa. an overview of the nagnag sites covered by the different transcript evidence pools is presented in table experimental confirmation of the nagnag as experiments were performed on candidate nagnags, as controls to see whether the splicing outcomes according to sanger and reads could be confirmed by a pcr based approach, and five on the basis of an orthologous alternative nagnag intron in a. thaliana. of the seven candidates with support for as from sanger or datasets, three were predicted to be alternative spliced with p values >. using sanger sequencing of cdna based pcr products, all three candidates were indeed verified as being alternatively spliced in p. patens protonema and gametophore tissue, respectively. eight candidate genes were used as potential negative controls, as their p predictions were and lower. all candidates showed support for the single predicted isoform by means of available transcript evidence and consequently only this single isoform could be detected during experimental validation. having support for both variants from either the sanger or the datasets, but a p <, four more candidates were chosen to be validated. nagnag as could be confirmed for the gene product phypa_ by sanger sequencing of cdna pcr products, although it has a low p of. the experimental validation is supported by the sanger dataset, where "e" variants as well as "i" variants could be identified. this is the only case where prediction from the naïve bayes classifier does not agree with the experimental results. in case of phypa_ and phypa_ only one of the two isoforms could be detected, reflecting the low p values. the sensitivity of sanger sequencing allows detection of as for ratios of the two isoforms of about 3: or lower, meaning that cases in which the minor isoform abundance is < 25%, as may go undetected even if present. therefore, validation using fluorescence labeled forward primers and fragment length detection on a capillary sequencer was used to detect the minor isoform abundance for two examples. in case of phypa_ the received data determined by sanger sequencing of pcr products could be confirmed by the more sensitive detection using the fluorescence labeled forward primers. the two isoforms with three nucleotides difference in length were detected using capillary separation and had a relative abundance of approximately 3:. in case of phypa_ only one of the two isoforms could be detected by sanger sequencing as well as in the more sensitive validation using fluorescence labeled primers. thus, a low p prediction for this candidate seems to be correct as is the case for phypa_ and phypa_ for which only one of the two isoforms could be identified as predicted. detection of both isoforms either in sanger datasets or in the datasets could be explained by the higher sensitivity of sequencing as compared to the pcr-based approach or by the fact that adult gametophores were used to generate the data, while the validation was carried out in the two principal tissues of the juvenile stage. thus it cannot not be excluded that these candidates are indeed alternatively spliced. gag acceptors twelve of the candidate genes possess a gag in the nagnag motif. using the above described methods, all of them are shown to be not alternatively spliced. therefore, gag seems not to be used as an alternative acceptor for as in p. patens in most cases, which is in line with the sequence logos. exceptions could be phypa_ and phypa_ which possess both isoforms regarding sanger and datasets. these two candidates may indeed use gag as acceptors for as, but this remains to be proven. rare usage of gags as acceptors in p. patens is in agreement with previous work which shows that functional acceptors are only very rarely gags - the order of preference for the nucleotide preceding the ag in functional acceptors is c > t > a > g, which has been shown both by experimental work as well as by in silico analyses of nagnag splicing. when we consider the est and evidence in p. patens, only % of gag-containing nagnags are alternative - filtering by transcript support to use only well-supported cases further reduces this to %. taken together, this strongly suggests that gags function only very rarely as functional acceptors in p. patens. using data for independent validation of predictions the classifier was trained based on previously existing sanger evidence, the additional evidence was used for independent validation. combining the and sanger datasets resulted in additional nagnag as events being detected - of these, had strong support for as in terms of satisfying the criteria used to define the training dataset. 62% of these were predicted to be alternative by the naïve bayes classifier. if we require ≥ reads per variant while keeping the threshold of minor variant abundance at ≥ 10%, the correct predictions rise to 75%. when considering as according to reads alone, 64% of the well-supported cases of as are predicted correctly, which increases to 79% if we require ≥ reads per variant while keeping the threshold of minor variant abundance at ≥ 10%. on the other hand, if we look at cases which are constitutive with a support of ≥ transcripts, according to the combined transcript dataset, only 1/ e cases and 0/ i cases are predicted to be alternative. the naïve bayes classifier predicts further cases of as in p. patens - the high specificity shown by nearly no predicted as in strongly supported constitutive nagnags combined with the sensitivity of 62% in detecting newly discovered strongly supported cases of as shows that there are potentially several hundred as yet undiscovered cases of nagnag as in p. patens. prediction of nagnag as in p. patens by a classifier trained on h. sapiens data we had earlier shown that a classifier trained on only h. sapiens nagnag data could predict nagnag splicing outcomes with near-identical accuracy on other vertebrate genomes, and with a slight drop in the case of d. melanogaster and caenorhabditis elegans. therefore, we also tried to predict nagnag as in p. patens using a naive bayes classifier trained on h. sapiens data and achieved an auc of, and for the ei, e and i forms, respectively. this was achieved using five features and is similar to that achieved on d. melanogaster earlier, reinforcing the notion that nagnag splicing in plants is similar to that in animals.  here we describe the first computational prediction of alternative splicing in a non-seed plant and find that nagnag as in p. patens, a moss, can be predicted with high accuracy. since the extent of nagnags in p. patens had not yet been reported, this work involved both characterization as well prediction of nagnag splicing in p. patens. using ests, we found that nagnag as is as widespread in the bryophyte p. patens as it is in the seed plants a. thaliana and o. sativa. thus, nagnag as is likely to be a common feature of as in all land plants, just as it is in animals. although we detected homologs with nagnag events among the two land plants p. patens and a. thaliana, nagnag splicing seems not to be conserved at the intron level. using carefully constructed training and test datasets, an in silico performance of auc =, and was achieved for the ei, e and i forms, respectively. the most informative features were the nucleotides in the nagnag and its immediate vicinity, and even a relatively simple classifier like the naïve bayes classifier could match the more sophisticated bayesian network and support vector machine. the performance achieved by a naïve bayes classifier trained on h. sapiens data was similar to that achieved on d. melanogaster earlier. this indicates that, as in animals, the mechanism behind nagnag as in plants is simple in nature and mostly dependent on the splice site neighborhood. independent validation of the predictions of the classifier using data showed that 64% of the well-supported cases of nagnag as could be predicted correctly. in total, seven candidates were chosen for independent experimental confirmation of the sanger and evidence of nagnag splicing. the experimental confirmation depends on detection of isoforms using sequence electropherograms and is less sensitive than size polymorphism detection using fluorescence-labeled primers. the latter method was used on two of the seven examples and confirmed the results of the previous method. while there is transcript support for alternative use of gag acceptors this could not be proven in our experimental validation. in addition, a further experiments were performed - six as negative controls, all of which agreed with the predictions, and five to check for possible conserved nagnag as with a. thaliana, which could not be detected. when additional transcript evidence was used to supplement the sanger est data, a total of alternative nagnags were found in p. patens. since the average coverage per constitutive nagnag was still only approximately ten ests, this number shall likely continue to rise with deeper coverage of the transcriptome. nevertheless, the results provide the first evidence that nagnag as is widespread in p. patens. our findings are in agreement with a recent study which showed that nagnag as shares common properties in a. thaliana and o. sativa and animals. this indicates that the mechanism behind nagnag as in land plants is similar to that in animals. the pervasiveness of nagnag as suggests that it may be a general feature of splicing in animals and plants, and possibly in all eukaryotes. 
using features and training and test datasets of constitutive and alternative nagnags, we trained a classifier to predict the splicing outcome at nagnag tandem splice sites. by generation of more transcript evidence from a next-generation sequencing platform, we found additional evidence for nagnag as, with altogether alternative nagnags being detected in p. patens using all currently available transcript evidence. using sanger data, we found alternatively used nagnag acceptors in p. patens. nagnag as is just as common in the moss p. patens as it is in the seed plants a. thaliana and o. sativa, and can be predicted with high accuracy. our results suggest that the mechanism behind nagnag as in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood. the most informative features are the nucleotides in the nagnag and in its immediate vicinity, along with the splice sites scores, as found earlier for nagnag as in animals. alternative splicing involving tandem acceptors that are separated by three nucleotides is an evolutionarily widespread class of as, which is well studied in homo sapiens and mus musculus. subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of nagnag splicing outcomes.
 alternative splicing involving tandem acceptors that are separated by three nucleotides is an evolutionarily widespread class of as, which is well studied in homo sapiens and mus musculus. it has also been shown to be common in the model seed plants arabidopsis thaliana and oryza sativa. in one of the first studies involving sequence-based prediction of as in plants, we performed a genome-wide identification and characterization of nagnag as in the model plant physcomitrella patens, a moss.  using sanger data, we found alternatively used nagnag acceptors in p. patens. using features and training and test datasets of constitutive and alternative nagnags, we trained a classifier to predict the splicing outcome at nagnag tandem splice sites. our classifier achieved a balanced specificity and sensitivity of ≥ 89%. subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of nagnag splicing outcomes. by generation of more transcript evidence from a next-generation sequencing platform, we found additional evidence for nagnag as, with altogether alternative nagnags being detected in p. patens using all currently available transcript evidence. the data also enabled us to validate the predictions of the classifier, with 64% of the well-supported cases of as being predicted correctly.  nagnag as is just as common in the moss p. patens as it is in the seed plants a. thaliana and o. sativa, and can be predicted with high accuracy. the most informative features are the nucleotides in the nagnag and in its immediate vicinity, along with the splice sites scores, as found earlier for nagnag as in animals. our results suggest that the mechanism behind nagnag as in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood. 