BACKGROUND
with only a few exceptions  <cit> , retrotransposons have been found in all analysed eukaryotic genomes.
although transcription of retrotransposons is an integral part of their life cycle, elements may be transcriptionally active without this resulting in proliferation of the elements within the host genome  <cit> .
transcriptional activity of retrotransposons has been detected in a range of organisms and conditions, and may involve a multitude of elements that are simultaneously transcribed  <cit> , or alternatively, single element loci driving transcription of nearby genes  <cit> .
the presence and transcriptional activity of retrotransposons may interfere with nearby genes  <cit> , and hence presumably are subject to negative selection  <cit> .
unfortunately, the intrinsic sequence redundancy of retrotransposons has limited the resolution by which activity can be assigned to specific elements  using genome-scale approaches  <cit> .
the recent advances in novel sequencing and hybridization technologies  <cit>  have permitted an unforeseen depth in detection of transcriptional activity.
recently, faulkner and colleagues reported that 6-30% of cap-selected mammalian transcripts were initiated in repetitive elements  <cit> .
we set out to test if transcriptome data could provide information on the transcriptional activity of presumably functional  retrotransposons, and turned our attention to the single-celled fission yeast schizosaccharomyces pombe.
the genome of s. pombe is highly compact and well annotated  <cit> , and harbours only a few families of long terminal repeat  retrotransposons  <cit> .
ltr retrotransposons are transposable elements that typically contain gag and pol genes required for transposition, are related to retroviruses, and have their name from the two repeated ltr sequences flanking them.
two ltr sequences may recombine resulting in a solitary ltr sequence.
all full-length ltr retrotransposons in the s. pombe reference genome  belong to the tf <dig> family, while all members of the other dominant ltr family, tf <dig>  are found as solitary ltr sequences  <cit> .
s. pombe ltr elements are predominantly inserted upstream of protein-coding genes  <cit> , where transcription activators are responsible for targeting the site of insertion  <cit> .
intriguingly, the tf <dig> elements were shown to harbour promoter regions restoring the regulatory functions that are disrupted by ltr integration  <cit> .
we have analysed the data from two recent studies: first, a high throughput sequencing of complementary dnas generating short reads  from s. pombe growth phase and five time points during meiosis from the bähler lab  <cit> .
this study is henceforth referred to as rna-seq.
second, a study from the cairns lab  <cit>  using a novel approach  in which rna from growth phase was directly hybridized to a whole-genome microarray with  <dig> base pair dna probes, followed by antibody procedures ensuring perfect matches as well as subsequent quantification by light emission  <cit> .
the direct hybridization approach hence allows the assignment of transcriptional activity to a specific strand.
we refer to this study simply as hybmap.
the available data samples are summarized in additional file 1; table s <dig>  from rna-seq sequence reads and signal intensities of hybmap array probes we recorded the transcriptional activity from ltr sequences, analysed the extent and orientation of ltr transcription, and how the expression profiles of ltr sequences correlate with nearby genes.
RESULTS
we retrieved  <dig> ltr sequences with lengths from  <dig> to  <dig> base pairs   from the s. pombe genome annotation.
of these,  <dig> ltr sequences were residing in full-length ltr retrotransposons.
this uneven number results from a single chimerical ltr retrotransposon with an  organization that potentially is a result of an ectopic recombination event.
two full-length ltr retrotransposons  are frame shifted and annotated as pseudogenes http://www.genedb.org/.
genomic coordinates of the ltr sequences are provided in additional file 1; table s <dig>  from a biological point of view, we are interested in distinguishing between transcriptional activity stemming from full-length ltr retrotransposons and from solitary ltrs.
analysis of transcriptional activity was therefore performed on these two sets of ltrs separately:  <dig> full-length ltr retrotransposons  and the remaining  <dig> solitary ltr sequences.
to assess the level of transcriptional activity using the hybmap approach, we collected the recorded signal intensities of hybmap probes mapping exclusively within the full-length ltr set and uniquely within the solitary ltr sets, and compared these to signal intensities for probes mapping to other genomic reference features.
these reference features include rna genes, protein-coding genes and background sequences  .
as expected, when plotting the signal intensity for genes  we observe higher levels of signal intensities for forward strand probes, and lower levels of intensities for reverse strand probes .
signal intensities for intergenic mapping probes are distributed around zero, in accordance with the normalization procedures carried out in the hybmap study  <cit> .
finally, low levels of signals are observed for intronic mapping probes, showing slightly higher intensities on the forward than on the reverse strands.
signal intensities for the two ltr sets are presented in detail below.
a) number of sequences in set
b) hybmap 60-mers mapping to sequence sets.
both forward and reverse strand probes included.
c) rna-seq sequence reads from growth stage mapping to sequence sets.
d) number provided is probes and reads mapping exclusively within sequence sets
full-length ltr retrotransposons
a total of  <dig>  hybmap probes are mapping exclusively to the set of full-length ltr retrotransposons.
of these, only  <dig> probes  are mapping uniquely to a single retrotransposon locus .
this means that for the vast majority of probes we cannot determine the number of loci contributing to any transcriptional activity.
in figure 2a, yellow curves depict the scenario where all retrotransposons contribute equally , and red curves depict the other extreme where the total signal of each probe is derived from a single loci.
as seen from figure 2a, the former scenario indicates that ltr retrotransposons are not transcribed at a level exceeding that of background transcription of intergenic sequence, whereas the latter scenario is equivalent to a few active ltr retrotransposon transcribed at a level comparable to histone genes.
when plotting the signal intensities of uniquely mapping probes , we see that these are highly skewed towards higher intensities, suggesting that only a small number of ltr retrotransposon loci contribute to the combined transcriptional activity, and concurrently, that these retrotransposons are transcribed at high levels.
the full-length uniquely mapping probes target  <dig> different ltr retrotransposons, of which  <dig> are the aforementioned pseudogenes .
thus, transcription is detectable from at least  <dig> retrotransposons with complete open reading frames.
we cannot, however, conclude anything about the individual levels of transcriptional activity from the remaining  <dig> full-length ltr retrotransposons that do not have any unique probes assigned to them.
we next mapped hybmap probes onto an alignment of the full-length ltr retrotransposons .
although data shown in figure  <dig> strongly suggest that a minority of ltr retrotransposons contribute to the overall transcriptional activity, in figure  <dig> probe intensities are divided by the number of possible mappings .
when ignoring the differences between those probes that are mapping uniquely and those that are not, transcription appears to be relatively equal across the retrotransposon internal sequence, consistent with transcription of the entire retrotransposon.
the notable exceptions of two areas covering parts of the reverse transcriptase and rnase h domains  coincide with regions where probes are mapping to a lower number of full-length ltr retrotransposons , providing additional support for the notion of a few highly transcriptionally active retrotransposons.
although high levels of reverse strand transcription is apparent, poly-enriched samples display a better resolution between the two strands, suggesting that polyadenylated full-length ltr retrotransposon transcripts are present in the samples .
the fact that two almost identical flanking ltrs are present in each full-length retrotransposon, and the likelihood that an ltr mapping probe will also map to ltr sequences outside the set of full-length elements , makes it difficult to compare directly the flanking ltrs and the internal sequence.
we would expect transcription to initiate at the transcription start site in the 5' ltr  and terminate in the 3' ltr.
in reality, as ltr-matching probes are often mapping to both flanking ltr sequences, we cannot establish from which end the activity stems, and for consistency we have simply mapped such probes to the 3' ltr in figure  <dig>  interestingly, no clear distinction between forward and reverse strand transcriptional activity is observed for the ltr sequences.
solitary ltr sequences
transcriptional activity is also detectable from solitary ltr sequences, although only a subset of the  <dig> probes mapping uniquely to solitary ltrs shows high levels of signal intensities .
further, transcriptional activity is similar from the forward and the reverse strand.
to establish if the divergent intensities of signals from solitary ltr probes are a result of differences between ltr loci, or between probes within ltr sequences, we calculated the average signal intensity for each ltr locus.
if the observed broad range of signal intensities in figure 2b was a result of ltr loci with equal levels of transcriptional activity, but with certain parts of the ltr sequences being transcribed and some not, we would expect the average signal intensity for individual ltr sequences to be relatively similar.
this is not the case , suggesting that individual solitary ltr loci differ in levels of transcriptional activity.
then why are some ltr loci apparently transcribed whereas others are not?
we do not find any noticeable patterns between highly and lowly transcribed ltr sequences in terms of orientation and distance with respect to nearest neighbouring protein-coding gene .
in fact, no correlation is found between the average signal intensity of ltr sequences and the average of the nearest gene .
finally, to test if the observed transcriptional differences between specific ltr sequences are due to simple stochastic variation due to the relatively small sample size, we performed a permutation analysis in which probe intensities were randomly assigned to an ltr sequence .
when repeating this analysis  <dig>  times, we find that the observed variance between the average signal intensities from each ltr sequence  fall well without the range of variances of the simulated sets  .
this strongly suggests that distinct differences in transcriptional activity levels exist between s. pombe solitary ltr sequence loci.
transcriptional activity stemming from solitary ltrs could be a result of transcription initiated outside the ltr sequences.
to assess this, we included genomic sequence flanking the ltr sequences and compared transcriptional activity within ltr sequences to activity in their genomic vicinity.
in practice, we collected a set of  <dig> easily alignable solitary ltr sequences , and for each ltr sequence we collected a maximum of  <dig> base pair flanking sequence both up- and downstream.
if the distance to another annotated feature  was lower than  <dig> bp, only half of this distance was used as flanking sequence.
if half the distance to nearest feature was smaller than  <dig> base pairs no flanking sequence was included.
the flanking sequences were then concatenated with the alignment of solitary ltrs, so that flanking sequence was not aligned but fixed by the borders of ltr sequences.
a schematic depiction of the procedure and statistics on sizes of flanking sequences are shown in additional file 2; figure s <dig>  as seen from figure  <dig>  a decrease in the level in transcriptional activity is observed immediately outside the ltr sequences.
the median level of signal intensities within ltr sequences  was found to be significantly higher than both flanking sequences upstream  and downstream .
therefore, the ltr transcriptional activity as indicated by hybmap probes does not appear to be a result of transcription continuing into ltrs from the flanking genomic regions, but seems remarkably confined to the ltr sequences.
hybmap analysis from alternative procedures and growth conditions
data from the cairns group hybmap study include hybridizations using poly-enriched rna samples, as well as samples from alternative growth conditions    <cit> .
in general, alternative growth conditions yield similar patterns of ltr transcriptional activity .
notably, a relative higher forward strand activity from full-length ltr retrotransposons is found in poly-enriched samples, consistent with polyadenylation of the full-length ltr retrotransposons .
in contrast, signal intensities from solitary ltr sequences are shifted markedly towards lower values in poly-enriched samples, suggesting that no polyadenylation takes place .
rna-seq analysis of ltr transcriptional activity
sequence reads from the rna-seq study were mapped to the ltr sequences and other genomic features in a manner similar to the hybmap probes.
contrary to the hybmap approach, rna-seq involves conversion of sampled rna into cdna  primer  <cit> ).
presumably this step is responsible for the observed bias of higher rna-seq read densities towards the 3' end of the protein-coding genes in our reference set - a bias that is not observed for the hybmap intensities .
consistent with this, when we plot the rna-seq reads mapping exclusively to full-length ltr retrotransposons onto the alignment, a pronounced 3' peak is observed .
again, this strongly suggests that s. pombe ltr retrotransposons are actively transcribed in full length and polyadenylated.
for the full-length ltr elements, we assigned the rna-seq reads using two complementary procedures.
first, reads mapping to multiple loci were assigned evenly between ltr loci .
second, multiple mapping reads were assigned to a single locus and the highest possible density of reads for a single locus was recorded.
similarly to the hybmap probe assignment procedure, these two alternatives reflect the two extremes of possible ltr transcription; all ltrs being transcribed at a similar level, or a minimum of ltr loci being responsible for all the detected transcription.
we then compared the transcriptional levels of all protein-coding genes as indicated by rna-seq and hybmap.
one initial observation is that calculating the density of rna-seq reads introduces a bias against longer genes .
this is of particular concern if we want to analyse transcription from full-length ltr retrotransposons, which with an average genomic length of  <dig> base pairs are among the  <dig> percent longest genes in s. pombe .
we therefore used the absolute number of rna-seq reads per gene loci , which show a clear correlation with hybmap levels , in an unbiased fashion with regards to length .
in figure  <dig>  the transcriptional activity from full-length ltr elements are shown as a rectangular space, in which the horizontal boundaries are determined by the median hybmap probe intensity assuming that all ltrs are transcribed equally , or assuming that the intensity for each probe stems from a minimal number of transcriptionally active loci .
the vertical boundaries are similarly defined for rna-seq read coverage.
the potential range of full-length ltr transcription follows the general distribution of protein-coding genes .
the transcriptional activity from solitary ltr sequences, as well as snornas and trnas are underestimated in the rna-seq analysis compared to the hybmap approach.
lower signals from these presumably non-polyadenylated transcripts would be expected from the rna-seq approach .
in summary, we find that the rna-seq data supports the finding of transcriptional activity from full-length ltr retrotransposons, and the notion that these transcripts are polyadenylated.
ltr transcription during meiosis
the rna-seq study includes time series samples during meiosis, and we attempted to evaluate the association between ltr transcription and the transcription of neighbouring protein-coding genes during meiosis.
we selected  <dig> ltrs with the highest uniquely mapping sequence read coverage and residing within  <dig> bp upstream of a protein-coding gene .
for each meiosis stage, we calculated the ratio between read density at meiosis and read density at growth phase, and compared these to the same ratios for the neighbouring protein-coding genes.
this approach allowed us to directly compare read levels without adjusting for sequence lengths and uniqueness.
as seen from figure  <dig>  an apparent correlation is observed between some ltrs and their neighbouring genes.
the scarcity of points makes correlation analysis problematic.
yet, we have attempted to assess the significance of the correlations using two different approaches.
first, we concatenated the  <dig> time series for ltrs and for genes and calculated a product moment correlation coefficient of  <dig> , which is significant below the  <dig>  confidence level.
second, we produced  <dig>  permutated sets of the  <dig> ltr expression profiles, in which the time points were shuffled independently and calculated correlation coefficients between the permutated ltr sets and the real gene sets .
we then compared the median of the product moment correlation coefficients from the real data  to the distribution of medians from the permutated data.
of the  <dig>  permutated sets, only a single set exceeded the real median, corresponding to a two-tailed significance level below  <dig> × 10- <dig> .
comparing the transcriptional activity of full-length ltr retrotransposons to solitary ltrs during meiosis shows no apparent correlation , supporting the notion that these two groups of sequences behave transcriptionally distinct.
it has recently been reported that cenp-b homologues of s. pombe are negatively regulating transcription by binding to ltr sequences  and facilitating chromatin modification  <cit> .
additionally, other apparently distinct protein pathways control ltr transcription in s. pombe  <cit> , although rna interference appears only to have a limited role  <cit> .
when comparing the meiosis expression profiles of proteins involved in transcription repression to the profiles for ltr sequences, no conclusive pattern of inverse correlation between repressor genes and ltr sequences is apparent .
on the other hand, the different transcription patterns of solitary and full-length ltrs and the indications from our analysis that large differences in transcriptional activity exist between ltr loci suggest that ltr transcription is not solely determined by a global mechanism exerting its effect across the entire genome.
CONCLUSIONS
our analysis of two independent transcriptome data sets clearly indicates that ltr retrotransposons are actively transcribed during growth phase, and that the retrotransposon transcripts are polyadenylated.
when considering the evolutionary dynamics of s. pombe ltr elements, kelly and levin  <cit>  speculated if only a few of the complete elements were responsible for majority of transposition events  as has been observed in saccharomyces cerevisiae  <cit> .
although we cannot conclude anything on the transcriptional levels of individual loci, our data strongly suggest that a minority of loci contribute the majority of the transcriptional output from ltr retrotransposons.
assuming that high levels of retrotransposition are detrimental to the host, selection will favour regulation of retrotransposition.
such regulation may either take place pre-transcriptionally  or post-transcriptionally .
the observed transcription of s. pombe ltr retrotransposons suggests that post-transcriptional mechanisms are the predominant type of regulation during regular growth phase.
the relatively high levels of transcriptional activity antisense to full-length ltr retrotransposons  may potentially be part of such post-transcriptional regulation pathways.
the targeting of ltr insertion upstream of genes  <cit>   could limit the potential of regulating retrotransposition pre-transcriptionally in s. pombe, as any interference with transcriptional regulation presumably would also affect neighbouring protein-coding genes.
from the hybmap data transcriptional activity is detectable from both strands of solitary ltr sequences, and the transcription appears to be confined to the ltr sequences themselves.
analysis of the rna-seq data suggests that transcription of solitary ltrs upstream of genes is correlated with the transcriptional activity of the neighbouring genes.
one might speculate that the presence of transcripts from both strands of the solitary ltrs could generate double-stranded rnas, similar to the double-stranded rnas involved in heterochromatin formation in s. pombe  <cit> .
however, this would suggest a negative correlation between the transcriptional activity of genes and ltr sequences.
the observed positive correlation rather suggests that the ltr transcripts represent transcription facilitated by a physical association with actively transcribed genes, in parallel to the observed co-expression of linked genes  <cit> .
our analysis indicates that a clear distinction exists between solitary ltr sequences and full-length ltr elements in terms of transcriptional activity.
transcription of full-length ltr retrotransposons is for the most part derived from the forward strand and transcripts are polyadenylated.
in contrast, solitary ltr transcription is found from both strands, with transcripts showing no signs of polyadenylation.
additionally, divergent expression levels during meiosis are observed between full-length ltr retrotransposons and solitary ltr sequences.
in hiv retroviruses, a number of transcription factors bind the internal retrotransposon downstream of the 5' ltr  <cit> .
it is possible that the absence of such binding sites around solitary ltrs contributes to their changed transcription patterns.
similarly, in a range of retrovirus ltrs, polyadenylation signals are present downstream of the transcription start sites, but a minimum distance is required for polyadenylation to take place  <cit> , so that transcription is initiated in the 5' ltr and polyadenylation only in the 3' ltr of full-length retroviruses.
certainly, this would preclude polyadenylation of transcripts initiated within solitary ltr sequences.
similar to the solitary ltr sequences, levels of transcriptional activity appear to be relatively similar from both strands of the ltrs from full-length retrotransposons.
one possible scenario is therefore that - compared to full-length retrotransposons - the solitary ltr sequences have changed their transcriptional patterns due to loss of regulatory motifs, and lost the ability to generate polyadenylated transcripts, but have retained the ability to generate transcription from both strands .
based on our analysis, we conclude that the application of large-scale transcriptome data allows the elucidation of retrotransposon transcriptional activity, but that the resolution by which transcription can be assigned to specific retrotransposon loci is still limited.
a recent rna-seq approach revealed an up-regulation of transposons in methylation-defective arabidopsis mutants  <cit> .
additionally, mapping of capped sequence reads demonstrate wide-spread, regulated transcription initiated with mammalian retrotransposons  <cit> .
hence, novel transcriptome analysis techniques will inevitably shed light on tissue-specific  and temporal expression patterns of retrotransposons facilitating an assessment of the dynamics and immediate impact of these long-term residents of eukaryotic genomes.

 with only a few exceptions, retrotransposons have been found in all analysed eukaryotic genomes. although transcription of retrotransposons is an integral part of their life cycle, elements may be transcriptionally active without this resulting in proliferation of the elements within the host genome. transcriptional activity of retrotransposons has been detected in a range of organisms and conditions, and may involve a multitude of elements that are simultaneously transcribed, or alternatively, single element loci driving transcription of nearby genes. the presence and transcriptional activity of retrotransposons may interfere with nearby genes, and hence presumably are subject to negative selection. unfortunately, the intrinsic sequence redundancy of retrotransposons has limited the resolution by which activity can be assigned to specific elements using genome-scale approaches. the recent advances in novel sequencing and hybridization technologies have permitted an unforeseen depth in detection of transcriptional activity. recently, faulkner and colleagues reported that 6-30% of cap-selected mammalian transcripts were initiated in repetitive elements. we set out to test if transcriptome data could provide information on the transcriptional activity of presumably functional retrotransposons, and turned our attention to the single-celled fission yeast schizosaccharomyces pombe. the genome of s. pombe is highly compact and well annotated, and harbours only a few families of long terminal repeat retrotransposons. ltr retrotransposons are transposable elements that typically contain gag and pol genes required for transposition, are related to retroviruses, and have their name from the two repeated ltr sequences flanking them. two ltr sequences may recombine resulting in a solitary ltr sequence. all full-length ltr retrotransposons in the s. pombe reference genome belong to the tf family, while all members of the other dominant ltr family, tf are found as solitary ltr sequences. s. pombe ltr elements are predominantly inserted upstream of protein-coding genes, where transcription activators are responsible for targeting the site of insertion. intriguingly, the tf elements were shown to harbour promoter regions restoring the regulatory functions that are disrupted by ltr integration. we have analysed the data from two recent studies: first, a high throughput sequencing of complementary dnas generating short reads from s. pombe growth phase and five time points during meiosis from the bähler lab. this study is henceforth referred to as rna-seq. second, a study from the cairns lab using a novel approach in which rna from growth phase was directly hybridized to a whole-genome microarray with base pair dna probes, followed by antibody procedures ensuring perfect matches as well as subsequent quantification by light emission. the direct hybridization approach hence allows the assignment of transcriptional activity to a specific strand. we refer to this study simply as hybmap. the available data samples are summarized in additional file 1; table s from rna-seq sequence reads and signal intensities of hybmap array probes we recorded the transcriptional activity from ltr sequences, analysed the extent and orientation of ltr transcription, and how the expression profiles of ltr sequences correlate with nearby genes.  we retrieved ltr sequences with lengths from to base pairs from the s. pombe genome annotation. of these, ltr sequences were residing in full-length ltr retrotransposons. this uneven number results from a single chimerical ltr retrotransposon with an organization that potentially is a result of an ectopic recombination event. two full-length ltr retrotransposons are frame shifted and annotated as pseudogenes http://www.genedb.org/. genomic coordinates of the ltr sequences are provided in additional file 1; table s from a biological point of view, we are interested in distinguishing between transcriptional activity stemming from full-length ltr retrotransposons and from solitary ltrs. analysis of transcriptional activity was therefore performed on these two sets of ltrs separately: full-length ltr retrotransposons and the remaining solitary ltr sequences. to assess the level of transcriptional activity using the hybmap approach, we collected the recorded signal intensities of hybmap probes mapping exclusively within the full-length ltr set and uniquely within the solitary ltr sets, and compared these to signal intensities for probes mapping to other genomic reference features. these reference features include rna genes, protein-coding genes and background sequences. as expected, when plotting the signal intensity for genes we observe higher levels of signal intensities for forward strand probes, and lower levels of intensities for reverse strand probes. signal intensities for intergenic mapping probes are distributed around zero, in accordance with the normalization procedures carried out in the hybmap study. finally, low levels of signals are observed for intronic mapping probes, showing slightly higher intensities on the forward than on the reverse strands. signal intensities for the two ltr sets are presented in detail below. a) number of sequences in set b) hybmap 60-mers mapping to sequence sets. both forward and reverse strand probes included. c) rna-seq sequence reads from growth stage mapping to sequence sets. d) number provided is probes and reads mapping exclusively within sequence sets full-length ltr retrotransposons a total of hybmap probes are mapping exclusively to the set of full-length ltr retrotransposons. of these, only probes are mapping uniquely to a single retrotransposon locus. this means that for the vast majority of probes we cannot determine the number of loci contributing to any transcriptional activity. in figure 2a, yellow curves depict the scenario where all retrotransposons contribute equally, and red curves depict the other extreme where the total signal of each probe is derived from a single loci. as seen from figure 2a, the former scenario indicates that ltr retrotransposons are not transcribed at a level exceeding that of background transcription of intergenic sequence, whereas the latter scenario is equivalent to a few active ltr retrotransposon transcribed at a level comparable to histone genes. when plotting the signal intensities of uniquely mapping probes, we see that these are highly skewed towards higher intensities, suggesting that only a small number of ltr retrotransposon loci contribute to the combined transcriptional activity, and concurrently, that these retrotransposons are transcribed at high levels. the full-length uniquely mapping probes target different ltr retrotransposons, of which are the aforementioned pseudogenes. thus, transcription is detectable from at least retrotransposons with complete open reading frames. we cannot, however, conclude anything about the individual levels of transcriptional activity from the remaining full-length ltr retrotransposons that do not have any unique probes assigned to them. we next mapped hybmap probes onto an alignment of the full-length ltr retrotransposons. although data shown in figure strongly suggest that a minority of ltr retrotransposons contribute to the overall transcriptional activity, in figure probe intensities are divided by the number of possible mappings. when ignoring the differences between those probes that are mapping uniquely and those that are not, transcription appears to be relatively equal across the retrotransposon internal sequence, consistent with transcription of the entire retrotransposon. the notable exceptions of two areas covering parts of the reverse transcriptase and rnase h domains coincide with regions where probes are mapping to a lower number of full-length ltr retrotransposons, providing additional support for the notion of a few highly transcriptionally active retrotransposons. although high levels of reverse strand transcription is apparent, poly-enriched samples display a better resolution between the two strands, suggesting that polyadenylated full-length ltr retrotransposon transcripts are present in the samples. the fact that two almost identical flanking ltrs are present in each full-length retrotransposon, and the likelihood that an ltr mapping probe will also map to ltr sequences outside the set of full-length elements, makes it difficult to compare directly the flanking ltrs and the internal sequence. we would expect transcription to initiate at the transcription start site in the 5' ltr and terminate in the 3' ltr. in reality, as ltr-matching probes are often mapping to both flanking ltr sequences, we cannot establish from which end the activity stems, and for consistency we have simply mapped such probes to the 3' ltr in figure interestingly, no clear distinction between forward and reverse strand transcriptional activity is observed for the ltr sequences. solitary ltr sequences transcriptional activity is also detectable from solitary ltr sequences, although only a subset of the probes mapping uniquely to solitary ltrs shows high levels of signal intensities. further, transcriptional activity is similar from the forward and the reverse strand. to establish if the divergent intensities of signals from solitary ltr probes are a result of differences between ltr loci, or between probes within ltr sequences, we calculated the average signal intensity for each ltr locus. if the observed broad range of signal intensities in figure 2b was a result of ltr loci with equal levels of transcriptional activity, but with certain parts of the ltr sequences being transcribed and some not, we would expect the average signal intensity for individual ltr sequences to be relatively similar. this is not the case, suggesting that individual solitary ltr loci differ in levels of transcriptional activity. then why are some ltr loci apparently transcribed whereas others are not? we do not find any noticeable patterns between highly and lowly transcribed ltr sequences in terms of orientation and distance with respect to nearest neighbouring protein-coding gene. in fact, no correlation is found between the average signal intensity of ltr sequences and the average of the nearest gene. finally, to test if the observed transcriptional differences between specific ltr sequences are due to simple stochastic variation due to the relatively small sample size, we performed a permutation analysis in which probe intensities were randomly assigned to an ltr sequence. when repeating this analysis times, we find that the observed variance between the average signal intensities from each ltr sequence fall well without the range of variances of the simulated sets. this strongly suggests that distinct differences in transcriptional activity levels exist between s. pombe solitary ltr sequence loci. transcriptional activity stemming from solitary ltrs could be a result of transcription initiated outside the ltr sequences. to assess this, we included genomic sequence flanking the ltr sequences and compared transcriptional activity within ltr sequences to activity in their genomic vicinity. in practice, we collected a set of easily alignable solitary ltr sequences, and for each ltr sequence we collected a maximum of base pair flanking sequence both up- and downstream. if the distance to another annotated feature was lower than bp, only half of this distance was used as flanking sequence. if half the distance to nearest feature was smaller than base pairs no flanking sequence was included. the flanking sequences were then concatenated with the alignment of solitary ltrs, so that flanking sequence was not aligned but fixed by the borders of ltr sequences. a schematic depiction of the procedure and statistics on sizes of flanking sequences are shown in additional file 2; figure s as seen from figure a decrease in the level in transcriptional activity is observed immediately outside the ltr sequences. the median level of signal intensities within ltr sequences was found to be significantly higher than both flanking sequences upstream and downstream. therefore, the ltr transcriptional activity as indicated by hybmap probes does not appear to be a result of transcription continuing into ltrs from the flanking genomic regions, but seems remarkably confined to the ltr sequences. hybmap analysis from alternative procedures and growth conditions data from the cairns group hybmap study include hybridizations using poly-enriched rna samples, as well as samples from alternative growth conditions. in general, alternative growth conditions yield similar patterns of ltr transcriptional activity. notably, a relative higher forward strand activity from full-length ltr retrotransposons is found in poly-enriched samples, consistent with polyadenylation of the full-length ltr retrotransposons. in contrast, signal intensities from solitary ltr sequences are shifted markedly towards lower values in poly-enriched samples, suggesting that no polyadenylation takes place. rna-seq analysis of ltr transcriptional activity sequence reads from the rna-seq study were mapped to the ltr sequences and other genomic features in a manner similar to the hybmap probes. contrary to the hybmap approach, rna-seq involves conversion of sampled rna into cdna primer ). presumably this step is responsible for the observed bias of higher rna-seq read densities towards the 3' end of the protein-coding genes in our reference set - a bias that is not observed for the hybmap intensities. consistent with this, when we plot the rna-seq reads mapping exclusively to full-length ltr retrotransposons onto the alignment, a pronounced 3' peak is observed. again, this strongly suggests that s. pombe ltr retrotransposons are actively transcribed in full length and polyadenylated. for the full-length ltr elements, we assigned the rna-seq reads using two complementary procedures. first, reads mapping to multiple loci were assigned evenly between ltr loci. second, multiple mapping reads were assigned to a single locus and the highest possible density of reads for a single locus was recorded. similarly to the hybmap probe assignment procedure, these two alternatives reflect the two extremes of possible ltr transcription; all ltrs being transcribed at a similar level, or a minimum of ltr loci being responsible for all the detected transcription. we then compared the transcriptional levels of all protein-coding genes as indicated by rna-seq and hybmap. one initial observation is that calculating the density of rna-seq reads introduces a bias against longer genes. this is of particular concern if we want to analyse transcription from full-length ltr retrotransposons, which with an average genomic length of base pairs are among the percent longest genes in s. pombe. we therefore used the absolute number of rna-seq reads per gene loci, which show a clear correlation with hybmap levels, in an unbiased fashion with regards to length. in figure the transcriptional activity from full-length ltr elements are shown as a rectangular space, in which the horizontal boundaries are determined by the median hybmap probe intensity assuming that all ltrs are transcribed equally, or assuming that the intensity for each probe stems from a minimal number of transcriptionally active loci. the vertical boundaries are similarly defined for rna-seq read coverage. the potential range of full-length ltr transcription follows the general distribution of protein-coding genes. the transcriptional activity from solitary ltr sequences, as well as snornas and trnas are underestimated in the rna-seq analysis compared to the hybmap approach. lower signals from these presumably non-polyadenylated transcripts would be expected from the rna-seq approach. in summary, we find that the rna-seq data supports the finding of transcriptional activity from full-length ltr retrotransposons, and the notion that these transcripts are polyadenylated. ltr transcription during meiosis the rna-seq study includes time series samples during meiosis, and we attempted to evaluate the association between ltr transcription and the transcription of neighbouring protein-coding genes during meiosis. we selected ltrs with the highest uniquely mapping sequence read coverage and residing within bp upstream of a protein-coding gene. for each meiosis stage, we calculated the ratio between read density at meiosis and read density at growth phase, and compared these to the same ratios for the neighbouring protein-coding genes. this approach allowed us to directly compare read levels without adjusting for sequence lengths and uniqueness. as seen from figure an apparent correlation is observed between some ltrs and their neighbouring genes. the scarcity of points makes correlation analysis problematic. yet, we have attempted to assess the significance of the correlations using two different approaches. first, we concatenated the time series for ltrs and for genes and calculated a product moment correlation coefficient of, which is significant below the confidence level. second, we produced permutated sets of the ltr expression profiles, in which the time points were shuffled independently and calculated correlation coefficients between the permutated ltr sets and the real gene sets. we then compared the median of the product moment correlation coefficients from the real data to the distribution of medians from the permutated data. of the permutated sets, only a single set exceeded the real median, corresponding to a two-tailed significance level below × 10-. comparing the transcriptional activity of full-length ltr retrotransposons to solitary ltrs during meiosis shows no apparent correlation, supporting the notion that these two groups of sequences behave transcriptionally distinct. it has recently been reported that cenp-b homologues of s. pombe are negatively regulating transcription by binding to ltr sequences and facilitating chromatin modification. additionally, other apparently distinct protein pathways control ltr transcription in s. pombe, although rna interference appears only to have a limited role. when comparing the meiosis expression profiles of proteins involved in transcription repression to the profiles for ltr sequences, no conclusive pattern of inverse correlation between repressor genes and ltr sequences is apparent. on the other hand, the different transcription patterns of solitary and full-length ltrs and the indications from our analysis that large differences in transcriptional activity exist between ltr loci suggest that ltr transcription is not solely determined by a global mechanism exerting its effect across the entire genome.  our analysis of two independent transcriptome data sets clearly indicates that ltr retrotransposons are actively transcribed during growth phase, and that the retrotransposon transcripts are polyadenylated. when considering the evolutionary dynamics of s. pombe ltr elements, kelly and levin speculated if only a few of the complete elements were responsible for majority of transposition events as has been observed in saccharomyces cerevisiae. although we cannot conclude anything on the transcriptional levels of individual loci, our data strongly suggest that a minority of loci contribute the majority of the transcriptional output from ltr retrotransposons. assuming that high levels of retrotransposition are detrimental to the host, selection will favour regulation of retrotransposition. such regulation may either take place pre-transcriptionally or post-transcriptionally. the observed transcription of s. pombe ltr retrotransposons suggests that post-transcriptional mechanisms are the predominant type of regulation during regular growth phase. the relatively high levels of transcriptional activity antisense to full-length ltr retrotransposons may potentially be part of such post-transcriptional regulation pathways. the targeting of ltr insertion upstream of genes could limit the potential of regulating retrotransposition pre-transcriptionally in s. pombe, as any interference with transcriptional regulation presumably would also affect neighbouring protein-coding genes. from the hybmap data transcriptional activity is detectable from both strands of solitary ltr sequences, and the transcription appears to be confined to the ltr sequences themselves. analysis of the rna-seq data suggests that transcription of solitary ltrs upstream of genes is correlated with the transcriptional activity of the neighbouring genes. one might speculate that the presence of transcripts from both strands of the solitary ltrs could generate double-stranded rnas, similar to the double-stranded rnas involved in heterochromatin formation in s. pombe. however, this would suggest a negative correlation between the transcriptional activity of genes and ltr sequences. the observed positive correlation rather suggests that the ltr transcripts represent transcription facilitated by a physical association with actively transcribed genes, in parallel to the observed co-expression of linked genes. our analysis indicates that a clear distinction exists between solitary ltr sequences and full-length ltr elements in terms of transcriptional activity. transcription of full-length ltr retrotransposons is for the most part derived from the forward strand and transcripts are polyadenylated. in contrast, solitary ltr transcription is found from both strands, with transcripts showing no signs of polyadenylation. additionally, divergent expression levels during meiosis are observed between full-length ltr retrotransposons and solitary ltr sequences. in hiv retroviruses, a number of transcription factors bind the internal retrotransposon downstream of the 5' ltr. it is possible that the absence of such binding sites around solitary ltrs contributes to their changed transcription patterns. similarly, in a range of retrovirus ltrs, polyadenylation signals are present downstream of the transcription start sites, but a minimum distance is required for polyadenylation to take place, so that transcription is initiated in the 5' ltr and polyadenylation only in the 3' ltr of full-length retroviruses. certainly, this would preclude polyadenylation of transcripts initiated within solitary ltr sequences. similar to the solitary ltr sequences, levels of transcriptional activity appear to be relatively similar from both strands of the ltrs from full-length retrotransposons. one possible scenario is therefore that - compared to full-length retrotransposons - the solitary ltr sequences have changed their transcriptional patterns due to loss of regulatory motifs, and lost the ability to generate polyadenylated transcripts, but have retained the ability to generate transcription from both strands. based on our analysis, we conclude that the application of large-scale transcriptome data allows the elucidation of retrotransposon transcriptional activity, but that the resolution by which transcription can be assigned to specific retrotransposon loci is still limited. a recent rna-seq approach revealed an up-regulation of transposons in methylation-defective arabidopsis mutants. additionally, mapping of capped sequence reads demonstrate wide-spread, regulated transcription initiated with mammalian retrotransposons. hence, novel transcriptome analysis techniques will inevitably shed light on tissue-specific and temporal expression patterns of retrotransposons facilitating an assessment of the dynamics and immediate impact of these long-term residents of eukaryotic genomes. 
retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. equal levels of transcriptional activity are observed from both strands of solitary ltr sequences. we have used recently published transcriptome data from the fission yeast schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from long terminal repeat retrotransposons. transcriptional activity was analysed for both full-length ltr retrotransposons and solitary ltrs. the finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a post-transcriptional level. alternatively, the transcriptional activity may signify a hitherto unrecognized activity level of retrotransposon proliferation. given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. ltr retrotransposons are normally flanked by two ltr sequences. two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from ltr retrotransposons in s. pombe during growth phase in rich medium. the redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the ltr retrotransposons contribute significantly to the detected transcription. presumably, the host organism negatively regulates proliferation of ltr retrotransposons. a considerable level of reverse strand transcription is also detected. however, the majority of ltr sequences in s. pombe exist as solitary ltrs, i.e. transcriptome data collected during meiosis suggests that transcription of solitary ltrs is correlated with the transcription of nearby protein-coding genes. our findings underline the usefulness of transcriptome data in elucidating dynamics in retrotransposon transcription.
 retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. the numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed - or near fixation - in the population or strain from which genetic material has been extracted for sequencing. one pre-requisite for retrotransposition is transcription of the elements. given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. we have used recently published transcriptome data from the fission yeast schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from long terminal repeat retrotransposons. ltr retrotransposons are normally flanked by two ltr sequences. however, the majority of ltr sequences in s. pombe exist as solitary ltrs, i.e. as single terminal repeat sequences not flanking a retrotransposon. transcriptional activity was analysed for both full-length ltr retrotransposons and solitary ltrs.  two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from ltr retrotransposons in s. pombe during growth phase in rich medium. the redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the ltr retrotransposons contribute significantly to the detected transcription. a considerable level of reverse strand transcription is also detected. equal levels of transcriptional activity are observed from both strands of solitary ltr sequences. transcriptome data collected during meiosis suggests that transcription of solitary ltrs is correlated with the transcription of nearby protein-coding genes.  presumably, the host organism negatively regulates proliferation of ltr retrotransposons. the finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a post-transcriptional level. alternatively, the transcriptional activity may signify a hitherto unrecognized activity level of retrotransposon proliferation. our findings underline the usefulness of transcriptome data in elucidating dynamics in retrotransposon transcription. 