BACKGROUND
the protozoan parasite plasmodium falciparum is responsible for the most lethal form of human malaria, leading to one million deaths annually.
the clinical symptoms of malaria are caused by the intraerythrocytic stages of the parasite, which multiply inside the host’s red blood cells .
during the past decades much research has focused on understanding how gene regulation is achieved in plasmodium.
the publication of the p. falciparum genome sequence in 2002
<cit>  was followed by transcriptome analyses using microarrays
<cit>  and, more recently, high-throughput sequencing of cdna
.
these analyses allowed determination of transcript levels for a large number of genes, helped to refine the original gene model and revealed a tight regulation of gene expression throughout the intraerythrocytic developmental cycle  of p. falciparum.
nevertheless, information regarding 5′ and 3′ untranslated regions  and the degree of antisense transcription is still missing for most genes.
transcription of non-coding rna  is common in eukaryotes, for example more than 90% of the human genome is transcribed
<cit>  whereas only ~ <dig> % of it encodes proteins.
based on their size and origin of transcription, ncrnas are generally referred to as small  or long ncrnas  and intronic, genic or intergenic ncrnas .
in addition, rnas that are complementary to other endogenous rnas, are referred to as natural antisense transcripts .
some nats result from incomplete transcription termination of adjacent genes and appear to represent transcriptional noise.
other nats are highly conserved and expressed in a developmentally regulated manner, with tissue-specific expression patterns
<cit> , providing strong arguments for their regulatory roles in biological processes such as gene expression.
work in several organisms has established the regulatory role of nats via the rna-interference  pathway, in which a specific group of nats, called microrna promote post-transcriptional gene silencing.
this pathway has been described in a large number of organisms but it is notably absent from saccharomyces cerevisiae and p. falciparum <cit> .
however, even in organisms lacking a functional rnai-machinery, nats have been shown to play important roles in regulating gene expression
<cit>  and many different mechanisms have been described by which nats can regulate gene expression in an rnai-independent manner.
at the transcriptional level, nats can cause transcriptional interference via physical collisions between transcribing polymerases
<cit> .
regulation at the post-transcriptional level can occur via the formation of sense-antisense duplex rna.
nuclear retention of nats is commonly observed and duplex formation may thus regulate gene expression by retaining mrna in the nucleus.
in the cytoplasm sense-antisense duplex formation has been shown to affect mrna stability and translation efficiency .
while most of the published transcriptome analyses in p. falciparum do not permit differentiation between sense and antisense transcripts, some studies provided a strand-specific analysis and revealed antisense transcription from multiple sites across the genome
<cit> .
however, no comprehensive analyses of potential correlations between sense and antisense rna transcript levels have been performed and a complete strand-specific transcriptome profile throughout the idc of the parasite, covering even the highly at-rich intergenic regions, is still lacking.
recent advances in rna-seq technology, in particular the ability to perform strand-specific analyses , the identification of a polymerase able to amplify even extremely at-rich regions
<cit>  and an increase in sequence read length, have motivated us to combine these improvements into one rna-seq protocol and determine the genome-wide, strand-specific transcriptional profile for p. falciparum.
using this protocol, we have generated strand-specific rna-seq libraries for parasites throughout the idc as well as for separately purified nuclear and cytosolic rna fractions resulting in the sequencing of close to  <dig> billion nucleotides.
our data demonstrate substantial antisense transcription for 24% of genes, developmental regulation of antisense transcripts, a strong bias of antisense transcription towards the 3′-end of genes and a complex picture of correlation between sense and antisense transcript levels.
in addition, the coverage of highly at-rich regions allowed us for the first time to map polyadenylation sites for  <dig> asexual blood stage genes.
RESULTS
preparation of strand-specific rna-seq libraries
to accurately determine rna levels in the parasite, any biases that are inadvertently introduced during the preparation of cdna libraries must be kept at a minimum while strand-specificity, coverage and sequencing depth should be maximized.
previously, a thorough comparison of different strand-specific rna-seq methods indicated that the least amount of ‘false’ antisense rna was generated when libraries were prepared by using the ‘rna-ligation method’ involving the sequential ligation of 3′-preadenylated and 5′-adapters to rna followed by reverse transcription using a primer complementary to the 3′-adapter
<cit>  .
therefore, we adopted this method for use in plasmodium although alternative strategies to generate strand-specific libraries may also be well suited
<cit> .figure  <dig>
flowchart of strand-specific rna-seq library preparation.
a) strand-specific sequencing libraries are prepared from total rna depleted of rrna by digestion of 5′-p-containing rna fragments with a 5′-phosphate-dependent exonuclease  or by enrichment for polyadenylated mrna.
subsequently, rrna-depleted rna is decapped, fragmented, dephosphorylated and re-phosphorylated to obtain rna fragments with 5′-p and 3′-oh groups.
next, the rna is ligated to 3′ and 5′-adapters, reverse-transcribed with a primer complementary to the 3′-adapter and the cdna is pcr amplified for 12- <dig> cycles using kapa hifi polymerase.
a recent analysis had compared amplification efficiencies of different polymerases for genomes ranging in gc-content from  <dig> %  to  <dig> %  and found kapa hifi  to provide the most consistent results for at-rich genomes  <cit> .
b) meta-gene coverage plots for libraries prepared from polya-enriched and tex-treated rna.
x-axis: scaled gene body, y-axis: scaled coverage.
to increase the sequencing coverage of non-ribosomal rna, we generated libraries from polya-enriched rna, unless indicated otherwise.
we found polya-enriched libraries to contain less rrna than libraries prepared from rna treated with the 5′-phosphate-dependent exonuclease  , an enzyme that specifically digests processed rnas with a 5′-monophosphate end .
furthermore, we noticed that the genome-wide coverage was substantially higher for libraries prepared from polya-enriched libraries than for libraries prepared from tex-treated rna.
this difference is probably a consequence of the lower percentage of rrna found in libraries prepared from polya-enriched rna .
next we compared the distribution of sequence reads across cdss for libraries prepared from polya-enriched and tex-treated rna and noticed a strong bias towards the 5′-end in the tex-treated library .
while this bias was not apparent for all genes, it was highly reproducible for individual genes and distinctly more pronounced in large genes.
therefore, to obtain more uniform sequencing coverage, we prepared all subsequent libraries from polya-enriched rna.figure  <dig>
rna-seq mapping statistics.
libraries were prepared either from polya-enriched  or tex-treated rna .
dna was amplified using the dna polymerases kapa hifi  or platinum® pfx .
using the rna-ligation-based protocol, we prepared sequencing libraries from rna extracted 10 h, 20 h, 30 h and 40 h post infection  , as well as from cytoplasmic and nucleic rna of parasites harvested at 20 h and 30 h p.i.
.
combining the data from these libraries with the data from libraries prepared for protocol development , our dataset consists of ~ <dig> million mapped strand-specific reads derived from  <dig> libraries .
data coverage, level of strand-specificity and prevalence of nats
although the existence of nats has been documented in p. falciparum based on sage, microarray and strand-specific high-throughput sequencing data
<cit> , the understanding of their genome-wide distribution is still incomplete due to the limited depth or uncertainties in the level of strand-specificity of the published datasets.
using strand-specific rna-seq data from ~ <dig> million mapped reads, lópez-barragán et al.
observed nat transcription in  <dig> coding genes
<cit> .
to gain more insight into the genome-wide distribution of nats, we took advantage of the unprecedented depth of our dataset  and combined the mapping results of  <dig> libraries to generate a coverage map.
we also re-analyzed the lópez-barragán et al.
dataset  in parallel for comparison of coverage and level of strand-specificity.
using the combined data from  <dig> libraries, we detected transcription of  <dig> % of the plasmodium genome .
keeping all parameters constant, re-analysis of the lópez-barragán et al.
dataset indicated transcription of ~ <dig> % of the plasmodium genome.
most of the transcribed genomic positions detected in the lópez-barragán et al.
dataset were also detected in this study , suggesting both datasets are consistent and that our dataset represents a substantially deeper coverage.
next, we tried to determine the level of strand-specificity of our datasets compared to that generated by lópez-barragán et al.
previously, a globally positive correlation between sense and antisense transcripts has been used as an indicator for potential presence of artifactual antisense transcripts
<cit> .
this approach is based on the fact that the observed antisense transcripts could be derived from the sense transcript during library preparation, e.g.
incomplete second-strand cdna digestion in dutp method
<cit> , spurious synthesis of second-strand cdna
<cit> , or other unidentified sources of artifacts.
while the correlation in our dataset was close to  <dig> , we observed a strong positive correlation for the lópez-barragán et al.
dataset , suggesting a relatively higher level of strand-specificity of our dataset.
likewise, the ratios of sense to antisense reads differ significantly between our  and the lópez-barragán et al.
dataset .
the relatively lower level of strand-specificity in lópez-barragán et al.
dataset maybe due to the imperfect second-strand cdna digestion in dutp method as the authors mentioned
<cit> , comparing to the rna ligation method used in this study.
lópez-barragán et al.
thus avoided false positives in nat identification by applying stringent cutoffs on both proportion of antisense read  and antisense read number
<cit> , which may have underestimated the prevalence of nats.
to estimate the prevalence of nats in plasmodium, we established two sets of thresholds with different stringency .
our data indicate that between ~24%  and ~45%  of all coding genes  are overlapping with nats .
in the following sections, unless mentioned otherwise, we restricted our analyses to the genes with nats defined at stringent thresholds .
transcription of nats is pervasive and developmentally regulated
numerous mechanisms have been described by which antisense rna can regulate gene expression even in the absence of a functional rnai-machinery.
common to these mechanisms is the ability of the respective organism to regulate antisense transcription independently of the complementary sense transcription.
previously, the distinct regulation of mrna levels during the idc has been described
<cit> .
the idc of p. falciparum takes 48 h to complete, thus, to evaluate developmental regulation of nats across the idc, we compared patterns of sense and antisense expression in parasites at evenly spaced intervals .
to determine whether transcription of nats is developmentally regulated, we searched for changes in nat levels across the idc.
using the software package edger, we observed significant changes  in antisense transcript levels for  <dig> of the  <dig> genes with nats in at least  <dig> of the  <dig> non-redundant time point pair comparisons.
for  <dig> genes with nats we observed significant changes in antisense transcript levels in at least  <dig> of the  <dig> non-redundant time point pair comparisons .
these findings suggest that at least ~10% of nats are regulated during the idc .
subsequently, we investigated whether the changes in sense and antisense transcript levels between times points are correlated and observed no global correlation .
thus, our data suggest that transcription of nats does not exert a globally positive or negative effect on the transcription of its sense counterpart.
to further investigate this observation visually, we sorted the genes based on patterns of antisense expression levels throughout the idc and plotted the antisense expression heat map , and observed a distinct life cycle-specific expression pattern of antisense transcripts.
then, we re-plotted the heat map using sense expression levels while keeping the gene order sorted using antisense expression levels.
in agreement with the above observation, no apparent pattern was observed .
it should be noted that sorting the genes based on sense transcript levels confirmed the previously observed regulation of sense transcript levels
<cit>  .
thus, both sense and antisense transcript levels are regulated during the idc but regulation is not synchronous.
an example of developmentally regulated antisense transcript levels is shown in figure
3b.figure  <dig>
antisense transcription is abundant and developmentally regulated.
a) heat map of genes containing sense and antisense transcripts throughout the idc  sorted based on antisense rna trends  and sense rna trends .
b) coverage plot for a gene containing developmentally regulated nats.
yellow box represents cds and green arrow indicates direction of transcription.
transcription of nats is biased towards the 3′-end of genes
to further characterize the newly identified nats, we mapped antisense transcript levels across the cdss of all genes and observed a striking enrichment of antisense transcripts at the 3′-ends of genes .
it has been suggested that nats could be a consequence of run-through transcription or transcription initiation of bi-directional promoters from adjacent genes
<cit> .
to determine if p. falciparum contains nats whose expression is regulated independently from that of adjacent genes, we investigated the correlation between the changes in mrna levels of the downstream genes and the changes in antisense transcript levels of the corresponding upstream genes during the idc.
we reasoned that, should antisense transcription be a consequence of active transcription of the downstream gene, changes in antisense transcript level across the idc should be positively correlated to those of the downstream mrna.
to this end, we performed  <dig> non-redundant time-point pair comparisons for each gene  and observed a positive correlation between the change of antisense transcript levels of one gene and that of mrna levels of the gene located downstream .
the observed correlation was independent of the orientation of the downstream gene, suggesting that both run-through transcription and bi-directional promoter activity contribute to the observed antisense transcription in plasmodium.
however, while we observed a positive correlation for  <dig> of  <dig> genes with overlapping nats , for  <dig> genes  we observed a negative correlation and for  <dig> genes  no clear correlation .
for  <dig>  genes we did not have enough data points or no gene was located downstream.
among the nats whose transcription did not correlate with the transcription of adjacent genes , i.e.
nats that appear to be independently regulated, we found ~13%  to be developmentally regulated .
taken together, these findings indicate that while run-through transcription and bi-directional promoter activity are likely to contribute to the observed antisense transcription, a significant number of nats seems to be regulated independently of mrna transcription.figure  <dig>
antisense transcription is strongly enriched at the 3′-end of cdss.
a) averaged coverage of antisense and sense rna levels for all annotated genes.
b) sense  and antisense  coverage for a representative gene .
green arrows indicate direction of transcription and yellow boxes represent cdss.
the grey triangles mark the site of a putative bi-directional promoter.
c) coverage plot for a representative gene  likely to be affected by run-through transcription from a neighboring gene .
green arrows indicate direction of transcription and yellow boxes represent cdss.
correlation of nats with orientation of downstream genes.
figure summarizing the correlation of nat levels of a reference gene  with the mrna levels of the gene located downstream.
nat levels often correlate with expression of downstream genes.
scatter plots showing fold-changes of anti-sense rpkm of a reference gene versus fold-changes of sense rpkm for the gene located downstream of the reference gene located on the same strand  or opposite strand .
time-point comparisons are plotted if the reference gene and downstream gene had significant anti-sense rpkms and sense rpkms for at least two time points.
up to six non-redundant time-point comparisons are plotted per gene.
nats do not accumulate in the nucleus or correlate with mrna levels
to understand the role of antisense transcription in plasmodium, we sought to determine whether our data provide evidence to support the known mechanisms for antisense-mediated regulation of gene expression.
collisions between rna polymerases transcribing the sense and antisense strands have been described to interfere with the transcription of mrnas as shown in escherichia coli <cit>  and s. cerevisiae <cit> .
if antisense transcription has a negative impact on sense transcription in plasmodium, an increase in antisense levels from one time point to the next should correlate with a decrease in sense levels and vice versa.
however, as mentioned above, comparison of changes in sense and antisense levels during four time-points showed a correlation close to  <dig> .
alternatively, duplex formation between sense and antisense rna molecules can result in nuclear retention of antisense transcripts and modulate gene expression.
if antisense transcripts played a role in retaining sense transcripts in the nucleus, an enrichment of antisense transcripts in the nucleus would be observed.
several studies have reported nuclear localization of ncrna in p. falciparum.
thus, even though polyadenylated nats are not commonly enriched in the nucleus, and our sequencing libraries were enriched for polyadenylated rna, we examined the possible nuclear enrichment of antisense reads in genes that showed significant levels of antisense transcription but are not likely to be affected by run-through transcription from neighboring genes .
we did not observe a significant enrichment of antisense reads in these genes  in the nucleus against both the total and the cytosolic fractions at 20 h p.i., while ncrna from telomere-associated repeated elements were enriched in the nuclear fraction .
a final mechanism we considered was antisense-mediated translational inhibition in which case overexpression of antisense transcripts leads to a reduction of proteins but not to reduction of sense rna levels
<cit> .
to this end, we correlated our rna-seq data with previously published stage-specific proteomic datasets
<cit> .
as observed by le roch et al., we saw a positive correlation between mrna abundance ) and protein abundance , thus an increase in sense rpkm correlates with an increase in peptide counts per kilobase for a given gene .
to evaluate the global effect of antisense transcripts on translation, we investigated the correlation between antisense transcript levels  and translation efficiency .
however, for all four time points the correlation was close to  <dig> , suggesting that the level of antisense transcription has no global effect on translation efficiency.
mapping of sense and antisense polyadenylation sites
the sequencing depth of our dataset combined with the use of a polymerase better suited for the amplification of extremely at-rich regions than previously used enzymes , allowed us to map for the first time polyadenylation sites  for p. falciparum.
despite the importance of 3′-utrs in the regulation of gene expression, the genome-wide characterization of 3′-utrs in plasmodium lags far behind that of coding sequences.
to determine pass and to address the question of whether the observed antisense transcripts are polyadenylated, we used the combined rna-seq data from all  <dig> libraries to map pass on a genome-wide scale.
due to the high abundance of coding mrnas compared to antisense rna , we expected most of the identified pass to correspond to coding mrnas and only a small fraction to correspond to nats.
to map genuine pass and to avoid false positive assignments derived from internal polya stretches or sequence reads of low quality, we followed a set of previously published criteria
<cit>  .
to assess whether the identified pass could represent genuine pass, we plotted the occurrence of pass around stop codons of all genes .
most of the identified pass fell within  <dig> nt downstream of the stop codon of genes, corresponding to the expected sites of polyadenylation and therefore validating our approach.
as microheterogeneity in pas selection is well documented
<cit> , we grouped individual pass located in close proximity to each other into so-called ‘pas-clusters’ with a maximum width of  <dig> nt.
setting the maximum 3′-utr length to  <dig> nt, we were able to assign  <dig> pas-clusters to  <dig> coding mrnas , yielding an average 3′-utr length of  <dig> nt  , for an example see figure
7b.
genome-wide we observed that  <dig> % genes  contained multiple pas-clusters compared to  <dig> % genes with multiple pass found in s. cerevisiae <cit> .figure  <dig>
mapping of polyadenylation sites.
a) distribution of mapped sense pass , distance  relative to the corresponding stop codon.
b) distribution of sequence reads and pass mapping to the upper  and lower  strand.
green arrows indicate direction of sense transcription for the corresponding cds and yellow boxes mark cdss.
c) distribution of antisense sequence reads and antisense pass mapping to the upper strand .
the green arrow indicates the direction of sense transcription for the corresponding cds and the yellow box marks the cds.
previously, some nats in plasmodium were shown to be transcribed by rna polymerase ii and it was suggested that nat stability may be regulated at the level of polyadenylation
<cit>  but no polyadenylation of nats has been described for p. falciparum.
thus, we decided to search for pass of nats, i.e.
antisense pas-clusters.
antisense pas-clusters were defined as those that 1) are located on the antisense strand within the coding region of an annotated gene, 2) contain at least two pass within the cluster, 3) are located at least  <dig> nt upstream of an annotated stop codon .
because most of the antisense transcripts exist in low abundance, the likelihood of identifying their pass are much lower than for coding mrnas.
nonetheless, based on the above criteria, we identified  <dig> antisense pas-clusters for  <dig> genes .
these data indicate that at least some of the observed nats are polyadenylated.
evidence of antisense spliced junctions
based on the splicing junctions identified from our datasets using hmmsplicer
<cit> , we found evidence for antisense spliced junctions in  <dig> of the  <dig> genes with nats .
thus, at least a portion of the observed nats  is spliced.
furthermore, we found  <dig> of the  <dig> antisense junction clusters to overlap with sense junctions on mrna .
these findings are consistent with a previous study, which has identified antisense introns and antisense junctions overlapping with sense junctions, using a similar approach
<cit> .
these observations further validate antisense transcription in p. falciparum to be a widespread phenomenon.
discussion
to perform a comprehensive strand-specific analysis of the p. falciparum transcriptome, we combined several recent improvements in rna-seq technology.
specifically, we took advantage of improvements made in the preparation of highly strand-specific sequencing libraries, we used a polymerase that allows amplification of highly at-rich dna sequences and increased the number and length of sequence reads obtained from high-throughput sequencers.
we generated a total of  <dig> strand-specific transcriptome profiles for parasites across the idc as well as for separately purified nuclear and cytosolic rna fractions.
nine of the  <dig> rna-seq libraries were prepared from polya-enriched rna because we found these libraries yielded a higher coverage and a more even distribution of sequence reads across the cdss than libraries prepared from tex-treated rna.
while polya-enrichment harbors the intrinsic problem of selecting against non-polyadenylated transcripts, for the following reasons we believe that our libraries contain at least some non-polyadenylated transcripts: 1) polya-enrichment is generally not 100% efficient and indeed, trna and rrna sequencing reads were present in our libraries.
2) the plasmodium genome is extremely at-rich and long stretches of as exist even within genes.
thus it is likely that polya-enrichment can also enrich for non-polyadenylated transcripts as long as they have internal stretches of as.
nevertheless, most likely our dataset lacks weakly transcribed rna without polya-tails.
our analysis revealed transcription from almost 80% of the plasmodium genome and exposed antisense transcription for 24% of genes many of which are developmentally regulated.
interestingly, we find that antisense transcripts are not uniformly distributed but strongly enriched at the 3′-ends of cdss.
such an enrichment of antisense transcripts at the 3′-end of cdss has been detected in both yeast and humans
<cit>  and it has been suggested that they can arise via pathways that copy mrna, from run-through transcription, bi-directional transcription initiation or promiscuous transcription initiation from nucleosome depleted regions
<cit> .
however, the biological function of these 3′-antisense transcripts remains unknown.
our analysis indicates that in plasmodium, for the majority of genes, antisense levels do not correlate with sense transcript levels, which would have hinted at an rna-copying mechanism or a role of ndrs.
in contrast to the corresponding sense mrna levels, for a large number of genes  changes in nats levels correlate with changes in mrna levels of the gene located downstream.
this correlation may be due to an overlap of different utrs or of utrs with cdss.
such an overlap of utrs raises the intriguing possibility that transcription of one gene may influence transcription of its neighboring gene and that the genomic location of a gene may be important for its correct regulation.
for example, a gene knockout followed by expression of the same gene from a different genomic locus may lead to different antisense transcript levels compared to those from the endogenous locus.
in addition, replacement of an endogenous gene with a resistance marker will not only lead to a loss of the mrna of the endogenous gene but also to a loss of the corresponding antisense rna, possibly leading to confusing consequences and complicating the interpretation of the results.
while our analysis revealed a positive correlation between antisense and sense transcript levels of neighboring genes, such a correlation could not be identified for all genes.
thus, for a significant number of genes  our data point to the existence of cryptic promoters at the 3′-end of genes that initiate transcription of nats independent of the promoter that controls transcription of sense rna.
reports on the role of antisense rna transcripts in p. falciparum have been conflicting, some studies found increased antisense levels to repress gene expression
, while others saw no effect
<cit> .
correlating our rna-seq data with previously published proteomics data
<cit>  we find a general correlation between mrna levels and peptide levels, however, we observed no consistent effect of antisense transcripts on protein levels.
thus it remains to be seen if antisense transcripts modulate gene expression in this parasite.
3′-utrs and polya tails have important roles in mrna localization, stability and regulation of translation .
such regulation can be mediated by cis-acting sequence elements that interact with rna-binding proteins.
in plasmodium little is known about how transcription initiation is regulated but numerous studies have reported post-transcriptional control, particularly at the level of translational repression
<cit> .
specific sequence motifs within the 3′-utr have been correlated with tr but genome-wide searches for regulatory motifs have not been performed for a lack of information on 3′ utrs’ length.
in this study we present the genome-wide assignment of pass in plasmodium.
we were able to map pass for 65% of genes , which we believe will represent a valuable resource to the community.
for example, the newly generated data should allow other researchers to perform systematic genome-wide searches for sequence motifs located in utrs that may help elucidate some of the secrets of post-transcriptional gene regulation in the blood stages of p. falciparum.
besides the mapping of mrna pass we investigated the presence of antisense pas-clusters.
the latter analysis was much more challenging than the mapping of sense pass but our data indicate that at least some of the observed antisense transcripts are polyadenylated.
this observation further strengthens the assumption that nats are generated by transcription of dna and not via transcription of mrna.
this finding is not unexpected given previously published findings that antisense transcription in plasmodium can be carried out by rna polymerase ii
<cit>  and that termination of rna pol ii transcription and polyadenylation are commonly coupled.
future deep sequencing projects with libraries derived from rna fragments enriched for the 3′-end of cdss should help determine if all nats are polyadenylated or just represent a small subset.
CONCLUSIONS
intergenic and non-coding regions of the p. falciparum genome have been particularly understudied due to high at-content, typically ranging between  <dig> and 95%.
using a protocol that permits the generation of highly strand-specific rna-seq libraries and kapa hifi dna polymerase, a polymerase that amplifies even extremely at-rich dna, we generated a comprehensive transcriptome dataset for p. falciparum, containing great detail about the origin of antisense rna.
given the large number of independently regulated nats, our work suggests that ncrna in p. falciparum is of biological significance and not merely a consequence of noisy transcriptional regulation.
importantly, our high coverage and long sequence reads allow us to provide for the first time an extensive list of polyadenylation sites for blood stage parasites.
this data should enable others to perform comprehensive searches for regulatory motifs in utrs and help to understand post-transcriptional regulation of gene expression in p. falciparum.

 the protozoan parasite plasmodium falciparum is responsible for the most lethal form of human malaria, leading to one million deaths annually. the clinical symptoms of malaria are caused by the intraerythrocytic stages of the parasite, which multiply inside the host’s red blood cells. during the past decades much research has focused on understanding how gene regulation is achieved in plasmodium. the publication of the p. falciparum genome sequence in 2002 was followed by transcriptome analyses using microarrays and, more recently, high-throughput sequencing of cdna. these analyses allowed determination of transcript levels for a large number of genes, helped to refine the original gene model and revealed a tight regulation of gene expression throughout the intraerythrocytic developmental cycle of p. falciparum. nevertheless, information regarding 5′ and 3′ untranslated regions and the degree of antisense transcription is still missing for most genes. transcription of non-coding rna is common in eukaryotes, for example more than 90% of the human genome is transcribed whereas only ~ % of it encodes proteins. based on their size and origin of transcription, ncrnas are generally referred to as small or long ncrnas and intronic, genic or intergenic ncrnas. in addition, rnas that are complementary to other endogenous rnas, are referred to as natural antisense transcripts. some nats result from incomplete transcription termination of adjacent genes and appear to represent transcriptional noise. other nats are highly conserved and expressed in a developmentally regulated manner, with tissue-specific expression patterns, providing strong arguments for their regulatory roles in biological processes such as gene expression. work in several organisms has established the regulatory role of nats via the rna-interference pathway, in which a specific group of nats, called microrna promote post-transcriptional gene silencing. this pathway has been described in a large number of organisms but it is notably absent from saccharomyces cerevisiae and p. falciparum. however, even in organisms lacking a functional rnai-machinery, nats have been shown to play important roles in regulating gene expression and many different mechanisms have been described by which nats can regulate gene expression in an rnai-independent manner. at the transcriptional level, nats can cause transcriptional interference via physical collisions between transcribing polymerases. regulation at the post-transcriptional level can occur via the formation of sense-antisense duplex rna. nuclear retention of nats is commonly observed and duplex formation may thus regulate gene expression by retaining mrna in the nucleus. in the cytoplasm sense-antisense duplex formation has been shown to affect mrna stability and translation efficiency. while most of the published transcriptome analyses in p. falciparum do not permit differentiation between sense and antisense transcripts, some studies provided a strand-specific analysis and revealed antisense transcription from multiple sites across the genome. however, no comprehensive analyses of potential correlations between sense and antisense rna transcript levels have been performed and a complete strand-specific transcriptome profile throughout the idc of the parasite, covering even the highly at-rich intergenic regions, is still lacking. recent advances in rna-seq technology, in particular the ability to perform strand-specific analyses, the identification of a polymerase able to amplify even extremely at-rich regions and an increase in sequence read length, have motivated us to combine these improvements into one rna-seq protocol and determine the genome-wide, strand-specific transcriptional profile for p. falciparum. using this protocol, we have generated strand-specific rna-seq libraries for parasites throughout the idc as well as for separately purified nuclear and cytosolic rna fractions resulting in the sequencing of close to billion nucleotides. our data demonstrate substantial antisense transcription for 24% of genes, developmental regulation of antisense transcripts, a strong bias of antisense transcription towards the 3′-end of genes and a complex picture of correlation between sense and antisense transcript levels. in addition, the coverage of highly at-rich regions allowed us for the first time to map polyadenylation sites for asexual blood stage genes.  preparation of strand-specific rna-seq libraries to accurately determine rna levels in the parasite, any biases that are inadvertently introduced during the preparation of cdna libraries must be kept at a minimum while strand-specificity, coverage and sequencing depth should be maximized. previously, a thorough comparison of different strand-specific rna-seq methods indicated that the least amount of ‘false’ antisense rna was generated when libraries were prepared by using the ‘rna-ligation method’ involving the sequential ligation of 3′-preadenylated and 5′-adapters to rna followed by reverse transcription using a primer complementary to the 3′-adapter. therefore, we adopted this method for use in plasmodium although alternative strategies to generate strand-specific libraries may also be well suited.figure flowchart of strand-specific rna-seq library preparation. a) strand-specific sequencing libraries are prepared from total rna depleted of rrna by digestion of 5′-p-containing rna fragments with a 5′-phosphate-dependent exonuclease or by enrichment for polyadenylated mrna. subsequently, rrna-depleted rna is decapped, fragmented, dephosphorylated and re-phosphorylated to obtain rna fragments with 5′-p and 3′-oh groups. next, the rna is ligated to 3′ and 5′-adapters, reverse-transcribed with a primer complementary to the 3′-adapter and the cdna is pcr amplified for 12- cycles using kapa hifi polymerase. a recent analysis had compared amplification efficiencies of different polymerases for genomes ranging in gc-content from % to % and found kapa hifi to provide the most consistent results for at-rich genomes. b) meta-gene coverage plots for libraries prepared from polya-enriched and tex-treated rna. x-axis: scaled gene body, y-axis: scaled coverage. to increase the sequencing coverage of non-ribosomal rna, we generated libraries from polya-enriched rna, unless indicated otherwise. we found polya-enriched libraries to contain less rrna than libraries prepared from rna treated with the 5′-phosphate-dependent exonuclease, an enzyme that specifically digests processed rnas with a 5′-monophosphate end. furthermore, we noticed that the genome-wide coverage was substantially higher for libraries prepared from polya-enriched libraries than for libraries prepared from tex-treated rna. this difference is probably a consequence of the lower percentage of rrna found in libraries prepared from polya-enriched rna. next we compared the distribution of sequence reads across cdss for libraries prepared from polya-enriched and tex-treated rna and noticed a strong bias towards the 5′-end in the tex-treated library. while this bias was not apparent for all genes, it was highly reproducible for individual genes and distinctly more pronounced in large genes. therefore, to obtain more uniform sequencing coverage, we prepared all subsequent libraries from polya-enriched rna.figure rna-seq mapping statistics. libraries were prepared either from polya-enriched or tex-treated rna. dna was amplified using the dna polymerases kapa hifi or platinum® pfx. using the rna-ligation-based protocol, we prepared sequencing libraries from rna extracted 10 h, 20 h, 30 h and 40 h post infection, as well as from cytoplasmic and nucleic rna of parasites harvested at 20 h and 30 h p.i.. combining the data from these libraries with the data from libraries prepared for protocol development, our dataset consists of ~ million mapped strand-specific reads derived from libraries. data coverage, level of strand-specificity and prevalence of nats although the existence of nats has been documented in p. falciparum based on sage, microarray and strand-specific high-throughput sequencing data, the understanding of their genome-wide distribution is still incomplete due to the limited depth or uncertainties in the level of strand-specificity of the published datasets. using strand-specific rna-seq data from ~ million mapped reads, lópez-barragán et al. observed nat transcription in coding genes. to gain more insight into the genome-wide distribution of nats, we took advantage of the unprecedented depth of our dataset and combined the mapping results of libraries to generate a coverage map. we also re-analyzed the lópez-barragán et al. dataset in parallel for comparison of coverage and level of strand-specificity. using the combined data from libraries, we detected transcription of % of the plasmodium genome. keeping all parameters constant, re-analysis of the lópez-barragán et al. dataset indicated transcription of ~ % of the plasmodium genome. most of the transcribed genomic positions detected in the lópez-barragán et al. dataset were also detected in this study, suggesting both datasets are consistent and that our dataset represents a substantially deeper coverage. next, we tried to determine the level of strand-specificity of our datasets compared to that generated by lópez-barragán et al. previously, a globally positive correlation between sense and antisense transcripts has been used as an indicator for potential presence of artifactual antisense transcripts. this approach is based on the fact that the observed antisense transcripts could be derived from the sense transcript during library preparation, e.g. incomplete second-strand cdna digestion in dutp method, spurious synthesis of second-strand cdna, or other unidentified sources of artifacts. while the correlation in our dataset was close to, we observed a strong positive correlation for the lópez-barragán et al. dataset, suggesting a relatively higher level of strand-specificity of our dataset. likewise, the ratios of sense to antisense reads differ significantly between our and the lópez-barragán et al. dataset. the relatively lower level of strand-specificity in lópez-barragán et al. dataset maybe due to the imperfect second-strand cdna digestion in dutp method as the authors mentioned, comparing to the rna ligation method used in this study. lópez-barragán et al. thus avoided false positives in nat identification by applying stringent cutoffs on both proportion of antisense read and antisense read number, which may have underestimated the prevalence of nats. to estimate the prevalence of nats in plasmodium, we established two sets of thresholds with different stringency. our data indicate that between ~24% and ~45% of all coding genes are overlapping with nats. in the following sections, unless mentioned otherwise, we restricted our analyses to the genes with nats defined at stringent thresholds. transcription of nats is pervasive and developmentally regulated numerous mechanisms have been described by which antisense rna can regulate gene expression even in the absence of a functional rnai-machinery. common to these mechanisms is the ability of the respective organism to regulate antisense transcription independently of the complementary sense transcription. previously, the distinct regulation of mrna levels during the idc has been described. the idc of p. falciparum takes 48 h to complete, thus, to evaluate developmental regulation of nats across the idc, we compared patterns of sense and antisense expression in parasites at evenly spaced intervals. to determine whether transcription of nats is developmentally regulated, we searched for changes in nat levels across the idc. using the software package edger, we observed significant changes in antisense transcript levels for of the genes with nats in at least of the non-redundant time point pair comparisons. for genes with nats we observed significant changes in antisense transcript levels in at least of the non-redundant time point pair comparisons. these findings suggest that at least ~10% of nats are regulated during the idc. subsequently, we investigated whether the changes in sense and antisense transcript levels between times points are correlated and observed no global correlation. thus, our data suggest that transcription of nats does not exert a globally positive or negative effect on the transcription of its sense counterpart. to further investigate this observation visually, we sorted the genes based on patterns of antisense expression levels throughout the idc and plotted the antisense expression heat map, and observed a distinct life cycle-specific expression pattern of antisense transcripts. then, we re-plotted the heat map using sense expression levels while keeping the gene order sorted using antisense expression levels. in agreement with the above observation, no apparent pattern was observed. it should be noted that sorting the genes based on sense transcript levels confirmed the previously observed regulation of sense transcript levels. thus, both sense and antisense transcript levels are regulated during the idc but regulation is not synchronous. an example of developmentally regulated antisense transcript levels is shown in figure  3b.figure antisense transcription is abundant and developmentally regulated. a) heat map of genes containing sense and antisense transcripts throughout the idc sorted based on antisense rna trends and sense rna trends. b) coverage plot for a gene containing developmentally regulated nats. yellow box represents cds and green arrow indicates direction of transcription. transcription of nats is biased towards the 3′-end of genes to further characterize the newly identified nats, we mapped antisense transcript levels across the cdss of all genes and observed a striking enrichment of antisense transcripts at the 3′-ends of genes. it has been suggested that nats could be a consequence of run-through transcription or transcription initiation of bi-directional promoters from adjacent genes. to determine if p. falciparum contains nats whose expression is regulated independently from that of adjacent genes, we investigated the correlation between the changes in mrna levels of the downstream genes and the changes in antisense transcript levels of the corresponding upstream genes during the idc. we reasoned that, should antisense transcription be a consequence of active transcription of the downstream gene, changes in antisense transcript level across the idc should be positively correlated to those of the downstream mrna. to this end, we performed non-redundant time-point pair comparisons for each gene and observed a positive correlation between the change of antisense transcript levels of one gene and that of mrna levels of the gene located downstream. the observed correlation was independent of the orientation of the downstream gene, suggesting that both run-through transcription and bi-directional promoter activity contribute to the observed antisense transcription in plasmodium. however, while we observed a positive correlation for of genes with overlapping nats, for genes we observed a negative correlation and for genes no clear correlation. for genes we did not have enough data points or no gene was located downstream. among the nats whose transcription did not correlate with the transcription of adjacent genes, i.e. nats that appear to be independently regulated, we found ~13% to be developmentally regulated. taken together, these findings indicate that while run-through transcription and bi-directional promoter activity are likely to contribute to the observed antisense transcription, a significant number of nats seems to be regulated independently of mrna transcription.figure antisense transcription is strongly enriched at the 3′-end of cdss. a) averaged coverage of antisense and sense rna levels for all annotated genes. b) sense and antisense coverage for a representative gene. green arrows indicate direction of transcription and yellow boxes represent cdss. the grey triangles mark the site of a putative bi-directional promoter. c) coverage plot for a representative gene likely to be affected by run-through transcription from a neighboring gene. green arrows indicate direction of transcription and yellow boxes represent cdss. correlation of nats with orientation of downstream genes. figure summarizing the correlation of nat levels of a reference gene with the mrna levels of the gene located downstream. nat levels often correlate with expression of downstream genes. scatter plots showing fold-changes of anti-sense rpkm of a reference gene versus fold-changes of sense rpkm for the gene located downstream of the reference gene located on the same strand or opposite strand. time-point comparisons are plotted if the reference gene and downstream gene had significant anti-sense rpkms and sense rpkms for at least two time points. up to six non-redundant time-point comparisons are plotted per gene. nats do not accumulate in the nucleus or correlate with mrna levels to understand the role of antisense transcription in plasmodium, we sought to determine whether our data provide evidence to support the known mechanisms for antisense-mediated regulation of gene expression. collisions between rna polymerases transcribing the sense and antisense strands have been described to interfere with the transcription of mrnas as shown in escherichia coli and s. cerevisiae. if antisense transcription has a negative impact on sense transcription in plasmodium, an increase in antisense levels from one time point to the next should correlate with a decrease in sense levels and vice versa. however, as mentioned above, comparison of changes in sense and antisense levels during four time-points showed a correlation close to. alternatively, duplex formation between sense and antisense rna molecules can result in nuclear retention of antisense transcripts and modulate gene expression. if antisense transcripts played a role in retaining sense transcripts in the nucleus, an enrichment of antisense transcripts in the nucleus would be observed. several studies have reported nuclear localization of ncrna in p. falciparum. thus, even though polyadenylated nats are not commonly enriched in the nucleus, and our sequencing libraries were enriched for polyadenylated rna, we examined the possible nuclear enrichment of antisense reads in genes that showed significant levels of antisense transcription but are not likely to be affected by run-through transcription from neighboring genes. we did not observe a significant enrichment of antisense reads in these genes in the nucleus against both the total and the cytosolic fractions at 20 h p.i., while ncrna from telomere-associated repeated elements were enriched in the nuclear fraction. a final mechanism we considered was antisense-mediated translational inhibition in which case overexpression of antisense transcripts leads to a reduction of proteins but not to reduction of sense rna levels. to this end, we correlated our rna-seq data with previously published stage-specific proteomic datasets. as observed by le roch et al., we saw a positive correlation between mrna abundance ) and protein abundance, thus an increase in sense rpkm correlates with an increase in peptide counts per kilobase for a given gene. to evaluate the global effect of antisense transcripts on translation, we investigated the correlation between antisense transcript levels and translation efficiency. however, for all four time points the correlation was close to, suggesting that the level of antisense transcription has no global effect on translation efficiency. mapping of sense and antisense polyadenylation sites the sequencing depth of our dataset combined with the use of a polymerase better suited for the amplification of extremely at-rich regions than previously used enzymes, allowed us to map for the first time polyadenylation sites for p. falciparum. despite the importance of 3′-utrs in the regulation of gene expression, the genome-wide characterization of 3′-utrs in plasmodium lags far behind that of coding sequences. to determine pass and to address the question of whether the observed antisense transcripts are polyadenylated, we used the combined rna-seq data from all libraries to map pass on a genome-wide scale. due to the high abundance of coding mrnas compared to antisense rna, we expected most of the identified pass to correspond to coding mrnas and only a small fraction to correspond to nats. to map genuine pass and to avoid false positive assignments derived from internal polya stretches or sequence reads of low quality, we followed a set of previously published criteria. to assess whether the identified pass could represent genuine pass, we plotted the occurrence of pass around stop codons of all genes. most of the identified pass fell within nt downstream of the stop codon of genes, corresponding to the expected sites of polyadenylation and therefore validating our approach. as microheterogeneity in pas selection is well documented, we grouped individual pass located in close proximity to each other into so-called ‘pas-clusters’ with a maximum width of nt. setting the maximum 3′-utr length to nt, we were able to assign pas-clusters to coding mrnas, yielding an average 3′-utr length of nt, for an example see figure  7b. genome-wide we observed that % genes contained multiple pas-clusters compared to % genes with multiple pass found in s. cerevisiae.figure mapping of polyadenylation sites. a) distribution of mapped sense pass, distance relative to the corresponding stop codon. b) distribution of sequence reads and pass mapping to the upper and lower strand. green arrows indicate direction of sense transcription for the corresponding cds and yellow boxes mark cdss. c) distribution of antisense sequence reads and antisense pass mapping to the upper strand. the green arrow indicates the direction of sense transcription for the corresponding cds and the yellow box marks the cds. previously, some nats in plasmodium were shown to be transcribed by rna polymerase ii and it was suggested that nat stability may be regulated at the level of polyadenylation but no polyadenylation of nats has been described for p. falciparum. thus, we decided to search for pass of nats, i.e. antisense pas-clusters. antisense pas-clusters were defined as those that 1) are located on the antisense strand within the coding region of an annotated gene, 2) contain at least two pass within the cluster, 3) are located at least nt upstream of an annotated stop codon. because most of the antisense transcripts exist in low abundance, the likelihood of identifying their pass are much lower than for coding mrnas. nonetheless, based on the above criteria, we identified antisense pas-clusters for genes. these data indicate that at least some of the observed nats are polyadenylated. evidence of antisense spliced junctions based on the splicing junctions identified from our datasets using hmmsplicer, we found evidence for antisense spliced junctions in of the genes with nats. thus, at least a portion of the observed nats is spliced. furthermore, we found of the antisense junction clusters to overlap with sense junctions on mrna. these findings are consistent with a previous study, which has identified antisense introns and antisense junctions overlapping with sense junctions, using a similar approach. these observations further validate antisense transcription in p. falciparum to be a widespread phenomenon. discussion to perform a comprehensive strand-specific analysis of the p. falciparum transcriptome, we combined several recent improvements in rna-seq technology. specifically, we took advantage of improvements made in the preparation of highly strand-specific sequencing libraries, we used a polymerase that allows amplification of highly at-rich dna sequences and increased the number and length of sequence reads obtained from high-throughput sequencers. we generated a total of strand-specific transcriptome profiles for parasites across the idc as well as for separately purified nuclear and cytosolic rna fractions. nine of the rna-seq libraries were prepared from polya-enriched rna because we found these libraries yielded a higher coverage and a more even distribution of sequence reads across the cdss than libraries prepared from tex-treated rna. while polya-enrichment harbors the intrinsic problem of selecting against non-polyadenylated transcripts, for the following reasons we believe that our libraries contain at least some non-polyadenylated transcripts: 1) polya-enrichment is generally not 100% efficient and indeed, trna and rrna sequencing reads were present in our libraries. 2) the plasmodium genome is extremely at-rich and long stretches of as exist even within genes. thus it is likely that polya-enrichment can also enrich for non-polyadenylated transcripts as long as they have internal stretches of as. nevertheless, most likely our dataset lacks weakly transcribed rna without polya-tails. our analysis revealed transcription from almost 80% of the plasmodium genome and exposed antisense transcription for 24% of genes many of which are developmentally regulated. interestingly, we find that antisense transcripts are not uniformly distributed but strongly enriched at the 3′-ends of cdss. such an enrichment of antisense transcripts at the 3′-end of cdss has been detected in both yeast and humans and it has been suggested that they can arise via pathways that copy mrna, from run-through transcription, bi-directional transcription initiation or promiscuous transcription initiation from nucleosome depleted regions. however, the biological function of these 3′-antisense transcripts remains unknown. our analysis indicates that in plasmodium, for the majority of genes, antisense levels do not correlate with sense transcript levels, which would have hinted at an rna-copying mechanism or a role of ndrs. in contrast to the corresponding sense mrna levels, for a large number of genes changes in nats levels correlate with changes in mrna levels of the gene located downstream. this correlation may be due to an overlap of different utrs or of utrs with cdss. such an overlap of utrs raises the intriguing possibility that transcription of one gene may influence transcription of its neighboring gene and that the genomic location of a gene may be important for its correct regulation. for example, a gene knockout followed by expression of the same gene from a different genomic locus may lead to different antisense transcript levels compared to those from the endogenous locus. in addition, replacement of an endogenous gene with a resistance marker will not only lead to a loss of the mrna of the endogenous gene but also to a loss of the corresponding antisense rna, possibly leading to confusing consequences and complicating the interpretation of the results. while our analysis revealed a positive correlation between antisense and sense transcript levels of neighboring genes, such a correlation could not be identified for all genes. thus, for a significant number of genes our data point to the existence of cryptic promoters at the 3′-end of genes that initiate transcription of nats independent of the promoter that controls transcription of sense rna. reports on the role of antisense rna transcripts in p. falciparum have been conflicting, some studies found increased antisense levels to repress gene expression, while others saw no effect. correlating our rna-seq data with previously published proteomics data we find a general correlation between mrna levels and peptide levels, however, we observed no consistent effect of antisense transcripts on protein levels. thus it remains to be seen if antisense transcripts modulate gene expression in this parasite. 3′-utrs and polya tails have important roles in mrna localization, stability and regulation of translation. such regulation can be mediated by cis-acting sequence elements that interact with rna-binding proteins. in plasmodium little is known about how transcription initiation is regulated but numerous studies have reported post-transcriptional control, particularly at the level of translational repression. specific sequence motifs within the 3′-utr have been correlated with tr but genome-wide searches for regulatory motifs have not been performed for a lack of information on 3′ utrs’ length. in this study we present the genome-wide assignment of pass in plasmodium. we were able to map pass for 65% of genes, which we believe will represent a valuable resource to the community. for example, the newly generated data should allow other researchers to perform systematic genome-wide searches for sequence motifs located in utrs that may help elucidate some of the secrets of post-transcriptional gene regulation in the blood stages of p. falciparum. besides the mapping of mrna pass we investigated the presence of antisense pas-clusters. the latter analysis was much more challenging than the mapping of sense pass but our data indicate that at least some of the observed antisense transcripts are polyadenylated. this observation further strengthens the assumption that nats are generated by transcription of dna and not via transcription of mrna. this finding is not unexpected given previously published findings that antisense transcription in plasmodium can be carried out by rna polymerase ii and that termination of rna pol ii transcription and polyadenylation are commonly coupled. future deep sequencing projects with libraries derived from rna fragments enriched for the 3′-end of cdss should help determine if all nats are polyadenylated or just represent a small subset.  intergenic and non-coding regions of the p. falciparum genome have been particularly understudied due to high at-content, typically ranging between and 95%. using a protocol that permits the generation of highly strand-specific rna-seq libraries and kapa hifi dna polymerase, a polymerase that amplifies even extremely at-rich dna, we generated a comprehensive transcriptome dataset for p. falciparum, containing great detail about the origin of antisense rna. given the large number of independently regulated nats, our work suggests that ncrna in p. falciparum is of biological significance and not merely a consequence of noisy transcriptional regulation. importantly, our high coverage and long sequence reads allow us to provide for the first time an extensive list of polyadenylation sites for blood stage parasites. this data should enable others to perform comprehensive searches for regulatory motifs in utrs and help to understand post-transcriptional regulation of gene expression in p. falciparum. 
advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. directional rna-seqplasmodium falciparumncrnaantisense rnanatural antisense transcriptsgenes3′ utrpolyadenylation sitesissue-copyright-statement© the author 2014 finally, we were able to identify the polyadenylation sites for a subset of nats, demonstrating that at least some nats are polyadenylated. moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of nats change during the intraerythrocytic developmental cycle, they do not correlate with the corresponding mrna levels. interestingly, antisense transcription is not evenly distributed across coding regions but strongly clustered towards the 3′-end of cdss. our strand-specific analysis of the p. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in p. falciparum. furthermore, for a significant subset of nats, transcript levels correlate with mrna levels of neighboring genes. to gain insights into the genome-wide distribution of nats in p. falciparum, we performed rna-ligation based strand-specific rna sequencing at unprecedented depth. in addition, the extensive novel data on 3′ utr length will allow others to perform searches for regulatory motifs in the utrs and help understand post-translational regulation in p. falciparum. likewise, the existence of nats has been observed in plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets. for a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other nats appear to be regulated independently of mrna transcription. our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ.
 advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. likewise, the existence of nats has been observed in plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets.  to gain insights into the genome-wide distribution of nats in p. falciparum, we performed rna-ligation based strand-specific rna sequencing at unprecedented depth. our data indicate that % of the genome is transcribed during blood-stage development. moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of nats change during the intraerythrocytic developmental cycle, they do not correlate with the corresponding mrna levels. interestingly, antisense transcription is not evenly distributed across coding regions but strongly clustered towards the 3′-end of cdss. furthermore, for a significant subset of nats, transcript levels correlate with mrna levels of neighboring genes. finally, we were able to identify the polyadenylation sites for a subset of nats, demonstrating that at least some nats are polyadenylated. we also mapped the pass of coding genes, yielding an average 3′ untranslated region length of 523 bp.  our strand-specific analysis of the p. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in p. falciparum. for a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other nats appear to be regulated independently of mrna transcription. our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. in addition, the extensive novel data on 3′ utr length will allow others to perform searches for regulatory motifs in the utrs and help understand post-translational regulation in p. falciparum. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords directional rna-seqplasmodium falciparumncrnaantisense rnanatural antisense transcriptsgenes3′ utrpolyadenylation sitesissue-copyright-statement© the author 2014 