BACKGROUND
histoplasma capsulatum is a dimorphic fungal pathogen that is thought to infect up to  <dig>  individuals per year in the u.s <cit> .
notably, h. capsulatum is a primary pathogen that causes significant morbidity in immunocompetent hosts <cit> .
normally found in a filamentous mycelial form in the soil of endemic regions, h. capsulatum converts to the pathogenic yeast form in the lungs of the host after inhalation of infectious particles .
in the laboratory, temperature is a sufficient signal to specify growth in either the mycelial form  or growth in the yeast form, which can be achieved by incubating cells at 37°c.
once introduced into the host, h. capsulatum colonizes host immune cells.
understanding both how h. capsulatum switches its growth program in response to temperature and how this pathogen subverts the innate immune system are major areas of inquiry.
the elucidation of h. capsulatum pathogenesis and biology has been greatly aided by the genome sequencing of h. capsulatum strains g217b and g186ar at the genome sequencing center  at washington university in st. louis and strains g186ar, wu <dig>  h <dig>  and h <dig> at the broad institute.
these sequenced genomes open up a wealth of possibilities for the h. capsulatum community, enabling or abetting tools such as expression arrays, insertional mutagenesis, and bioinformatic analysis.
however, these approaches are limited by the gene annotations associated with the genome assemblies.
this limitation is pronounced in h. capsulatum given this eukaryote's sparse gene structure and a limited set of known transcripts with which to train gene prediction algorithms.
accordingly, although the gsc used a variety of tools to generate a set of predicted genes for g217b and g186ar http://genome.wustl.edu/genomes/view/histoplasma_capsulatum/, these predictions are based on limited experimental data.
in other systems where the gene finding problem has presented itself, whole genome tiling has proven a reliable technique for direct observation of the transcriptome <cit> .
to this end, we generated a set of tiling microarrays spanning the non-repetitive regions of the g217b genome and hybridized these arrays with a pool of cdna derived from yeast-form histoplasma growing under a diverse set of conditions.
the resultant data give an unbiased measure of expression level as a function of genome position, and thus identify the locations and boundaries of expressed genes.
the results of this study are available, along with tools for interactive exploration of the data, at http://histo.ucsf.edu.
RESULTS
whole-genome tiling array expression profiling
to survey the transcriptome of g217b, we designed a set of  <dig> unique tiling microarrays .
the g217b genome contains a large number of repeat regions, including the maggy retrotransposon <cit> , which were excluded from the tiling microarray probes.
both strands of the remaining sequence were tiled with  <dig> mer probes at an average frequency of one probe every  <dig> base pairs .
these arrays were hybridized with a pool of fluorescently labeled cdna generated from cells grown under a variety of conditions.
because technical limitations did not allow us to isolate sufficient poly-adenylated-rna from filamentous cells , we focused on the pathogenic yeast form.
g217b yeast cells were subjected to numerous growth conditions  which had previously been observed to elicit potent transcriptional responses <cit> .
tiles that passed an empirically determined detection threshold were merged into tars, as described in the materials and methods.
detection of predicted genes
the gsc predicted that the g217b genome contains  <dig>  genes, but  <dig>  of these gene predictions contain repeat sequence, including the maggy transposon, and were excluded from further analysis.
of the remaining  <dig>  predictions,  <dig>  were detected in our tiling microarrays .
60% of the gene predictions have some correspondence to the detected tars: 47% of the predictions were cleanly detected only on the predicted strand , 7% were detected only on the antisense strand , and 6% had tiling and/or prediction support for transcription on both strands , leaving 26% of the predicted set unsupported by our tiling data .
detection on both strands is consistent with the presence of sense and/or antisense transcripts in one or more of the growth conditions profiled by this experiment.
it has been shown that the dna-dependent dna polymerase activity of reverse transcriptase can generate false positive opposite strand signal in tiling experiments; e.g., two thirds of putative antisense transcripts in a saccharomyces cerevisiae tiling experiment were not detected in the presence of actinomycin d <cit> .
therefore, the number of sense/antisense pairs observed in our experiment is likely to be an overestimate.
detection on only the antisense strand may correspond to incorrect predictions coinciding with bona fide transcripts on the opposite strand  or to true genes that are repressed by an antisense transcript in our pooled yeast sample.
due to this ambiguity, genes in this category were not considered "detected".
an additional  <dig> novel transcripts, which were not present in the predicted set, were also detected , as described below.
as part of the web database associated with this study, the detected transcript set can be viewed in the context of the raw tiling signal and predicted gene set , allowing human estimation of transcript set accuracy on a case by case basis.
features of transcribed regions in the h. capsulatum genome
as is common for tiling data, the boundaries of tars did not correspond precisely with the boundaries of the predicted genes.
there were two common instances of this pattern.
first, in many cases, additional transcription was detected 5' and 3' of the predicted gene .
this was most likely due to untranslated  sequences which are missed by the gene model and resulted in a longer length distribution for the tars compared to the predicted genes .
second, it was not uncommon for a single long transcript to span multiple predictions.
in some cases, this was due to the sequence encoding a single tar being incorrectly predicted to contain multiple genes.
in others, this was due to multiple genes being incorrectly detected as a single transcript, either due to spurious or pathological background signal or due to intergenic regions too small to be distinguished from introns.
in the case of the saccharomyces cerevisiae genome, multi-gene detected transcripts could be segmented based on sharp transitions in the intensity of the tiling signal <cit> .
such analysis would be difficult in the present study, primarily because the tiling sample is a pool of cdnas corresponding to multiple transcriptional states of the h. capsulatum yeast phase, each of which may contain transcript isoforms that differ by splicing and transcriptional start site .
ultimately, we attempted to minimize this limitation of the tiling array method by selecting transcript detection parameters that distinguish the mostly small introns from the mostly large intergenic regions.
the majority of tars that did not overlap with gene predictions corresponded to unpredicted utr sequences.
for example, 29% of non-overlapping tar sequence can be interpreted as 5'utr , and 35% as 3'utr .
additionally, 33% of non-overlapping tars corresponded to the intervening sequence between two predictions .
tiling arrays revealed  <dig> novel genes
one advantage of a tiling strategy is that it can uncover novel tars that do not correspond to the predicted genes.
our tiling analysis detected  <dig> such loci that were not represented in the gsc predicted gene set for g217b .
tars were designated as "novel" if  they had no overlap with gene predictions on either strand,  they did not fall into the "5'utr", "3'utr", or "intervening" classifications described above , and  they had no overlap with repeat regions.
<dig> tars that did not coincide with the predicted gene set were chosen for experimental validation by rt-pcr.
<dig> of these tars were detected in a first-pass analysis with a single primer pair, giving a validation rate of 84%.
a representative sampling of rt-pcr results is shown in figure  <dig>
to determine whether the novel loci correspond to conserved sequences in other genomes and, if so, if these homologous loci have been independently annotated as transcribed , we searched for conserved sequences in other dimorphic fungal pathogens within the order onygenales .
a blastx search of the isolated novel sequences against the predicted protein sets yielded a number of hits in other genomes .
to increase the sensitivity of this search, we performed an inparanoid-based <cit>  mapping of syntenic loci that flanked each novel locus .
genes in  <dig> kb windows on either side of the novel tar could be independently mapped to orthologs on a common contig in at least  <dig> other genomes for  <dig> of the isolated novel sequences.
of the  <dig> isolated novel sequences with blastx hits,  <dig> could be mapped to syntenic loci, and, for  <dig> of these, the blastx hit coincided with the mapped locus.
a tblastx  comparison of the isolated novel sequence to the mapped locus yielded a significant alignment  for at least  <dig> h. capsulatum strains in  <dig> cases, for both b. dermatitidis strains in  <dig> cases, for at least two p. brasiliensis strains in  <dig> cases, and for the reference c. immitis strain in  <dig> cases.
in general, the tblastx results were consistent with evolutionary distance from g217b .
taken together, these results suggest that: 1) the isolated novel sequences are conserved at the sequence level, and, therefore, likely to be transcribed, relative to the other h. capsulatum strains in most cases, and relative to b. dermatitidis for about half of the cases; 2) transcripts with deeply conserved sequence across the onygenales also tend to be predicted as genes in most of these fungi; and 3) for about half of the isolated novel sequences, a corresponding gene prediction exists in another genome, highlighting differences in the prediction pipelines, while the other half represent truly novel discoveries of this tiling experiment.
using standard expression profiling and sequence homology to enrich gene validation
to complement our tiling arrays, we took advantage of our archive of expression data compiled across several distinct growth conditions, including iron limitation, and all three morphologies .
we surveyed whether gene predictions were detected in these expression profiling experiments, which employed whole-genome oligonucleotide microarrays where each prediction was represented by one or two gene-optimized  <dig> mer probes.
additionally, we used inparanoid <cit>  to determine if gene predictions had homologs in other fungi.
this validation by inferred homology to genes in other fungi relied on sequence conservation independent of expression pattern.
the validation criteria for each strategy are given in the methods section and the results are summarized in figure  <dig> .
by these criteria,  <dig>  non-repeat predicted proteins were validated by gene expression and  <dig>  were validated by sequence homology.
genes that were validated by tiling, gene expression, and sequence homology represented the largest category of predictions  and accounted for 56% of the non-repeat predicted gene set.
the next largest category was  <dig>  genes validated by gene-expression and sequence conservation but not by the tiling experiment , followed by  <dig> genes  validated only by expression array, and  <dig> genes  validated by expression and tiling but not sequence conservation.
<dig>  gene predictions  were unvalidated by any of the three methods.
in the following discussion, predicted genes are referred to by their common names.
additional file  <dig>  table s <dig> gives the corresponding systematic names.
genes that were missed by tiling array showed enriched expression in the mycelial form
as expected, gene predictions that were not detected by tiling tended to show reduced expression in the yeast phase and enhanced expression in the mycelial form.
examples include tyr <dig> and abc <dig>  both previously identified as highly enriched in the mycelial phase  <cit> ; velc, a mycelial-enriched paralog of the morphological regulators ryp <dig> and ryp <dig>  <cit> ; and the ortholog of bdbg_ <dig>  which is paralogous to the b. dermatitidis gene bys <dig>  <cit> .
other notable categories of genes not detected by tiling include genes in heavily repeat-masked regions of the genome  and genes with weak expression that did not give significant signal over background on tiling arrays.
genes that were not detected by homology represented short or interrupted predictions
for genes not detected by homology, there were two related trends:  the predicted lengths were short, on the order of those genes not detected by any method ; and/or  a single tar was inappropriately split into multiple predicted genes.
for example, the copper-repressed gene eli <dig>  which is known to be expressed as a single mrna <cit> , is split into two predictions.
both predictions are detected by expression and tiling, but only the 3' prediction, which contains the coding sequence, is detected by homology, whereas the 5' prediction, which likely contains 5'utr, is not.
short predictions are difficult to detect as homologs for two reasons: short runs of sequence similarity are likely to occur by chance, resulting in lower blastp p-values for hits to these predictions; and inparanoid requires 50% reciprocal coverage between orthologs, resulting in rejection of genes where the predicted length is significantly smaller than that of the corresponding homologs.
the same issues arise for split predictions, with the additional restriction that inparanoid will make an ortholog assignment for only one member of a split pair, automatically resulting in rejection of the other member.
in all of these cases, the discrepancy between the experimental and sequence based results is a useful indication that the predicted gene model should be revised.
in many cases, comparison of the transcript detected by tiling array to the results of less stringent sequence searches  is a useful starting point for such revision.
genes not detected by homology also tend to show enriched expression in conidia, the vegetative spores generated by h. capsulatum filaments.
h. capsulatum conidia, or their counterparts in any closely related fungi, have not been extensively studied; thus, the homologs of these genes may be unpredicted or entirely absent in the comparison genomes.
genes that were validated only by homology have restricted expression profiles
the category of genes with orthologs in other fungi but no direct observation in our experimental data was relatively small  and is predicted to contain genes that are expressed only under very restricted conditions that were not sampled in our expression data.
consistent with this hypothesis, we find ste <dig>  the a-factor receptor whose expression has been observed only in mutants of g217b <cit> ; the ortholog of n. crassa rid, which is required for the rip process and therefore expected to be expressed only during meiosis <cit> ; and the ortholog of t. reesei axe <dig>  a hemicellulolytic enzyme whose expression is dependent on carbon source <cit> .
empirical redesign of microarray probes
our tiling arrays and homology predictions can be used to inform future design of microarray probes.
because the expression experiments draw from a more diverse set of samples than the tiling experiments, detection of a predicted gene by homology and tiling but not by expression suggested a platform-specific defect in the  <dig> mer probe designed to detect that gene on our whole-genome oligonucleotide arrays .
our analyses support this hypothesis.
in particular, the 70-mer probes for genes that failed to be detected by expression array tend to lie outside of the transcribed locus detected by tiling , or span a predicted intron not supported by the tiling data .
we are currently augmenting the expression array platform with new  <dig> mers for these genes, based on the coincidence of tiling transcripts with predicted exons.
genes that failed to be validated by any method
we were unable to validate  <dig>  predictions, or 11% of the non-redundant genes, by any method.
this group primarily corresponds to wholly undetected predictions but may also include a small number of correct predictions for which the 5' end is undetected due to the 3' bias of the tiling experiment.
the unvalidated genes are significantly shorter than the detected genes .
this observation could be due to false negatives in the tiling data  or false gene predictions .
we note that genes validated only by expression  are significantly shorter than genes validated by all methods but significantly longer than the unvalidated genes, lending weight to both explanations.
CONCLUSIONS
we probed the transcriptome of h. capsulatum with a large set of tiling arrays, and combined the results with gene-targeted expression profiling and sequence homology, yielding a high confidence set of validated gene predictions for g217b with  <dig>  gene predictions being validated by at least two of the three methods.
in addition, the unbiased approach of the tiling arrays allowed us to detect  <dig> novel transcripts that are now being incorporated into our oligo expression arrays, directly extending the sensitivity of that platform.
additionally, the results of this study are available at http://histo.ucsf.edu in an interactive format intended to facilitate expression, insertional mutagenesis, and bioinformatics based studies.
thus, the transcript sets resulting from this study represent an enhancement of the previously available h. capsulatum gene set and a starting point for the experimental and theoretical characterization of the molecular biology of this important intracellular pathogen.

 histoplasma capsulatum is a dimorphic fungal pathogen that is thought to infect up to individuals per year in the u.s. notably, h. capsulatum is a primary pathogen that causes significant morbidity in immunocompetent hosts. normally found in a filamentous mycelial form in the soil of endemic regions, h. capsulatum converts to the pathogenic yeast form in the lungs of the host after inhalation of infectious particles. in the laboratory, temperature is a sufficient signal to specify growth in either the mycelial form or growth in the yeast form, which can be achieved by incubating cells at 37°c. once introduced into the host, h. capsulatum colonizes host immune cells. understanding both how h. capsulatum switches its growth program in response to temperature and how this pathogen subverts the innate immune system are major areas of inquiry. the elucidation of h. capsulatum pathogenesis and biology has been greatly aided by the genome sequencing of h. capsulatum strains g217b and g186ar at the genome sequencing center at washington university in st. louis and strains g186ar, wu h and h at the broad institute. these sequenced genomes open up a wealth of possibilities for the h. capsulatum community, enabling or abetting tools such as expression arrays, insertional mutagenesis, and bioinformatic analysis. however, these approaches are limited by the gene annotations associated with the genome assemblies. this limitation is pronounced in h. capsulatum given this eukaryote's sparse gene structure and a limited set of known transcripts with which to train gene prediction algorithms. accordingly, although the gsc used a variety of tools to generate a set of predicted genes for g217b and g186ar http://genome.wustl.edu/genomes/view/histoplasma_capsulatum/, these predictions are based on limited experimental data. in other systems where the gene finding problem has presented itself, whole genome tiling has proven a reliable technique for direct observation of the transcriptome. to this end, we generated a set of tiling microarrays spanning the non-repetitive regions of the g217b genome and hybridized these arrays with a pool of cdna derived from yeast-form histoplasma growing under a diverse set of conditions. the resultant data give an unbiased measure of expression level as a function of genome position, and thus identify the locations and boundaries of expressed genes. the results of this study are available, along with tools for interactive exploration of the data, at http://histo.ucsf.edu.  whole-genome tiling array expression profiling to survey the transcriptome of g217b, we designed a set of unique tiling microarrays. the g217b genome contains a large number of repeat regions, including the maggy retrotransposon, which were excluded from the tiling microarray probes. both strands of the remaining sequence were tiled with mer probes at an average frequency of one probe every base pairs. these arrays were hybridized with a pool of fluorescently labeled cdna generated from cells grown under a variety of conditions. because technical limitations did not allow us to isolate sufficient poly-adenylated-rna from filamentous cells, we focused on the pathogenic yeast form. g217b yeast cells were subjected to numerous growth conditions which had previously been observed to elicit potent transcriptional responses. tiles that passed an empirically determined detection threshold were merged into tars, as described in the materials and methods. detection of predicted genes the gsc predicted that the g217b genome contains genes, but of these gene predictions contain repeat sequence, including the maggy transposon, and were excluded from further analysis. of the remaining predictions, were detected in our tiling microarrays. 60% of the gene predictions have some correspondence to the detected tars: 47% of the predictions were cleanly detected only on the predicted strand, 7% were detected only on the antisense strand, and 6% had tiling and/or prediction support for transcription on both strands, leaving 26% of the predicted set unsupported by our tiling data. detection on both strands is consistent with the presence of sense and/or antisense transcripts in one or more of the growth conditions profiled by this experiment. it has been shown that the dna-dependent dna polymerase activity of reverse transcriptase can generate false positive opposite strand signal in tiling experiments; e.g., two thirds of putative antisense transcripts in a saccharomyces cerevisiae tiling experiment were not detected in the presence of actinomycin d. therefore, the number of sense/antisense pairs observed in our experiment is likely to be an overestimate. detection on only the antisense strand may correspond to incorrect predictions coinciding with bona fide transcripts on the opposite strand or to true genes that are repressed by an antisense transcript in our pooled yeast sample. due to this ambiguity, genes in this category were not considered "detected". an additional novel transcripts, which were not present in the predicted set, were also detected, as described below. as part of the web database associated with this study, the detected transcript set can be viewed in the context of the raw tiling signal and predicted gene set, allowing human estimation of transcript set accuracy on a case by case basis. features of transcribed regions in the h. capsulatum genome as is common for tiling data, the boundaries of tars did not correspond precisely with the boundaries of the predicted genes. there were two common instances of this pattern. first, in many cases, additional transcription was detected 5' and 3' of the predicted gene. this was most likely due to untranslated sequences which are missed by the gene model and resulted in a longer length distribution for the tars compared to the predicted genes. second, it was not uncommon for a single long transcript to span multiple predictions. in some cases, this was due to the sequence encoding a single tar being incorrectly predicted to contain multiple genes. in others, this was due to multiple genes being incorrectly detected as a single transcript, either due to spurious or pathological background signal or due to intergenic regions too small to be distinguished from introns. in the case of the saccharomyces cerevisiae genome, multi-gene detected transcripts could be segmented based on sharp transitions in the intensity of the tiling signal. such analysis would be difficult in the present study, primarily because the tiling sample is a pool of cdnas corresponding to multiple transcriptional states of the h. capsulatum yeast phase, each of which may contain transcript isoforms that differ by splicing and transcriptional start site. ultimately, we attempted to minimize this limitation of the tiling array method by selecting transcript detection parameters that distinguish the mostly small introns from the mostly large intergenic regions. the majority of tars that did not overlap with gene predictions corresponded to unpredicted utr sequences. for example, 29% of non-overlapping tar sequence can be interpreted as 5'utr, and 35% as 3'utr. additionally, 33% of non-overlapping tars corresponded to the intervening sequence between two predictions. tiling arrays revealed novel genes one advantage of a tiling strategy is that it can uncover novel tars that do not correspond to the predicted genes. our tiling analysis detected such loci that were not represented in the gsc predicted gene set for g217b. tars were designated as "novel" if they had no overlap with gene predictions on either strand, they did not fall into the "5'utr", "3'utr", or "intervening" classifications described above, and they had no overlap with repeat regions. tars that did not coincide with the predicted gene set were chosen for experimental validation by rt-pcr. of these tars were detected in a first-pass analysis with a single primer pair, giving a validation rate of 84%. a representative sampling of rt-pcr results is shown in figure to determine whether the novel loci correspond to conserved sequences in other genomes and, if so, if these homologous loci have been independently annotated as transcribed, we searched for conserved sequences in other dimorphic fungal pathogens within the order onygenales. a blastx search of the isolated novel sequences against the predicted protein sets yielded a number of hits in other genomes. to increase the sensitivity of this search, we performed an inparanoid-based mapping of syntenic loci that flanked each novel locus. genes in kb windows on either side of the novel tar could be independently mapped to orthologs on a common contig in at least other genomes for of the isolated novel sequences. of the isolated novel sequences with blastx hits, could be mapped to syntenic loci, and, for of these, the blastx hit coincided with the mapped locus. a tblastx comparison of the isolated novel sequence to the mapped locus yielded a significant alignment for at least h. capsulatum strains in cases, for both b. dermatitidis strains in cases, for at least two p. brasiliensis strains in cases, and for the reference c. immitis strain in cases. in general, the tblastx results were consistent with evolutionary distance from g217b. taken together, these results suggest that: 1) the isolated novel sequences are conserved at the sequence level, and, therefore, likely to be transcribed, relative to the other h. capsulatum strains in most cases, and relative to b. dermatitidis for about half of the cases; 2) transcripts with deeply conserved sequence across the onygenales also tend to be predicted as genes in most of these fungi; and 3) for about half of the isolated novel sequences, a corresponding gene prediction exists in another genome, highlighting differences in the prediction pipelines, while the other half represent truly novel discoveries of this tiling experiment. using standard expression profiling and sequence homology to enrich gene validation to complement our tiling arrays, we took advantage of our archive of expression data compiled across several distinct growth conditions, including iron limitation, and all three morphologies. we surveyed whether gene predictions were detected in these expression profiling experiments, which employed whole-genome oligonucleotide microarrays where each prediction was represented by one or two gene-optimized mer probes. additionally, we used inparanoid to determine if gene predictions had homologs in other fungi. this validation by inferred homology to genes in other fungi relied on sequence conservation independent of expression pattern. the validation criteria for each strategy are given in the methods section and the results are summarized in figure. by these criteria, non-repeat predicted proteins were validated by gene expression and were validated by sequence homology. genes that were validated by tiling, gene expression, and sequence homology represented the largest category of predictions and accounted for 56% of the non-repeat predicted gene set. the next largest category was genes validated by gene-expression and sequence conservation but not by the tiling experiment, followed by genes validated only by expression array, and genes validated by expression and tiling but not sequence conservation. gene predictions were unvalidated by any of the three methods. in the following discussion, predicted genes are referred to by their common names. additional file table s gives the corresponding systematic names. genes that were missed by tiling array showed enriched expression in the mycelial form as expected, gene predictions that were not detected by tiling tended to show reduced expression in the yeast phase and enhanced expression in the mycelial form. examples include tyr and abc both previously identified as highly enriched in the mycelial phase; velc, a mycelial-enriched paralog of the morphological regulators ryp and ryp; and the ortholog of bdbg_ which is paralogous to the b. dermatitidis gene bys. other notable categories of genes not detected by tiling include genes in heavily repeat-masked regions of the genome and genes with weak expression that did not give significant signal over background on tiling arrays. genes that were not detected by homology represented short or interrupted predictions for genes not detected by homology, there were two related trends: the predicted lengths were short, on the order of those genes not detected by any method; and/or a single tar was inappropriately split into multiple predicted genes. for example, the copper-repressed gene eli which is known to be expressed as a single mrna, is split into two predictions. both predictions are detected by expression and tiling, but only the 3' prediction, which contains the coding sequence, is detected by homology, whereas the 5' prediction, which likely contains 5'utr, is not. short predictions are difficult to detect as homologs for two reasons: short runs of sequence similarity are likely to occur by chance, resulting in lower blastp p-values for hits to these predictions; and inparanoid requires 50% reciprocal coverage between orthologs, resulting in rejection of genes where the predicted length is significantly smaller than that of the corresponding homologs. the same issues arise for split predictions, with the additional restriction that inparanoid will make an ortholog assignment for only one member of a split pair, automatically resulting in rejection of the other member. in all of these cases, the discrepancy between the experimental and sequence based results is a useful indication that the predicted gene model should be revised. in many cases, comparison of the transcript detected by tiling array to the results of less stringent sequence searches is a useful starting point for such revision. genes not detected by homology also tend to show enriched expression in conidia, the vegetative spores generated by h. capsulatum filaments. h. capsulatum conidia, or their counterparts in any closely related fungi, have not been extensively studied; thus, the homologs of these genes may be unpredicted or entirely absent in the comparison genomes. genes that were validated only by homology have restricted expression profiles the category of genes with orthologs in other fungi but no direct observation in our experimental data was relatively small and is predicted to contain genes that are expressed only under very restricted conditions that were not sampled in our expression data. consistent with this hypothesis, we find ste the a-factor receptor whose expression has been observed only in mutants of g217b; the ortholog of n. crassa rid, which is required for the rip process and therefore expected to be expressed only during meiosis; and the ortholog of t. reesei axe a hemicellulolytic enzyme whose expression is dependent on carbon source. empirical redesign of microarray probes our tiling arrays and homology predictions can be used to inform future design of microarray probes. because the expression experiments draw from a more diverse set of samples than the tiling experiments, detection of a predicted gene by homology and tiling but not by expression suggested a platform-specific defect in the mer probe designed to detect that gene on our whole-genome oligonucleotide arrays. our analyses support this hypothesis. in particular, the 70-mer probes for genes that failed to be detected by expression array tend to lie outside of the transcribed locus detected by tiling, or span a predicted intron not supported by the tiling data. we are currently augmenting the expression array platform with new mers for these genes, based on the coincidence of tiling transcripts with predicted exons. genes that failed to be validated by any method we were unable to validate predictions, or 11% of the non-redundant genes, by any method. this group primarily corresponds to wholly undetected predictions but may also include a small number of correct predictions for which the 5' end is undetected due to the 3' bias of the tiling experiment. the unvalidated genes are significantly shorter than the detected genes. this observation could be due to false negatives in the tiling data or false gene predictions. we note that genes validated only by expression are significantly shorter than genes validated by all methods but significantly longer than the unvalidated genes, lending weight to both explanations.  we probed the transcriptome of h. capsulatum with a large set of tiling arrays, and combined the results with gene-targeted expression profiling and sequence homology, yielding a high confidence set of validated gene predictions for g217b with gene predictions being validated by at least two of the three methods. in addition, the unbiased approach of the tiling arrays allowed us to detect novel transcripts that are now being incorporated into our oligo expression arrays, directly extending the sensitivity of that platform. additionally, the results of this study are available at http://histo.ucsf.edu in an interactive format intended to facilitate expression, insertional mutagenesis, and bioinformatics based studies. thus, the transcript sets resulting from this study represent an enhancement of the previously available h. capsulatum gene set and a starting point for the experimental and theoretical characterization of the molecular biology of this important intracellular pathogen. 
the data is available for interactive exploration at http://histo.ucsf.edu. to further enrich our analysis, we incorporated expression data from whole-genome oligonucleotide microarrays. a functional interrogation of the histoplasma genome provides critical support for continued investigation into the biology and pathogenesis of h. capsulatum and related fungi. we employed a three-pronged approach to provide a functional annotation for the h. capsulatum g217b strain. these analyses yielded a high confidence set of validated gene predictions for h. capsulatum. these expression data included profiling under growth conditions that were not represented in the tiling experiment, and validated an additional gene predictions. these data defined transcriptionally active regions, providing validation of gene predictions. additionally, we detected transcription of novel genes not present in the original gene predictions. finally, we compared the g217b gene predictions to other available fungal genomes, and observed that an additional gene predictions had an ortholog in a different fungal species, suggesting that they represent genuine coding sequences.
 the fungal pathogen histoplasma capsulatum is thought to be the most common cause of fungal respiratory infections in immunocompetent humans, yet little is known about its biology. here we provide the first genome-wide studies to experimentally validate its genome annotation. a functional interrogation of the histoplasma genome provides critical support for continued investigation into the biology and pathogenesis of h. capsulatum and related fungi.  we employed a three-pronged approach to provide a functional annotation for the h. capsulatum g217b strain. first, we probed high-density tiling arrays with labeled cdnas from cells grown under diverse conditions. these data defined transcriptionally active regions, providing validation of gene predictions. interestingly, 22% of these predictions showed evidence of anti-sense transcription. additionally, we detected transcription of novel genes not present in the original gene predictions. to further enrich our analysis, we incorporated expression data from whole-genome oligonucleotide microarrays. these expression data included profiling under growth conditions that were not represented in the tiling experiment, and validated an additional gene predictions. finally, we compared the g217b gene predictions to other available fungal genomes, and observed that an additional gene predictions had an ortholog in a different fungal species, suggesting that they represent genuine coding sequences.  these analyses yielded a high confidence set of validated gene predictions for h. capsulatum. the transcript sets resulting from this study are a valuable resource for further experimental characterization of this ubiquitous fungal pathogen. the data is available for interactive exploration at http://histo.ucsf.edu. 