BACKGROUND
the complete arabidopsis transcriptome microarray  consortium  <cit>  was created in  <dig> to take advantage of the available arabidopsis genome sequence to enable novel functional genomics approaches.
eight european plant genomics research groups teamed up to produce a comprehensive set of gene-specific sequence tags  originally designed for microarray transcript profiling.
these gsts were 150– <dig> base pairs in length and were selected to have no significant similarity with any other sequence in the genome  <cit> .
the specific primer and amplicon design software  was written to automate the design of these tags  <cit> .
the resulting gst amplicons can be used as features on spotted microarrays for transcript profiling experiments.
indeed, such catma arrays performed as well as, if not better than, the affymetrix  and agilent  platforms in terms of specificity, sensitivity and gene coverage  <cit> .
as an academic initiative, catma provide the research community with an independent and flexible alternative to commercial arrays.
furthermore, the gsts can be utilized for posttranscriptional gene silencing when cloned into hairpin rna expression vectors  <cit> .
the agrikola consortium has converted the catma gst repertoire into hairpin rna expression vectors  <cit>  and over  <dig>  such silencing constructs have been transformed into arabidopsis to produce knock-down lines  <cit> .
here, we describe a major effort to create a comprehensive dna tag repertoire effectively targeting nearly all protein-encoding genes in arabidopsis.
the pre-existing catmav <dig> repertoire was first mapped to recent arabidopsis genome sequence annotations, tigr release  <dig>   and eugène  <dig> , with the aim of identifying a gst of the highest possible quality for each documented protein-encoding gene model.
eugène results were taken into account because annotation projects focusing on the genome of various species  <cit>  have confirmed the quality of the eugène annotation algorithm  <cit>  and no single algorithm can be perfectly accurate.
we implemented the improved and alternative algorithms for the design of gsts of most of the remaining 'orphan' arabidopsis protein-encoding genes.
RESULTS
mapping of catma gsts and gene classification
to keep up with evolving and increasingly more accurate genome annotations, continued efforts are needed to synchronize probe repertoires with changes in the list of annotated genes.
the first catma gst design rounds  <cit>  were based on earlier arabidopsis genome annotation releases, namely eugène  <dig>  tigr <dig> and tigr <dig>  that were outdated at the time the present work was initiated.
therefore, we first determined which genes described in the more recent eugène  <dig> and tigr <dig>  annotation releases were still unambiguously tagged by pre-existing gsts to identify the list of 'orphan' genes that should be considered for upgrading and expanding the gst repertoire .
we finally mapped all newly designed gsts onto the tair <dig>  gene models.
this work is part of our ongoing efforts to assure the comprehensive nature of the catma resources .
the first step of the procedure was to position the coordinates of the amplified catmav <dig> gsts with respect to both the tigr <dig> and eugène  <dig> gene models.
briefly, a gene was considered 'tagged' when it contained one or more exonic regions that sufficiently overlapped with at least one gst.
a gene was 'uniquely tagged' when this gst had no significant overlap with any other gene.
the following criteria were applied to classify a gene as being 'uniquely tagged' by a gst or, vice versa, to classify a gst as 'uniquely tagging' a gene:
a) the gene overlaps with the primary blast hit of the gsts so that the percentage sequence identity of the gst with the corresponding gene region must be at least 99%.
b) at least  <dig> bp of this matching gene region must be inside an exon.
c) at most  <dig> bp of the whole gst sequence might overlap with an exonic region of another gene.
d) the gst must not have a significant secondary blast hit, i.e.
the percentage sequence identity of the gst with any exon of any other gene should be lower than 70%.
five non-overlapping classes of genes  and gsts  were defined based on these rules .
class ge <dig> and gst <dig> contain the uniquely tagged genes and the uniquely tagging gsts, respectively.
the flowchart for the classification procedure is presented in additional file  <dig>
the mapping of the catmav <dig> gsts onto the  <dig>  tigr <dig> protein-encoding gene models and the subsequent classification resulted in  <dig>   genes classified as ge <dig>  mapping onto the  <dig>  eugène models resulted in  <dig>   ge <dig> genes.
the class distribution statistics, including mapping against the tair <dig>  genome annotation release, is detailed in table  <dig>  the gst/gene correspondence  is listed in additional file  <dig>
the numbers refer either to protein-encoding genes of the nuclear genome  or to catmav <dig> gsts that were successfully amplified .
spads algorithm adjustments
the gst design procedure presented below was based on the previously described spads algorithm  <cit> .
the successive steps encoded in spads  are described in methods.
the script of spads  <dig> .
<dig> was debugged and optimized, in particular with regard to  the verification of uniqueness of pcr primer binding sites and  the program's performance in identifying novel gsts.
the adjusted spads version was numbered  <dig> .
<dig>
to obtain a quantitative measure of the performance increase between the spads versions, we compared their efficiency on the same set of 'orphan' gene models, consisting of  <dig>  tigr <dig> protein-encoding genes located on chromosome  <dig> and untagged by the catmav <dig> repertoire.
the efficiency of gst design was improved from 57% to 69%, for spads  <dig> .
<dig> and  <dig> .
<dig>  respectively.
the increased efficiency was at the cost of a slightly reduced specificity.
for details, see 'characteristics of the catmav <dig> addition'.
design of the catmav <dig> addition
to design novel gsts for all the genes not yet uniquely tagged in the catmav <dig> repertoire , we first produced an optimized target gene set.
this 'orphan' set totaled  <dig>  gene models, consisting of  <dig>  tigr <dig> sequences and  <dig>  models predicted additionally by eugène  <dig> that shared no overlap with any genic region in tigr <dig> .
the orphan gene models were slightly modified prior to spads processing:  as in previous gst design efforts  <cit> , gene models that ended with the orf stop codon were extended by  <dig> bp, as a conservative prediction for the presence of a 3' utr; this extension is henceforth referred to as the 'artificial 3' utr' ;  regions shared by two overlapping genes were removed  to avoid gsts tagging of more than one gene;  in the case of splice variants, the gene model presented to spads was trimmed to contain the exons present in all variants .
with spads  <dig> .
<dig>  a gst was successfully designed for  <dig>  out of  <dig>  target genes .
table  <dig> presents a breakdown of results according to the four gene classes  requiring the design of novel tags.
the new set of gsts, combined with the catmav <dig> repertoire, constituted catmav <dig>  their correspondence with gene models  is listed in additional file  <dig>  as a first step toward their use in microarray transcript profiling and gene silencing experiments, the tags of this new set were amplified by pcr as described  <cit>  from arabidopsis genomic dna, with spads-selected primer pairs.
the size of all pcr products was analyzed by dna gel electrophoresis.
in total,  <dig>  gsts  were detected as products with the predicted size.
these results are comparable to the two previous synthesis campaigns.
the cloning of these amplified gsts is described elsewhere  <cit> .
all newly designed tags are listed in additional file  <dig>  this information is also available on line via the catma database  <cit> .
characteristics of the catmav <dig> addition
two parameters were analyzed to investigate whether the new gst set  has properties similar to the previously designed tags  and to assess the consistency of the subsequent gst batches:  the relative position of the tag within the gene , with the 3' location being preferable to 5', because transcript labeling techniques often include oligo-dt priming steps;  the distribution of gst specificity measured as the percentage sequence identity of a tag with the next best non-trivial genome blast hit , indicating the likelihood that gst microarray features might yield unspecific hybridization.
the comparative analysis indicated that both sets are similar with regard to the relative gst position.
the catmav <dig> addition did not show an overall decrease in specificity.
however, from figure  <dig>  a larger fraction of the next best non-trivial blast hits has a homology closer to 70%  in v <dig> than in v <dig>  although it is reasonable to assume that the performance of the majority of the added features would be similar to that of the catmav <dig> repertoire in microarray experiments  <cit> , we examined more closely the cross-hybridization potential for all gsts.
for this purpose, we calculated the melting temperature of the dna heteroduplex between each gst and their best non-target blast hit sequence with the baldino formula  <cit> , in commonly adopted catma microarray hybridization conditions .
conservatively, a gst was flagged for potential cross-hybridization when this predicted melting temperature was equal to or higher than 45°c.
this information is available via the catma database  <cit> .
the percentages of flagged gsts were  <dig> %, and  <dig> % for catmav <dig> and catmav <dig>  respectively.
experimental validation of gst classification
we reasoned that hybridization signal values would be differently distributed for gst features of different classes , reflecting their efficacy in detecting expressed sequences.
compared to the best feature class , sequences similar to multiple genes  might hybridize to more than one transcript and, thus display on average a higher signal.
inversely, features with insufficient gene coverage  or no discernable gene model  would have a lower signal, with gst <dig> features theoretically reporting background.
to test this hypothesis, we analyzed the results of  <dig> catma microarray experiments performed in the context of the european fp <dig> compendium of arabidopsis gene expression  project  <cit> .
the corresponding biological samples consist of various arabidopsis anatomical parts, harvested at different development stages, after different treatments or stresses, and from mutants , including links to arrayexpress experiment accessions  <cit> .
figure  <dig> shows the number of experiments in which a gst feature  was associated with statistically significant foreground signal values  in the  <dig> microarray data sets.
the five top graphs represent the experimentally observed hybridization characteristics for each of the five gst classes.
as expected, the median number of significant foreground value experiments per probe for each class could be ranked in the following order: gst <dig>  < gst <dig>  < gst <dig>  < gst <dig> .
it is important to note that class size varied significantly.
the gst <dig> curve was irregular and difficult to rank because it was constructed with data collected from very few features.
remarkably, gst <dig> and gst <dig> medians were far from negligible.
this observation could be explained in different ways:  the hybridization conditions might yield unspecific signals;  gst <dig> and gst <dig> tags might still yield residual signal because of partial overlap with actual transcription units;  regions currently defined as intergenic, but nevertheless transcribed, might hybridize to gst <dig> or gst <dig> tags  <cit> .
we favor the latter two explanations because the cy <dig> hybridization signal pattern observed for the lucidea  <cit>  background control spikes printed on the catma array had a very low median number of experiments with a significant foreground signal , distinctly below the median number for probe classes gst <dig> and gst <dig>  to summarize, our observations confirm that the distribution of hybridization signal values is in agreement with the gst classification established on the basis of dna sequence analysis and that this analysis correctly predicts the quality of the gst features in microarray experiments.
design of the catmav <dig> addition including gene family tags
in a final effort to reach comprehensive genome coverage of the catma repertoire, we had to resort to alternative strategies to design a tag for the remaining  <dig>  untouched arabidopsis nucleus-encoded tair <dig> cdna sequences.
many of these genes lacked a matching gst because they belonged to gene families with high sequence similarity .
to establish their relationship, the untouched genes were compared with each other and with the catmav3-tagged genes with ncbi-blastn  <cit>  and grouped in gene families.
the gene families were delimited to minimize the number of catmav3-tagged members they contained and the number of genes adhering to multiple families.
for each family, a representative sequence was defined as a fragment from one of the members of the family, sharing at least 70% identity with all other untouched members and containing at least 50% of exon sequence.
using the primer <dig> software  <cit> , we designed sequence tags based on the representative family sequences.
tags corresponding to multi-member gene families were referred to as gene family tags , whereas tags corresponding to singleton genes were referred to as additional gsts.
an example of sequence alignment leading to the design of a gft is presented in additional file  <dig>
to increase the chance of identifying gfts or gsts, the minimum amplicon length was lowered from  <dig> to  <dig> bp.
such tags still yield satisfactory hybridization signals   <cit> , whereas shorter tags are difficult to purify from a pcr mix.
as shown in figure  <dig>  the length of most newly designed tags was between  <dig> and  <dig> bp, justifying the choice to lower the length threshold as it greatly improved the ability for the tag design algorithm to identify appropriate sequences.
other criteria whose relaxation markedly affected the design included extension of primer gc content range to 20% to 95%  and restricting the specificity calculation only to cdna sequences.
in all  <dig>  genes, only three genes  did not yield a satisfactory tag because of an unusually high proportion of gc or at repeats.
all additional  <dig> gfts and  <dig> gsts are listed in additional file  <dig> and constitute the catmav <dig> addition.
this information is also available online  <cit> .
in addition, we assessed the specificity of the catmav <dig> gsts, essentially as described above for the catmav <dig> and catmav <dig> probes.
for 31% of these gsts, a flag was added in the catma database, warning for potential off-target hybridization.
a first amplification round for a subset of the catmav <dig> repertoire addition had an amplification success rate of 95% .
note that 65% of the gfts tagged only two or three genes , so that a positive hybridization result corresponding to a given feature in practice could easily be assigned to the responsible gene in a follow-up study.
CONCLUSIONS
we launched a significant effort to extend the existing catmav <dig> repertoire with additional gsts to reach comprehensive genome coverage.
the catmav <dig> gst repertoire was synchronized with two recent arabidopsis genome annotations to identify 'orphan' genes.
in two complementary design rounds, we could increase the number of sequence tags to  <dig> .
first, spads adjustments significantly increased the success of gst design at the cost of only a limited lower specificity.
subsequently, a sequence tag for nearly all remaining genes was selected by applying a markedly different design algorithm in which key design criteria were relaxed and single sequences allowed tagging of multiple genes of the same gene family.
counting all the genes satisfactorily tagged by a catmav <dig> gst  and the additional genes mapped by a v <dig> or v <dig> tag, the catma repository addresses  <dig>  nuclear protein-coding tair <dig> annotated genes.
catmav <dig> gsts were carefully classified to predict and test their performance in microarray experiments.
the analysis of a large set of microarray experiments validated both the correctness of this classification as well as the robustness of the catma microarrays.
interestingly, the analysis also revealed a substantial number of gsts that now map to intergenic regions, but in hybridizations appear to be capable to detect transcripts in microarray experiments.
as these gsts were once designed to target annotated gene models, these gene models might have been incorrectly declared obsolete during subsequent genomic re-annotations.
this work concludes a unique voluntary collaboration of european laboratories funded by national government agencies to generate a valuable community resource that would not have been possible to create on a national basis.

 the complete arabidopsis transcriptome microarray consortium was created in to take advantage of the available arabidopsis genome sequence to enable novel functional genomics approaches. eight european plant genomics research groups teamed up to produce a comprehensive set of gene-specific sequence tags originally designed for microarray transcript profiling. these gsts were 150– base pairs in length and were selected to have no significant similarity with any other sequence in the genome. the specific primer and amplicon design software was written to automate the design of these tags. the resulting gst amplicons can be used as features on spotted microarrays for transcript profiling experiments. indeed, such catma arrays performed as well as, if not better than, the affymetrix and agilent platforms in terms of specificity, sensitivity and gene coverage. as an academic initiative, catma provide the research community with an independent and flexible alternative to commercial arrays. furthermore, the gsts can be utilized for posttranscriptional gene silencing when cloned into hairpin rna expression vectors. the agrikola consortium has converted the catma gst repertoire into hairpin rna expression vectors and over such silencing constructs have been transformed into arabidopsis to produce knock-down lines. here, we describe a major effort to create a comprehensive dna tag repertoire effectively targeting nearly all protein-encoding genes in arabidopsis. the pre-existing catmav repertoire was first mapped to recent arabidopsis genome sequence annotations, tigr release and eugène, with the aim of identifying a gst of the highest possible quality for each documented protein-encoding gene model. eugène results were taken into account because annotation projects focusing on the genome of various species have confirmed the quality of the eugène annotation algorithm and no single algorithm can be perfectly accurate. we implemented the improved and alternative algorithms for the design of gsts of most of the remaining'orphan' arabidopsis protein-encoding genes.  mapping of catma gsts and gene classification to keep up with evolving and increasingly more accurate genome annotations, continued efforts are needed to synchronize probe repertoires with changes in the list of annotated genes. the first catma gst design rounds were based on earlier arabidopsis genome annotation releases, namely eugène tigr and tigr that were outdated at the time the present work was initiated. therefore, we first determined which genes described in the more recent eugène and tigr annotation releases were still unambiguously tagged by pre-existing gsts to identify the list of'orphan' genes that should be considered for upgrading and expanding the gst repertoire. we finally mapped all newly designed gsts onto the tair gene models. this work is part of our ongoing efforts to assure the comprehensive nature of the catma resources. the first step of the procedure was to position the coordinates of the amplified catmav gsts with respect to both the tigr and eugène gene models. briefly, a gene was considered'tagged' when it contained one or more exonic regions that sufficiently overlapped with at least one gst. a gene was'uniquely tagged' when this gst had no significant overlap with any other gene. the following criteria were applied to classify a gene as being'uniquely tagged' by a gst or, vice versa, to classify a gst as'uniquely tagging' a gene: a) the gene overlaps with the primary blast hit of the gsts so that the percentage sequence identity of the gst with the corresponding gene region must be at least 99%. b) at least bp of this matching gene region must be inside an exon. c) at most bp of the whole gst sequence might overlap with an exonic region of another gene. d) the gst must not have a significant secondary blast hit, i.e. the percentage sequence identity of the gst with any exon of any other gene should be lower than 70%. five non-overlapping classes of genes and gsts were defined based on these rules. class ge and gst contain the uniquely tagged genes and the uniquely tagging gsts, respectively. the flowchart for the classification procedure is presented in additional file the mapping of the catmav gsts onto the tigr protein-encoding gene models and the subsequent classification resulted in genes classified as ge mapping onto the eugène models resulted in ge genes. the class distribution statistics, including mapping against the tair genome annotation release, is detailed in table the gst/gene correspondence is listed in additional file the numbers refer either to protein-encoding genes of the nuclear genome or to catmav gsts that were successfully amplified. spads algorithm adjustments the gst design procedure presented below was based on the previously described spads algorithm. the successive steps encoded in spads are described in methods. the script of spads. was debugged and optimized, in particular with regard to the verification of uniqueness of pcr primer binding sites and the program's performance in identifying novel gsts. the adjusted spads version was numbered. to obtain a quantitative measure of the performance increase between the spads versions, we compared their efficiency on the same set of'orphan' gene models, consisting of tigr protein-encoding genes located on chromosome and untagged by the catmav repertoire. the efficiency of gst design was improved from 57% to 69%, for spads. and. respectively. the increased efficiency was at the cost of a slightly reduced specificity. for details, see'characteristics of the catmav addition'. design of the catmav addition to design novel gsts for all the genes not yet uniquely tagged in the catmav repertoire, we first produced an optimized target gene set. this'orphan' set totaled gene models, consisting of tigr sequences and models predicted additionally by eugène that shared no overlap with any genic region in tigr. the orphan gene models were slightly modified prior to spads processing: as in previous gst design efforts, gene models that ended with the orf stop codon were extended by bp, as a conservative prediction for the presence of a 3' utr; this extension is henceforth referred to as the'artificial 3' utr'; regions shared by two overlapping genes were removed to avoid gsts tagging of more than one gene; in the case of splice variants, the gene model presented to spads was trimmed to contain the exons present in all variants. with spads. a gst was successfully designed for out of target genes. table presents a breakdown of results according to the four gene classes requiring the design of novel tags. the new set of gsts, combined with the catmav repertoire, constituted catmav their correspondence with gene models is listed in additional file as a first step toward their use in microarray transcript profiling and gene silencing experiments, the tags of this new set were amplified by pcr as described from arabidopsis genomic dna, with spads-selected primer pairs. the size of all pcr products was analyzed by dna gel electrophoresis. in total, gsts were detected as products with the predicted size. these results are comparable to the two previous synthesis campaigns. the cloning of these amplified gsts is described elsewhere. all newly designed tags are listed in additional file this information is also available on line via the catma database. characteristics of the catmav addition two parameters were analyzed to investigate whether the new gst set has properties similar to the previously designed tags and to assess the consistency of the subsequent gst batches: the relative position of the tag within the gene, with the 3' location being preferable to 5', because transcript labeling techniques often include oligo-dt priming steps; the distribution of gst specificity measured as the percentage sequence identity of a tag with the next best non-trivial genome blast hit, indicating the likelihood that gst microarray features might yield unspecific hybridization. the comparative analysis indicated that both sets are similar with regard to the relative gst position. the catmav addition did not show an overall decrease in specificity. however, from figure a larger fraction of the next best non-trivial blast hits has a homology closer to 70% in v than in v although it is reasonable to assume that the performance of the majority of the added features would be similar to that of the catmav repertoire in microarray experiments, we examined more closely the cross-hybridization potential for all gsts. for this purpose, we calculated the melting temperature of the dna heteroduplex between each gst and their best non-target blast hit sequence with the baldino formula, in commonly adopted catma microarray hybridization conditions. conservatively, a gst was flagged for potential cross-hybridization when this predicted melting temperature was equal to or higher than 45°c. this information is available via the catma database. the percentages of flagged gsts were %, and % for catmav and catmav respectively. experimental validation of gst classification we reasoned that hybridization signal values would be differently distributed for gst features of different classes, reflecting their efficacy in detecting expressed sequences. compared to the best feature class, sequences similar to multiple genes might hybridize to more than one transcript and, thus display on average a higher signal. inversely, features with insufficient gene coverage or no discernable gene model would have a lower signal, with gst features theoretically reporting background. to test this hypothesis, we analyzed the results of catma microarray experiments performed in the context of the european fp compendium of arabidopsis gene expression project. the corresponding biological samples consist of various arabidopsis anatomical parts, harvested at different development stages, after different treatments or stresses, and from mutants, including links to arrayexpress experiment accessions. figure shows the number of experiments in which a gst feature was associated with statistically significant foreground signal values in the microarray data sets. the five top graphs represent the experimentally observed hybridization characteristics for each of the five gst classes. as expected, the median number of significant foreground value experiments per probe for each class could be ranked in the following order: gst < gst < gst < gst. it is important to note that class size varied significantly. the gst curve was irregular and difficult to rank because it was constructed with data collected from very few features. remarkably, gst and gst medians were far from negligible. this observation could be explained in different ways: the hybridization conditions might yield unspecific signals; gst and gst tags might still yield residual signal because of partial overlap with actual transcription units; regions currently defined as intergenic, but nevertheless transcribed, might hybridize to gst or gst tags. we favor the latter two explanations because the cy hybridization signal pattern observed for the lucidea background control spikes printed on the catma array had a very low median number of experiments with a significant foreground signal, distinctly below the median number for probe classes gst and gst to summarize, our observations confirm that the distribution of hybridization signal values is in agreement with the gst classification established on the basis of dna sequence analysis and that this analysis correctly predicts the quality of the gst features in microarray experiments. design of the catmav addition including gene family tags in a final effort to reach comprehensive genome coverage of the catma repertoire, we had to resort to alternative strategies to design a tag for the remaining untouched arabidopsis nucleus-encoded tair cdna sequences. many of these genes lacked a matching gst because they belonged to gene families with high sequence similarity. to establish their relationship, the untouched genes were compared with each other and with the catmav3-tagged genes with ncbi-blastn and grouped in gene families. the gene families were delimited to minimize the number of catmav3-tagged members they contained and the number of genes adhering to multiple families. for each family, a representative sequence was defined as a fragment from one of the members of the family, sharing at least 70% identity with all other untouched members and containing at least 50% of exon sequence. using the primer software, we designed sequence tags based on the representative family sequences. tags corresponding to multi-member gene families were referred to as gene family tags, whereas tags corresponding to singleton genes were referred to as additional gsts. an example of sequence alignment leading to the design of a gft is presented in additional file to increase the chance of identifying gfts or gsts, the minimum amplicon length was lowered from to bp. such tags still yield satisfactory hybridization signals, whereas shorter tags are difficult to purify from a pcr mix. as shown in figure the length of most newly designed tags was between and bp, justifying the choice to lower the length threshold as it greatly improved the ability for the tag design algorithm to identify appropriate sequences. other criteria whose relaxation markedly affected the design included extension of primer gc content range to 20% to 95% and restricting the specificity calculation only to cdna sequences. in all genes, only three genes did not yield a satisfactory tag because of an unusually high proportion of gc or at repeats. all additional gfts and gsts are listed in additional file and constitute the catmav addition. this information is also available online. in addition, we assessed the specificity of the catmav gsts, essentially as described above for the catmav and catmav probes. for 31% of these gsts, a flag was added in the catma database, warning for potential off-target hybridization. a first amplification round for a subset of the catmav repertoire addition had an amplification success rate of 95%. note that 65% of the gfts tagged only two or three genes, so that a positive hybridization result corresponding to a given feature in practice could easily be assigned to the responsible gene in a follow-up study.  we launched a significant effort to extend the existing catmav repertoire with additional gsts to reach comprehensive genome coverage. the catmav gst repertoire was synchronized with two recent arabidopsis genome annotations to identify'orphan' genes. in two complementary design rounds, we could increase the number of sequence tags to. first, spads adjustments significantly increased the success of gst design at the cost of only a limited lower specificity. subsequently, a sequence tag for nearly all remaining genes was selected by applying a markedly different design algorithm in which key design criteria were relaxed and single sequences allowed tagging of multiple genes of the same gene family. counting all the genes satisfactorily tagged by a catmav gst and the additional genes mapped by a v or v tag, the catma repository addresses nuclear protein-coding tair annotated genes. catmav gsts were carefully classified to predict and test their performance in microarray experiments. the analysis of a large set of microarray experiments validated both the correctness of this classification as well as the robustness of the catma microarrays. interestingly, the analysis also revealed a substantial number of gsts that now map to intergenic regions, but in hybridizations appear to be capable to detect transcripts in microarray experiments. as these gsts were once designed to target annotated gene models, these gene models might have been incorrectly declared obsolete during subsequent genomic re-annotations. this work concludes a unique voluntary collaboration of european laboratories funded by national government agencies to generate a valuable community resource that would not have been possible to create on a national basis. 
to cover the remaining untagged genes, we identified additional gsts using less stringent design criteria and designed sequence tags matching multiple members of gene families to cover any remaining untagged genes. these gst amplicons can easily be reamplified and shared, subsets can be picked at will to print dedicated arrays, and the gsts can be cloned and used for other functional studies. to update the catma gst repertoire, we designed additional sequence tags, bringing the total number of tagged tair6-annotated arabidopsis nuclear protein-coding genes to. to complete the catma repertoire, all gene models for which no gst had yet been designed were processed with an adjusted version of the specific primer and amplicon design software. gsts from the catma version repertoire were mapped onto the gene models from two independent arabidopsis nuclear genome annotation efforts, tigr and psb-eugène, to consolidate a list of genes that were targeted by previously designed catma tags. a total of gene models were not tagged by any amplified catmav gst, and amplified gsts were no longer predicted to tag an updated gene model. it comprises unique amplified sequences that tag and protein-encoding nuclear gene models in the tair and eugène genome annotations, respectively. to validate the efficacy of gst mapping criteria and design rules, the predicted and experimentally observed hybridization characteristics associated to gst features were correlated in transcript profiling datasets obtained with the catmav microarray, confirming the reliability of this platform. the complete arabidopsis transcript microarray initiative combines the efforts of laboratories in eight european countries to deliver gene-specific sequence tags for the arabidopsis research community. all information about the resulting updated catma repertoire is available through the catma database http://www.catma.org. a total of novel gsts were designed and amplified by pcr from genomic dna.
 the complete arabidopsis transcript microarray initiative combines the efforts of laboratories in eight european countries to deliver gene-specific sequence tags for the arabidopsis research community. the catma initiative offers the power and flexibility to regularly update the gst collection according to evolving knowledge about the gene repertoire. these gst amplicons can easily be reamplified and shared, subsets can be picked at will to print dedicated arrays, and the gsts can be cloned and used for other functional studies. this ongoing initiative has already produced approximately gsts that have been made publicly available for spotted microarray printing and rna interference.  gsts from the catma version repertoire were mapped onto the gene models from two independent arabidopsis nuclear genome annotation efforts, tigr and psb-eugène, to consolidate a list of genes that were targeted by previously designed catma tags. a total of gene models were not tagged by any amplified catmav gst, and amplified gsts were no longer predicted to tag an updated gene model. to validate the efficacy of gst mapping criteria and design rules, the predicted and experimentally observed hybridization characteristics associated to gst features were correlated in transcript profiling datasets obtained with the catmav microarray, confirming the reliability of this platform. to complete the catma repertoire, all gene models for which no gst had yet been designed were processed with an adjusted version of the specific primer and amplicon design software. a total of novel gsts were designed and amplified by pcr from genomic dna. together with the pre-existing gst collection, this new addition constitutes the catmav repertoire. it comprises unique amplified sequences that tag and protein-encoding nuclear gene models in the tair and eugène genome annotations, respectively. to cover the remaining untagged genes, we identified additional gsts using less stringent design criteria and designed sequence tags matching multiple members of gene families to cover any remaining untagged genes. these latter features constitute the catmav addition.  to update the catma gst repertoire, we designed additional sequence tags, bringing the total number of tagged tair6-annotated arabidopsis nuclear protein-coding genes to. this resource is used both for the production of spotted microarrays and the large-scale cloning of hairpin rna silencing vectors. all information about the resulting updated catma repertoire is available through the catma database http://www.catma.org. 