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CATMA, a comprehensive genome-scale resource for silencing and transcript profiling of Arabidopsis genes.

Sclep G, Allemeersch J, Liechti R, De Meyer B, Beynon J, Bhalerao R, Moreau Y, Nietfeld W, Renou JP, Reymond P, Kuiper MT, Hilson P - BMC Bioinformatics (2007)

Bottom Line: 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 CATMAv2 microarray, confirming the reliability of this platform.These latter 1,533 features constitute the CATMAv4 addition.This resource is used both for the production of spotted microarrays and the large-scale cloning of hairpin RNA silencing vectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium. gert.sclep@tecnoparco.org

ABSTRACT

Background: The Complete Arabidopsis Transcript MicroArray (CATMA) initiative combines the efforts of laboratories in eight European countries 1 to deliver gene-specific sequence tags (GSTs) 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 24,000 GSTs that have been made publicly available for spotted microarray printing and RNA interference.

Results: GSTs from the CATMA version 2 repertoire (CATMAv2, created in 2002) were mapped onto the gene models from two independent Arabidopsis nuclear genome annotation efforts, TIGR5 and PSB-EuGène, to consolidate a list of genes that were targeted by previously designed CATMA tags. A total of 9,027 gene models were not tagged by any amplified CATMAv2 GST, and 2,533 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 CATMAv2 microarray, confirming the reliability of this platform. To complete the CATMA repertoire, all 9,027 gene models for which no GST had yet been designed were processed with an adjusted version of the Specific Primer and Amplicon Design Software (SPADS). A total of 5,756 novel GSTs were designed and amplified by PCR from genomic DNA. Together with the pre-existing GST collection, this new addition constitutes the CATMAv3 repertoire. It comprises 30,343 unique amplified sequences that tag 24,202 and 23,009 protein-encoding nuclear gene models in the TAIR6 and EuGène genome annotations, respectively. To cover the remaining untagged genes, we identified 543 additional GSTs using less stringent design criteria and designed 990 sequence tags matching multiple members of gene families (Gene Family Tags or GFTs) to cover any remaining untagged genes. These latter 1,533 features constitute the CATMAv4 addition.

Conclusion: To update the CATMA GST repertoire, we designed 7,289 additional sequence tags, bringing the total number of tagged TAIR6-annotated Arabidopsis nuclear protein-coding genes to 26,173. 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.

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Distribution of probe length for both GSTs and GFTs. The distribution of the probe length is shown for all CATMAv4 addition features. The height of each bar corresponds to the number of probes in the length bins. Upper and lower panels represent the length distribution of the GFTs and GSTs, respectively.
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Figure 4: Distribution of probe length for both GSTs and GFTs. The distribution of the probe length is shown for all CATMAv4 addition features. The height of each bar corresponds to the number of probes in the length bins. Upper and lower panels represent the length distribution of the GFTs and GSTs, respectively.

Mentions: To increase the chance of identifying GFTs or GSTs, the minimum amplicon length was lowered from 150 to 100 bp. Such tags still yield satisfactory hybridization signals (data not shown) [24], whereas shorter tags are difficult to purify from a PCR mix. As shown in Figure 4, the length of most newly designed tags was between 100 and 150 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% (from 30% to 80%) and restricting the specificity calculation only to cDNA sequences. In all 2,338 genes, only three genes (At2g03937, At3g50250 and At4g19270) did not yield a satisfactory tag because of an unusually high proportion of GC or AT repeats. All additional 990 GFTs and 543 GSTs are listed in Additional File 7 and constitute the CATMAv4 addition. This information is also available online [14,25]. In addition, we assessed the specificity of the CATMAv4 GSTs, essentially as described above for the CATMAv2 and CATMAv3 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 CATMAv4 repertoire addition had an amplification success rate of 95% (data not shown). Note that 65% of the GFTs tagged only two or three genes (Figure 5), so that a positive hybridization result corresponding to a given feature in practice could easily be assigned to the responsible gene(s) in a follow-up study.


CATMA, a comprehensive genome-scale resource for silencing and transcript profiling of Arabidopsis genes.

Sclep G, Allemeersch J, Liechti R, De Meyer B, Beynon J, Bhalerao R, Moreau Y, Nietfeld W, Renou JP, Reymond P, Kuiper MT, Hilson P - BMC Bioinformatics (2007)

Distribution of probe length for both GSTs and GFTs. The distribution of the probe length is shown for all CATMAv4 addition features. The height of each bar corresponds to the number of probes in the length bins. Upper and lower panels represent the length distribution of the GFTs and GSTs, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2147040&req=5

Figure 4: Distribution of probe length for both GSTs and GFTs. The distribution of the probe length is shown for all CATMAv4 addition features. The height of each bar corresponds to the number of probes in the length bins. Upper and lower panels represent the length distribution of the GFTs and GSTs, respectively.
Mentions: To increase the chance of identifying GFTs or GSTs, the minimum amplicon length was lowered from 150 to 100 bp. Such tags still yield satisfactory hybridization signals (data not shown) [24], whereas shorter tags are difficult to purify from a PCR mix. As shown in Figure 4, the length of most newly designed tags was between 100 and 150 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% (from 30% to 80%) and restricting the specificity calculation only to cDNA sequences. In all 2,338 genes, only three genes (At2g03937, At3g50250 and At4g19270) did not yield a satisfactory tag because of an unusually high proportion of GC or AT repeats. All additional 990 GFTs and 543 GSTs are listed in Additional File 7 and constitute the CATMAv4 addition. This information is also available online [14,25]. In addition, we assessed the specificity of the CATMAv4 GSTs, essentially as described above for the CATMAv2 and CATMAv3 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 CATMAv4 repertoire addition had an amplification success rate of 95% (data not shown). Note that 65% of the GFTs tagged only two or three genes (Figure 5), so that a positive hybridization result corresponding to a given feature in practice could easily be assigned to the responsible gene(s) in a follow-up study.

Bottom Line: 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 CATMAv2 microarray, confirming the reliability of this platform.These latter 1,533 features constitute the CATMAv4 addition.This resource is used both for the production of spotted microarrays and the large-scale cloning of hairpin RNA silencing vectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium. gert.sclep@tecnoparco.org

ABSTRACT

Background: The Complete Arabidopsis Transcript MicroArray (CATMA) initiative combines the efforts of laboratories in eight European countries 1 to deliver gene-specific sequence tags (GSTs) 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 24,000 GSTs that have been made publicly available for spotted microarray printing and RNA interference.

Results: GSTs from the CATMA version 2 repertoire (CATMAv2, created in 2002) were mapped onto the gene models from two independent Arabidopsis nuclear genome annotation efforts, TIGR5 and PSB-EuGène, to consolidate a list of genes that were targeted by previously designed CATMA tags. A total of 9,027 gene models were not tagged by any amplified CATMAv2 GST, and 2,533 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 CATMAv2 microarray, confirming the reliability of this platform. To complete the CATMA repertoire, all 9,027 gene models for which no GST had yet been designed were processed with an adjusted version of the Specific Primer and Amplicon Design Software (SPADS). A total of 5,756 novel GSTs were designed and amplified by PCR from genomic DNA. Together with the pre-existing GST collection, this new addition constitutes the CATMAv3 repertoire. It comprises 30,343 unique amplified sequences that tag 24,202 and 23,009 protein-encoding nuclear gene models in the TAIR6 and EuGène genome annotations, respectively. To cover the remaining untagged genes, we identified 543 additional GSTs using less stringent design criteria and designed 990 sequence tags matching multiple members of gene families (Gene Family Tags or GFTs) to cover any remaining untagged genes. These latter 1,533 features constitute the CATMAv4 addition.

Conclusion: To update the CATMA GST repertoire, we designed 7,289 additional sequence tags, bringing the total number of tagged TAIR6-annotated Arabidopsis nuclear protein-coding genes to 26,173. 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.

Show MeSH
Related in: MedlinePlus