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A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization.

Talla E, Tekaia F, Brino L, Dujon B - BMC Genomics (2003)

Bottom Line: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences.The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected.The OliD program is available from authors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures (URA 2171 CNRS, UFR 927 Université PM Curie), 25 rue du Docteur Roux, F-75724 Paris cedex 15, France. etalla@pasteur.fr

ABSTRACT

Background: Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes. The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data.

Results: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences. An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames. The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected. A total of 5660 novel probes (more than 97% of the yeast genes) were designed. For the remaining 143 genes, cross-hybridization was unavoidable. Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure. Sensitivity, reproducibility and dynamic range of these new microarrays have been measured. Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences.

Conclusions: A validated procedure to predict cross-hybridization in microarray probe design was defined in this work. Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed. Arrays are available at Eurogentec S. A. Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays. The OliD program is available from authors.

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Comparison of transcript level measurements between duplicated probe spots within a single array from Batch616. A scatter plot of Cy5 intensities at duplicated probe spots from a single hybridization is shown. cDNA targets were prepared from total RNA isolated from wild-type BY4742 (Cy3 labelled) and Δydr225w (Cy5 labelled) strain as described in "Methods". The 2 × limits are shown.
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Figure 2: Comparison of transcript level measurements between duplicated probe spots within a single array from Batch616. A scatter plot of Cy5 intensities at duplicated probe spots from a single hybridization is shown. cDNA targets were prepared from total RNA isolated from wild-type BY4742 (Cy3 labelled) and Δydr225w (Cy5 labelled) strain as described in "Methods". The 2 × limits are shown.

Mentions: We have determined the sensitivity and specificity of our microarrays from results of hybridization experiments described in the next section. Under our experimental conditions, 82–92% of all probe spots produce detectable transcript signals i.e. showing Cy3 or Cy5 intensities higher than the 95% upper limit of the distribution of empty spot signals. This is indicative of the high sensitivity of our system. An example (for Cy5 channel) of the dynamic range and reproducibility of the duplicate measurements of an array is shown in Figure 2. As can be seen, transcript intensities can be measured over a 3-log range and the reproducibility of measurements of the two duplicates is high (slope close to 1 with r value of 0.98). Similar results were obtained for the Cy3 channel (data not shown).


A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization.

Talla E, Tekaia F, Brino L, Dujon B - BMC Genomics (2003)

Comparison of transcript level measurements between duplicated probe spots within a single array from Batch616. A scatter plot of Cy5 intensities at duplicated probe spots from a single hybridization is shown. cDNA targets were prepared from total RNA isolated from wild-type BY4742 (Cy3 labelled) and Δydr225w (Cy5 labelled) strain as described in "Methods". The 2 × limits are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC239980&req=5

Figure 2: Comparison of transcript level measurements between duplicated probe spots within a single array from Batch616. A scatter plot of Cy5 intensities at duplicated probe spots from a single hybridization is shown. cDNA targets were prepared from total RNA isolated from wild-type BY4742 (Cy3 labelled) and Δydr225w (Cy5 labelled) strain as described in "Methods". The 2 × limits are shown.
Mentions: We have determined the sensitivity and specificity of our microarrays from results of hybridization experiments described in the next section. Under our experimental conditions, 82–92% of all probe spots produce detectable transcript signals i.e. showing Cy3 or Cy5 intensities higher than the 95% upper limit of the distribution of empty spot signals. This is indicative of the high sensitivity of our system. An example (for Cy5 channel) of the dynamic range and reproducibility of the duplicate measurements of an array is shown in Figure 2. As can be seen, transcript intensities can be measured over a 3-log range and the reproducibility of measurements of the two duplicates is high (slope close to 1 with r value of 0.98). Similar results were obtained for the Cy3 channel (data not shown).

Bottom Line: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences.The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected.The OliD program is available from authors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures (URA 2171 CNRS, UFR 927 Université PM Curie), 25 rue du Docteur Roux, F-75724 Paris cedex 15, France. etalla@pasteur.fr

ABSTRACT

Background: Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes. The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data.

Results: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences. An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames. The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected. A total of 5660 novel probes (more than 97% of the yeast genes) were designed. For the remaining 143 genes, cross-hybridization was unavoidable. Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure. Sensitivity, reproducibility and dynamic range of these new microarrays have been measured. Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences.

Conclusions: A validated procedure to predict cross-hybridization in microarray probe design was defined in this work. Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed. Arrays are available at Eurogentec S. A. Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays. The OliD program is available from authors.

Show MeSH
Related in: MedlinePlus