A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus.
BACKGROUND
DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus.


RESULTS
We describe an efficient procedure for selecting 40-60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling.


CONCLUSIONS
This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions.
Charbonnier
Yvan
Y
Genomic Research Laboratory, University Hospitals of Geneva, rue Micheli-du-Crest 24, CH-1211 Geneva 14, Switzerland. yvan.charbonnier@genomic.ch
Gettler
Brian
B
François
Patrice
P
Bento
Manuela
M
Renzoni
Adriana
A
Vaudaux
Pierre
P
Schlegel
Werner
W
Schrenzel
Jacques
J
D000465:Algorithms; D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods; D015342:DNA Probes / Q000737:chemistry; D017353:Gene Deletion; D015870:Gene Expression; D020869:Gene Expression Profiling; D016678:Genome; D016680:Genome, Bacterial; D023281:Genomics; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software; D013211:Staphylococcus aureus / Q000235:genetics*; D013816:Thermodynamics; D014158:Transcription, Genetic
D015342:DNA Probes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides