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Design, Validation, and Application of a Seven-Strain Staphylococcus aureus PCR Product Microarray for Comparative Genomics

Adam A. Witney,¹ Gemma L. Marsden,^1, Matthew T. G. Holden,² Richard A. Stabler,^1, Sarah E. Husain,¹ J. Keith Vass,³ Philip D. Butcher,¹ Jason Hinds,¹ and Jodi A. Lindsay^4*

Bacterial Microarray Group,¹ Infectious Diseases, Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, London SW17 0RE,⁴ The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA,² Beatson Institute for Cancer Research, Garscube Estate, Bearsden G61 1BD, United Kingdom³

Received 8 February 2005/ Accepted 15 June 2005

Bacterial comparative genomics has been revolutionized by microarrays, but the power of any microarray is dependent on the number and diversity of gene reporters it contains. Staphylococcus aureus is an important human pathogen causing a wide range of invasive and toxin-mediated diseases, and more than 20% of the genome of any isolate consists of variable genes. Seven whole-genome sequences of S. aureus are available, and we exploited this rare opportunity to design, build, and validate a comprehensive, nonredundant PCR product microarray carrying reporters that represent every predicted open reading frame (3,623 probes). Such a comprehensive microarray necessitated a novel design strategy. Validation with the seven sequenced strains showed correct identification of 93.9% of genes present or absent/divergent but was dependent on the method of analysis chosen. Microarray data were highly reproducible, reducing the need for many replicate slides. Interpretation of microarray data was enhanced by focusing on the major areas of variation—the presence or absence of mobile genetic elements (MGEs). We compiled "composite genomes" of every individual MGE and visualized their distribution. This allowed the sensitive discrimination of related isolates, including the first clear description of how isolates of the same clone of epidemic methicillin-resistant S. aureus differ substantially in their carriage of MGEs. These MGEs carry virulence and resistance genes, suggesting differences in pathogenic potential. The novel methods of design and interpretation of data generated from this microarray will enable further studies of S. aureus evolution, epidemiology, and pathogenesis.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.

Present address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.

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