BMC Genomics Methodology article Optimal control and analysis of two-color genomotyping experiments using bacterial multistrain arrays Francisco R Pinto*, Sandra I Aguiar, J Melo-Cristino and Mário Ramirez BioMed Central Open Access Address: Instituto de Microbiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Av. Professor Egas Moniz, 1649-028 Lisboa, Portugal Email: Francisco R Pinto* - fpinto@fm.ul.pt; Sandra I Aguiar - siaguiar@fm.ul.pt; J Melo-Cristino - melo_cristino@fm.ul.pt; Mário Ramirez - ramirez@fm.ul.pt * Corresponding author Published: 19 May 2008 BMC Genomics 2008, 9:230 doi:10.1186/1471-2164-9-230 This article is available from: http://www.biomedcentral.com/1471-2164/9/230 Received: 27 February 2008 Accepted: 19 May 2008 © 2008 Pinto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Microarray comparative genomic hybridization (aCGH) evaluates the distribution of genes of sequenced bacterial strains among unsequenced strains of the same or related species. As genomic sequences from multiple strains of the same species become available, multistrain microarrays are designed, containing spots for every unique gene in all sequenced strains. To perform two-color aCGH experiments with multistrain microarrays, the choice of control sample can be the genomic DNA of one strain or a mixture of all the strains used in the array design. This important problem has no universally accepted solution. Results: We performed a comparative study of the two control sample options with a Streptococcus pneumoniae microarray designed with three fully sequenced strains. We separately hybridized two of these strains (R6 and G54) as test samples using the third strain alone (TIGR4) or a mixture of the three strains as control. We show that for both types of control it is advantageous to analyze spots in separate sets according to their expected control channel signal (5–15% AUC increase). Following this analysis, the use of a mix control leads to higher accuracies (5% increase). This enhanced performance is due to gains in sensitivity (21% increase, p = 0.001) that compensate minor losses in specificity (5% decrease, p = 0.014). Conclusion: The use of a single strain control increases the error rate in genes that are part of the accessory genome, where more variation across unsequenced strains is expected, further justifying the use of the mix control. Background Microarray technology is commonly used to monitor gene expression profiles. However, microarrays have also become an established technique in bacterial comparative genomics, in which the assessment of gene inventories is made at the genomic DNA level. This technique is some- times referred to as microarray genomotyping [1,2] but also as array based comparative genomic hybridizations (aCGH) [3,4]. Until recently it was widely assumed that adaptive evolution in bacteria occurred mainly by muta- tion and restricted recombination among members of the same species [5]. However, the availability of fully sequenced prokaryotic genomes changed this view since they provided ample evidence for extensive lateral gene transfer and it became clear that this was a major source of variation and fundamental to understand adaptive evolu- tion in prokaryotes [6]. The ability to compare microbial genomes using microarrays has been used in evolutionary Page 1 of 11 (page number not for citation purposes)