BMC Genomics 2008, 9:230 http://www.biomedcentral.com/1471-2164/9/230 rate detection of the presence of genes that are part of the accessory genome and these would benefit greatly from a mixed control approach. Since single strain controls are frequently used in these studies [9,14], this may lead to the incorrect identification of the absence of genes associ- ated with the accessory genome and may contribute to the variable success of this technique in identifying different groups of strains within the same species. Although we believe our results to be generally applicable to all multistrain microarrays used in aCGH, it is impor- tant to note that the magnitude of the observed effects might differ according to the array technology, quality of oligonucleotide design and the relative number of spots that are specific for only one or a subset of the strains used in the array design. Arrays with cDNA probes, for exam- ple, are expected to have similar sensitivity to 70 mer probes but lower specificity, which can lead to more cross- hybridization. Additionally, depending on the degree of sequence divergence between the strains used in the array design, probes targeting ORFs present in multiple strains can have lower sensitivity, lower specificity or both. In fact, the oligos in our arrays can have different sensitivities and specificities when hybridizing with the R6 or G54 strains, respectively. Additionnally, the G54 strain has more genes with spots specific for one strain only (more G spots than R spots) and less genes targeted by spots rec- ognizing two strains (more TR spots than TG spots, Table 1), which according to the previous discussion should improve the quality of G54 results. Notwithstanding, our analysis shows that, independently of these differences, we can say that using a single strain as control will lead to higher error rates for spots that recognize genes that are not present in all reference strains. Moreover, if the mix control is used and spots are analyzed in separate classes higher sensitivities are achieved, meaning that less false absent call are produced and a higher fraction of the truly present genes are recovered from the analysis. Snipen and colleagues discuss that in aCGH experiments, the study focus of the researcher can be primarily to identify diver- gent or absent genes or, on the other hand, the focus can be on the present genes, for example, when estimating the conserved core genome over all strains [4]. In the first sit- uation, it may be more important to require higher sensi- tivities, that is, less false absent calls, while in the second situation specificity may be more relevant, attempting to avoid false present calls. In published studies the first sit- uation is more common, which led Repsilber and col- leagues to state that in a typical genomotyping experiment high sensitivities, around 0.9, are desirable to prevent high rates of false absent calls [1]. We have shown here, that those levels are attainable for multistrain microarrays if a mix control is applied and spots are analyzed in two different classes. If spots are analyzed in a single class, high sensitivities are also observed, but accompanied by undesirable very low specificities. If the main objective is to define a core genome and high specificity is required, analyzing spots in separate classes is mandatory whichever control is used. Again, the mix control provides the best-balanced performance since the use of the single strain control, although associated with slightly better specificities, presents substantial losses in sensitivity. Independently of the method chosen for the analysis of aCGH data, a suboptimal choice of control sample may compromise the quality of the final results as well as the possibility to compare different studies. Our study thus provides an important source of information to support the design of bacterial genomotyping experiments with multistrain microarrays. Methods Strain growth and DNA preparation Strains included in the present study were R6 (kindly pro- vided by A. Tomasz), TIGR4 (obtained from the American Type Culture Collection) and G54 (kindly provided by J.F. Garcia-Bustos). Strains were grown overnight on blood agar plates at 37°C in 5% CO2. A few colonies were inoc- ulated in 6 ml of Brain Heart Infusion medium and the culture was incubated at 37°C for approximately 6 h. Total DNA extraction was performed using the CTAB pro- tocol [24]. Genome Labeling and Microarray processing Microarray experiments were performed using whole- genome S. pneumoniae oligonucleotide microarrays (ver- sion 5) obtained from the Pathogen Functional Genomic Resource center at The Institute for Genomic Research (TIGR) [25]. Briefly, the microarray contains 3425 oligos (70 mers) replicated four times targeting S. pneumoniae open reading frames (ORFs) and 10 amplicons and 500 oligos targeting ORFs of Arabidopsis thaliana as a negative control. 2445 of the S. pneumoniae oligos target genes present in the TIGR4 genome, 2289 in the R6 genome and 2233 in the G54 genome. Oligos were designed based on ORF sequences across all the three genomes simultaneously, attempting to provide coverage for every non-redundant ORF present in the three genomic sequences. There are oligos that bind specifically to ORFs present in two or in all of the three genomes (T, R and G). According to their specificity to one, two or three of the reference genomes, spots (or oli- gos) were classified in 7 sets, listed in Table 1. Annotation files provided by The Pathogen Functional Genomic Resource Center [26] contain the results of BLASTN searches of each designed oligo against each of the three Page 8 of 11 (page number not for citation purposes)