BMC Genomics 2008, 9:230 http://www.biomedcentral.com/1471-2164/9/230 Results Expected values and empirical distribution of LR Since the strains hybridized as test samples in our experi- ments have their genomes fully sequenced, we know a pri- ori which spots should identify genes as present or absent. These expected results are compiled in Table 1. Following the accepted methodologies in processing the results of two-color microarray experiments, the output variable of analysis is the ratio of the signal intensity of the test chan- nel by the signal intensity of the control channel. For sim- plicity, the expected signal intensity of single fluorescence channels indicated in Table 1 was assumed to take only the values 0, 1, 2 or 3. In single strain control experiments, when a spot identifies a gene present both in the test sam- ple and in the control, both channels have maximum sig- nal intensity, which is 3, and a resulting signal ratio of 1. If the spot identifies a gene present in the test but not in the control, the test channel and control channel will have intensities of 3 and 0, respectively. The resulting signal ratio would tend to infinity in the absence of random noise in the control channel. Still, the resulting signal ratio will be larger as compared with other spot sets. In the reverse situation, when the spot is absent in the test but present in the control, the test channel and control chan- nel will have intensities of 0 and 3, respectively. The signal ratio is expected to be 0, and once again, due to random noise, exact zeros will not be observed, resulting in small ratio values instead. If a mixture of the 3 strains represented in the array is used as control, the relative intensity of the signal in the control channel can take the value of 1 if the gene identified by the spot is present in a single strain (T, R and G spots), 2 if the gene is present in two of the strains (TR, TG and RG spots) and 3 when the gene is shared by all three strains (TRG spots). If the spot identifies a gene present in the test sample, the resulting signal ratios will be 3, 3/2 or 1, respectively. If the spot identifies a gene absent in the test strain, all the signal ratios are expected to be 0, in the absence of random noise. In practice, the three spot classes described above will produce decreasing ratio val- ues, represented in Table 1 as 0/1, 0/2 and 0/3, respec- tively. The main problem of using the strain with the larger number of genes spotted in the array as control (T) is read- ily detected. There are three spot classes with an expected ratio of 1, two of which have a present call while the other should lead to an absent call. In the two classes with a present call, the unity ratio results from maximal signal intensity in both test and control, while for the absent spot class, both channels should produce a signal. In expression arrays this would not be a problem as all three classes contribute to the bulk of non-differentially expressed genes and consequently, is of no interest to the researcher. Contrastingly, in aCGH experiments it is cru- cial to differentiate these two outputs, the absent and the present call. One possible solution for this problem is to analyze the spots with oligos specific for T (T, TR, TG and TRG spots) separately from the ones with oligos not hybridizing with T genes. But Table 1 also suggests another way to circumvent this problem by using the mixture of the three reference genomes (T+R+G) as control. In these hybridizations all the spot classes with absent calls have expected signal ratios lower than 1. Using the mix control produces five different expected ratio values, with three possible levels of control signal. We then expect to improve the detection of absent/present genes by performing the analysis sepa- rately for spots that are specific for one control strain (T, R and G spots), two control strains (TR, TG and RG spots) and all control strains (TRG spots). Figure 1 presents the actual distribution of LR values (base 10) for the distinct hybridizations performed. Each panel is the combined distribution of four replicates. Median values are in good agreement with respect to the loga- Expected signal ratio R6 G54 Table 1: Correct calls (identification as absent or present gene) and expected test/control signal ratios for the four types of hybridization performed. Spots specific for: T4 (T) R6 (R) G54 (G) T4, R6 (TR) T4, G54 (TG) R6, G54 (RG) T4, R6, G54 (TRG) Number of unique spots Call vs T4 vs Mix Call vs T4 vs Mix 374 230 258 209 125 113 1737 A P A P A P P 0/3 3/0 1 1 0/3 3/0 1 0/1 3/1 0/1 3/2 0/2 3/2 1 A A P A P P P 0/3 1 3/0 0/3 1 3/0 1 0/1 0/1 3/1 0/2 3/2 3/2 1 Page 3 of 11 (page number not for citation purposes)