1168 Nucleic Acids Research, 2006, Vol. 34, No. 4 analysis (Supplementary Tables S1 and S2). For timepoints with multiple microarrays, ratio values were averaged. Fast Fourier transform (FFT) of data To analyze the three strains simultaneously, we required that for each oligonucleotide (oligo) measured by the microarray, data must be present in at least 60% of the timepoints: for 3D7 (31 of 53), Dd2 (29 of 50), HB3 (27 of 46). These comprise the quality control datasets (Supplementary Tables S3–S5). The ﬁltered datasets were then normalized, log2 transformed and smoothed using the LOESS function from the statistical pack- age R (http://www.R-project.org) with weighting restricted at 30%. Missing data were estimated based on nearest neighbors using the PREDICT function. Fast Fourier analysis was per- formed as described previously (10) on the continuous smoothed dataset. For all strain datasets FFT phase values ranged from p to +p. For FFT phase comparison between strains, both the absolute value of the difference between the two phases ABS(phase1-phase2) and 2p-ABS(phase1-phase2) were calculated. The minimum difference was then used as the actual phase difference. This was necessary because expres- sion proﬁles with FFT phase values that are phase shifted by 2p are mathematically identical; a phase of p is equivalent to a phase of +p. Two exactly identical expression proﬁles there- fore have a phase difference of zero whereas two completely out of phase proﬁles have a phase difference of p. Pearson correlations of data Pearson correlations were determined by analyzing the smoothed data from above (FFT analysis) using the COR function from R for each pairwise combination of oligo probes. We ﬁrst determined the global median shift value for a given set of strains by calculating the Pearson correlation of every probe to every other probe along a sliding timescale window starting with the ﬁrst timepoint (of one strain probe) and the ﬁnal timepoint (of the second strain probe) until all possible pairwise correlations were computed. The difference in timepoints resulting in the best inter-strain correlation for each probe was noted and the global median value of this difference was then used to shift the proﬁles in the longer dataset. Finally, the longer proﬁles were trimmed to match the shorter proﬁle before calculating the pairwise Pearson correlation. RESULTS Overview of the data To identify potential inter-strain variability in the transcrip- tional program of different P.falciparum strains we character- ized the complete IDC transcriptome for the Dd2 and 3D7 strains and compared them to our previous study of HB3 (10). To measure the IDC transcriptomes of these strains, we used a highly synchronous large-scale culturing methodology featur- ing sampling every hour throughout the 48 h IDC. For each strain, gene expression was measured through one cycle of the IDC using our custom oligo DNA microarray consisting of 7470 spotted elements [described previously (10)]. Time courses were measured for 53, 50 and 48 h, respectively for 3D7, Dd2 and HB3. Gene expression proﬁles from individual oligos on the array were excluded from further analysis if data were lacking for more than 40% of the total time course (Supplementary Tables S3–S5). The resulting dataset con- sisted of 6287 gene expression proﬁles for 3D7, 5294 proﬁles for Dd2, and 6415 proﬁles for HB3. To directly compare the previous HB3 strain data with the new results, we re-examined all of the data by the following two different approaches. Transcription profiles are highly conserved Variation in the gene expression proﬁles among the three IDCs manifested both in changes in amplitude and in the time of maximal expression. We utilized two methodologies to ascer- tain the concordance of individual expression proﬁles for sim- ilar probes between all possible strain pairs. First, given the inherent periodicity in the data, we used a FFT to calculate the relative timing (phase) and amplitude modulation in the expression proﬁles for all genes in the three strains (see Materials and Methods). Most of the FFT results demonstrate very few frequency components contributing to the measured expression proﬁle signal and we used this property of the data to calculate a percent power score for all proﬁles (10). This score, which is equivalent to the percentage of the total power spectrum present in the two primary FFT frequency compon- ents, reﬂects the global periodic nature of the expression pro- ﬁles: genes with a higher percent score are expressed with a period nearing one during the timescale of the IDC ( 48 h). An overview of the highest quality data consisting of all proﬁles with power scores greater than 75% ordered by FFT phase are shown in Figure 1 for the three strains (Supplement- ary Tables S6–S8). The transcriptional program of the P.fal- ciparum IDC is remarkably conserved. The majority of (>75% of all probes) gene expression proﬁles were measured in all three strains as depicted in Table 1. Genes known to belong to the functional categories glycolysis, cytoplasmic transcription, translation, the tricarboxylic acid cycle, the proteasome and plastid biosynthesis are tightly correlated in their gene expres- sion proﬁles as well as in the timing of maximal expression during the IDC. We do note that a global phase shift in gene expression for each strain is apparent due to the initiation point of each time course. As observed in the HB3 strain, the vast majority of the gene transcripts in our datasets for the 3D7 and Dd2 strains are expressed in a highly periodic manner during the IDC (7). The maxima of gene induction occur in a logical order consistent with cellular functions relevant to the pro- gression of the parasitic life cycle. The FFT phase we measure for any periodic wave is always within the range of p to p and reﬂects the timing of periodic gene expression. By measuring the difference in the calculated FFT phase for each pair of oligos between the strains, an estimate of global shift and the true inter-strain variation was analyzed (see Materials and Methods). As expected, the calculated FFT phases do not match exactly, but rather are distributed about a global experimental phase shift, with outliers representing signiﬁcant inter-strain differences in gene expression (Figure 2A). Between HB3 and 3D7, the median phase shift is relatively small at 0.47 or 3/20*p. If 2p repres- ents an 48 h IDC, then this is equivalent to an average timing difference of 3.6 h. Surprisingly, between these two strains we measure a phase difference greater than p/2 ( 12 h) for probes from only 69 open reading frames (ORFs). However, many of