BMC Genomics 2009, 10:640 http://www.biomedcentral.com/1471-2164/10/640 number in all bacteria is 1024, which corresponds to the median of 2511 bp/window. Therefore, if a genome is suf- ficiently large, use of 1024 windows (2511 bp/window) produces the most accurate gGCSI value. Although the basic concept of integrating the Fourier power spectrum to capture the "shape" of the GC skew graph and the Euclidean distance between base composi- tions of leading and lagging strands remain unchanged in the gGCSI, this new index introduces several new calcula- tion methodologies compared with the original GCSI, such as the use of a geometric mean and the calculation of SA without taking the S/N ratio. To test whether this new index can be used interchangeably with the original index, we have plotted the gGCSI value against the GCSI value for 822 complete bacterial chromosomes, using 4096 windows for the calculation of both indices (Figure 1). The two indices are highly correlated (Pearson product moment correlation coefficient, r = 0.993; Spearman rho rank correlation coefficient, ρ = 0.997), and therefore sev- eral criteria identified in a previous analysis (e.g., visible GC skew when GCSI > 0.1 and the absence of GC skew when GCSI < 0.05) can be applied to the gGCSI. SA and dist are generally correlated, and majority of the genomes exhibit dist/SA ratio of around 0.184 (Supple- mental Figure S2 [see Additional File 1]. However, this ratio varies by about 10-fold among the genomes, so that the geometric mean better captures the balance between 0.7 0.6 0.5 )w0.4 e n ( ISC0.3 G g 0.2 0.1 0 0 0.3 0.4 GCSI (old) 0.1 0.2 0.5 0.6 0.7 FCiogrurreelat1ion of old and new GC skew index (GCSI) values Correlation of old and new GC skew index (GCSI) values. Plot of GCSI (X-axis) and gGCSI (Y-axis) values for 822 bacterial chromosomes with 4096 windows. the two indices than the arithmetic mean: (10x + x)/2 = 5.5x, whereas 10x × x ≈ 3.3x . When GC skew continu- ously exists along one strand of the genome and does not shift its polarity, the strand results in extremely high dist while SA is low, deviating from the above dist/SA ratio. The genomes of Pseudoalteromonas haloplanktis TAC125 and Halorhodospira halophila SL1 are good examples for such continuously biased genomes, that show gGCSI < 0.1 with geometric mean, but exceed this threshold when calculated with arithmetic mean. This deviation is more pronounced with RCR plasmids that have the same non- shifting GC skew. Sixteen RCR plasmids used in this work showed gGCSI > 1.0 (with maximum of 1.544) when cal- culated with arithmetic mean, but the use of geometric mean limits to only one genome exceeding gGCSI > 1.0, with 1.069. Difference in GC skew strength between eubacteria and archaea with different types of replication machinery As an application of the comparative capabilities of gGCSI, we investigated the effects of replication machin- ery on the degree of genomic compositional asymmetry. Genomic polarity in circular eubacterial genomes is attrib- uted to bi-directional replication machinery starting from a finite single origin of replication, and thus GC skew is not observable in most archaeal genomes that contain multiple replication origins [32]. We have plotted the gGCSI values and corresponding z-scores for 822 eubacte- ria and archaea using 512 windows (Figure 2). Archaeal chromosomes represented by closed red circles are clus- tered around the lower left corner where gGCSI < 0.1 and z-score < 5, indicating the lack of selection pressure caused by bi-directional replication. Of the top ten archaeal chro- mosomes with high gGCSI values, only seven were signif- icant (p < 0.01), including two human intestinal archaea Methanobrevibacter smithii(gGCSI = 0.315, z = 13.5) and Methanosphaera stadtmanae (gGCSI = 0.117, z = 7.06) that are reported to have visible GC skew, suggesting a single origin of replication for each [33]. Two Halobacterium spe- cies (gGCSI = 0.121, z = 18.2 and 17.1) had significantly high gGCSI values; for these species, multiple replication origins were suggested by computational analyses [34,35], but experimental validation through insertion of putative origins into non-replicating plasmid confirmed only one to be active in vivo [36]. Pyrococcus horikoshii (gGCSI = 0.140, z = 7.01) and Pyrococcus abyssi (gGCSI = 0.074, z = 3.11), for which the existence of only a single origin of replication has been extensively studied [37-41], also had significantly high gGCSI values. Although Meth- anococcus aeolicus (gGCSI = 0.107, z = 4.62) has no pub- lished evidence suggesting or confirming a single origin of replication, its gGCSI score suggests a high likelihood of bi-directional replication, which is supported by the V- Page 5 of 10 (page number not for citation purposes)