BMC Genomics 2009, 10:640 http://www.biomedcentral.com/1471-2164/10/640 (6) (7) where k1 = 1/6000 and k2 = 1/600 as in GCSI. SA is the normalised spectral amplitude at 1-Hz, which is equiva- lent to PS(1). SA = k4(k3PS(1))a where k3 = 600000, k4 = 40, and α = 0.4, as calculated by regression analysis. Normalised dist, dist(norm), is calculated as follows: dist(norm) = dist × 4096 / W where W is the number of windows used in the analysis. Calculation of z-score and p-value Because the gGCSI is independent of the window size and number of windows, the significance of the gGCSI value should be noted to determine whether the number of windows used in the analysis is statistically sufficient to give the resulting value. Therefore, the significance meas- ure is calculated from the distribution of gGCSI values for a shuffled input signal. For a given discrete GC skew signal f(n), 100 randomly shuffled series f'(n) are generated for which the gGCSI is calculated. Iteration size of 100 is cho- sen by default for computational efficiency, and this number can be configured when necessary. Then, the sig- nificance of the gGCSI based on the original GC skew sig- nal f(n) is statistically assessed using the z-score based on the shuffled iterations, from which the p-value is obtained. Normal distribution of shuffled iterations was confirmed with Kolmovorov-Smirnov-Lillifors test with p < 0.001, for all genomes used in this work. Because re- sampling methods change the necessary window num- bers/sizes and the coordination of genomic loci and because purely random values ignore the effects of diverse GC content, we have chosen this parametric statistic. List of abbreviations dist: Euclidean distance; FFT: fast Fourier transform; GCSI: GC skew index; gATSI: generalised AT skew index; gGCSI: generalised GC skew index; gKetoSI: generalised; Keto skew index; gPurineSI: generalised Purine skew index; RCR: rolling circle replication; SA: spectral amplitude; SR: spectral ratio; S/N: signal to noise ratio; r: Pearson product moment correlation coefficient; ρ: Spearman rho rank correlation coefficient. Authors' contributions KA developed and validated the gGCSI and carried out the analysis with RCR plasmids. HS analysed the correlation of gGCSI of plasmids and their host chromosomes. MT supervised the project. All authors have read and approved the final manuscript. Additional material Additional file 1 Supplemental Figures Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-640-S1.PDF] Acknowledgements This research is supported by the Grant-in-Aid for Young Scientists No.20710158 from the Japan Society for the Promotion of Science (JSPS), as well as funds from the Yamagata Prefectural Government and Tsuruoka City. References 1. Couturier E, Rocha EP: Replication-associated gene dosage effects shape the genomes of fast-growing bacteria but only for transcription and translation genes. Mol Microbiol 2006, 59(5):1506-1518. 2. Rocha EP: The replication-related organization of bacterial genomes. Microbiology 2004, 150(Pt 6):1609-1627. 3. Lobry JR, Louarn JM: Polarisation of prokaryotic chromosomes. Curr Opin Microbiol 2003, 6(2):101-108. 4. Arakawa K, Uno R, Nakayama Y, Tomita M: Validating the signifi- cance of genomic properties of Chi sites from the distribu- tion of all octamers in Escherichia coli. 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