BMC Genomics 2009, 10:640 http://www.biomedcentral.com/1471-2164/10/640 terminus [3]. Accordingly, many genomic features exhibit characteristic replication-related organisation, including the nucleotide compositional bias, distribution of signal oligonucleotides such as Chi sites [4,5] and KOPS motifs [6,7], as well as gene positioning and strand preference [8]. Nucleotide compositional asymmetry in the leading and lagging strands has been extensively studied using GC skew analysis, which calculates the excess of C over G nor- malised to the GC content ([C-G]/[C+G]) along the chro- mosome [9,10]. In many bacterial genomes, GC skew graphs "shift" their polarity between the two replichores, and thus the shift points of GC skew correspond to the replication origin and terminus. Analysis of the GC skew of a bacterial chromosome is therefore useful for the pre- diction of its replication origin and terminus [11] and, subsequently, its leading and lagging strands. The putative position of the replication origin predicted by computa- tional methods based on GC skew is frequently used to define the first base position of circular genome sequences in many genome projects as an accurate and effective alternative to experimental means. Moreover, the polari- sation of nucleotide composition is suggested to affect the replication-directed architecture of genomes. This includes the aforementioned replication-oriented sequence elements and gene orientation [13]; therefore, the degree of strand-specific mutational bias observed with GC skew analysis can be used as a reference for muta- tion or selection pressures that a genome receives due to the replication machinery [12-15]. Bacterial species exhibit highly diverse GC skew [16]. Many fast-growing bacteria show extremely biased GC skew, whereas only weak skew can be discerned in the chromosomes of slow-growing bacteria [17-19]. There- fore, the prediction of the replication origin with GC skew could be erroneous in genomes with only weak skew, requiring a quantitative confidence measure of GC skew strength. In order to allow comparative study of the degree of GC skew in bacterial genomes, we have previously reported the GC skew index (GCSI), which quantifies the strength of GC skew in given bacterial chromosomes and can be used as a confidence measure for GC skew-based predictions or for the comparative study of replication- related mutation or selection pressures in bacterial chro- mosomes [20]. The GCSI ranges from 0 to 1 and is calcu- lated as an arithmetic mean of two indices: spectral ratio (SR) and dist. SR is the signal/noise (S/N) ratio of the 1 Hz signal in the Fourier power spectrum of a GC skew graph; it captures the fitness of the shape of the GC skew graph to be partitioned into two segments of opposite polarity having equal length (a discrete sine curve) [21], and dist measures the Euclidean distance between the two vertices in cumulative GC skew graphs. SR is essential for accurate quantification of a weak GC skew whose dist is affected by local regions of biased nucleotide content, such as large insertions. In order to eliminate the effects of biased nucleotide composition in coding regions, the GCSI is cal- culated with a fixed number of windows (4096, consider- ing an average gene length of 1 kbp and a genome size of 2 to 4 Mbp). This use of a fixed number of windows limits the applicability of the GCSI to bacterial chromosomes and does not allow it to be used for shorter sequences, such as plasmids. Many plasmids are circular DNA mole- cules that exhibit nucleotide compositional asymmetry. GC skew is therefore frequently utilised for the prediction of replication origins in plasmids, which creates a need for extended applicability of the GCSI. Circular plasmids can be categorised into two groups according to their replication machineries: theta and roll- ing circle replication (RCR). Theta replication requires the Rep protein and characteristic origins as well as DNA polymerase I from the host bacterium [22]. When there is only one origin of replication, theta replication results in two replichores of opposite polarity due to bi-directional replication forks, and hence these plasmids exhibit GC skew. Therefore, the shift points of the GC skew are indic- ative of the positions of the replication origin and termi- nus. The other type of replication, RCR, requires the RepABC family of proteins, and replication occurs through strand displacement [23-25]. In RCR, one of the two strands is always the template, and therefore plasmids that undergo RCR usually do not show significant GC skew. Instead, RCR plasmids show continuously biased nucleotide composition, resulting in linear cumulative GC skew, as opposed to the V-shaped graph observed in genomes with GC skew that indicates the existence of clear shift points. It has been suggested that any genetic elements that repro- duce inside the cell (chromosomes, plasmids, and phages) using the same replication machinery might have the same nucleotide composition and that recently acquired elements with unusual nucleotide compositions would drift towards the average nucleotide composition of the host genome by amelioration [26,27]. To investi- gate the evolution of plasmids in their hosts, comparisons have been made at the levels of GC content [28,29] and dinucleotide composition [30,31], but not from the view- point of replication strand asymmetry. To this end, here we report a novel quantitative measure of GC skew strength called the generalised GC skew index (gGCSI) that is independent of window size and is there- fore applicable to comparative studies of genomes of any length. Using this new index, we show discriminant crite- ria for the replication machinery of plasmids and the cor- relation of the degree of replication-related mutation or selection pressures in the host chromosome and plas- mids. Page 2 of 10 (page number not for citation purposes)