Bohlin et al. BMC Genomics 2010, 11:464 http://www.biomedcentral.com/1471-2164/11/464 Page 3 of 8 Figure 1 The distributions of GC difference within genomes. The histograms show the distribution of GC difference, Di, (Equation (1) in the Methods section) for eight different microbial genomes. The blue curves are empirical density estimates, while the red curves are Gaussian densities using based on the same means and standard deviations as the empirical estimates. The upper panels show the statistical distributions for four AT rich genomes, while the lower panels show the distributions for four GC rich genomes. within each of the described genomes. With the excep- tion of Carsonella rudii, one of the smallest bacterial gen- omes currently sequenced (~160 kbp), all empirical distributions follow the bell shaped Gaussian curve. This indicates that GC difference within prokaryotic genomes appears to be a sum of many independent processes, giv- ing a Gaussian like distribution according to the central limit theorem (see, for instance, [32]). Thus, it seems likely that for most prokaryotic genomes intra-genomic GC content variation appears to follow a random, white- noise like pattern, devoid of any complex and long-range interacting factors. The GCVAR regression model We define GCVAR as a measure of the intra-genomic GC variation in a genome. A linear regression model was fitted to data for 526 prokaryote chromosomes with GCVAR as the response and with GC content, size, phylum, oxygen requirement, growth temperature, pathogenicity and habi- tat as covariates (Equation (3) in the Methods section). The results of the GCVAR regression model can be observed in Table 1, and in Figure 2 we show the 95% confidence intervals for the significant effects. The vari- ables: size, growth temperature, pathogenicity and habitat had no significant influence on GCVAR, and were there- fore discarded from further analyses. GCVAR in phyla Table 1 shows that GCVAR is significantly influenced by phylum. We find that 10 phyla have GCVAR signifi- cantly above the average phylum, and 4 phyla have GCVAR significantly below average. The two phyla, Cre- narchaeota and Euryarcheota, (both archaea) are among the groups with an above average GCVAR. The archaea domain, consisting predominantly of organisms living in extreme environments, had a significantly higher GCVAR than bacteria (p < 0.001). The highest GCVAR are found in the aquatic group Cyanobacteria, which is largely populated with species capable of photosynthesis [33]. The lowest GCVAR are found in the phylum of the aquatic Planctomycetes, but this group is only based on one single genome, therefore no conclusions can be assumed at the phylum level. Environmental factors and phylogenetic bias To examine how GCVAR was affected by phylogenetic bias a regression model similar to the one described above was fitted, i.e. GCVAR was the response variable,