PAGE 9 OF 16 Nucleic Acids Research, 2005, Vol. 33, No. 13 e115 were analyzed. The signiﬁcance of the observations was estim- ated by calculating the Z-values based on normal distribution. The Z-values indicate the statistical bias in each position for the proportion of each base type. The nucleotide distributions follow well the Chargaff’s ﬁrst parity rule for duplex DNA (%A = %T and %C = %G) (49) and the Chargaff’s second parity rule for single-stranded DNA (50,51) (Figure 3A–D). It is of interest that the curves pass through almost a single point when traversing from T to C ratio. The major differences to the parity rules is the genome of A.fulgidus, which is the only one where the ratios for A and T, and C and G are not close to each other. The use of criteria to choose for valid oligos biases the dis- tribution in B.burgdorferi, which has quite low C+G content. This seems to be related to nucleotide composition because the distribution of Buchera sp., which has the lowest C+G content among the analyzed genomes, has also slightly biased U-shaped distribution. When looking at the actual differences compared to normal distributions, the biggest change can be seen in C.acetobutylicum and the other genomes with extreme C+G values (Figure 3E and F). The criteria for valid oligos signiﬁcantly reduce the number of oligos (Table 2). Valid/unique oligo ratio is from 0.47 to 0.79. Valid/invalid oligo ratio for genome data is from 0.036 to 1.11. Further analysis of the nucleotide numbers in oligonuc- leotides indicated that the distribution in the majority of bac- terial and archaeal genomes was as expected (Figure 4). The major exceptions were M.tuberculosis and C.acetobutylicum. Of these, M.tuberculosis has the highest C+G content among the analyzed genomes. It has more than expected number of oligonucleotides with 4–11 A, or 5–10 T, or 6–7 C, or 5–8 G bases among the valid oligos for genome data. On the other hand, oligonucleotides with large numbers of C or G are in fact underrepresented. The distributions of yeast, C.elegans and A.thaliana are all very biased. Common to all these is the underrepresentation of small numbers of nucleotides in oligos, overrepresentation usually in the range 3–8 and again underrepresentation in the range 9–19 nt. The actual borders of these patterns vary between nucleotide types and organisms. Interestingly, the B