e115 Nucleic Acids Research, 2005, Vol. 33, No. 13 Different computer setups were used for calculations and analysis. The genomes of prokaryotes as well as of S.cerevisiae were processed on a single PC with 1 GB RAM. The use of large enough memory facilitated storage of the complete genome and avoidance of excessive I/O operations. The two eukaryotes with larger genomes, C.elegans and A.thaliana, were processed in parallel on a Linux cluster of 10 PCs. The processing time was 3 days for A.thaliana and somewhat <2 days for C.elegans. It is thus feasible to search unique and valid probes for any organism. Unique and valid probes The analysis was divided into two parts to obtain full picture of the properties and distribution of unique and valid oligos in a single strand and in both strands. We determined unique and valid oligos both for CDS regions (in eukaryotes together with 50 and 30 extensions of 100 bp) and for the complete genome. If not otherwise stated, the results refer to genome-wide analysis. The proportion of unique oligonucleotides varied between 18.2 and 59.4% (25.3 and 83.6%) depending on the organism A PAGE 8 OF 16 being smaller for the larger genomes. The corresponding values for valid oligos are 3.5 and 52.5 (18.2 and 52.3%). The numbers in parentheses are for CDS regions. Unique and valid probes were found for at least 92% of the genes, which is in agreement with the theoretical calculations based on the size of the genome and density of the genes (data not shown). The number of redundant probes exceeded signiﬁc- antly the number of valid oligos for all the eukaryotes analyzed as well as A.tumefaciens, N.meningitidis and M.tuberculosis. The total number of valid oligos per gene was high, the aver- age varying from 51 to 537. The use of annealing and com- position criteria clearly reduced the number of valid oligos compared to unique ones. Naturally, the use of more stringent edit distance has similar effect (Figure 2). C+G content has no direct effect on the number or ratios of valid and redundant oligos. Nucleotide distribution To analyze the properties of the oligos, the distribution of nucleotides within the unique and valid oligonucleotides