e115 Nucleic Acids Research, 2005, Vol. 33, No. 13 PAGE 12 OF 16 Figure 8. Distribution of amino acids within the valid oligonucleotides in genome data. Effects on amino acid level The 25mers from coding regions were further studied on amino acid level. Depending on the location within codons, the oligo-encoded sequence matched with either 7 or 8 amino acids in the protein sequence. The encoded amino acids of the oligonucleotides were compared to general amino acid compositions in each organism. Z-values (Figure 8) indicate strong bias from general pattern. As already seen in the codon usage, C.acetobutylicum and M.tuberculosis along with B.burgdorferi have the largest Z-values. Otherwise, the bacterial genomes have rather even distribution. The yeast, nematode and plant genomes have the largest variation and there are in fact only a few residue types that have normal distribution in these organisms. When looking at the effect of C+G content on the amino acid bias, it is evident that most of the residues have normal distribution. However, there is quite linear correlation between the decrease in alanine and glycine along increased C+G con- tent and opposite effect in lysine. The distribution within the genes was further investigated by calculating the distribution within eight equally sized gene segments (Figure 9). Certain residues such as C, D, H, M, Q and S have very equal distribution in all sections. G has generally somewhat pronounced underrepresentation in the middle of the sequence when compared to the termini. Also in this data, the organ- isms that have greatest bias in the other features are biased, namely the three eukaryotes and C.acetobutylicum, and M.tuberculosis of prokaryotes. CONCLUSIONS A new method for searching unique and valid oligonucleotides from complete genomes was developed. Despite the exhaust- ive analysis approach, genomes of any size could be analyzed. The presented method can be modiﬁed to permit other probe lengths and/or error thresholds. This may affect the run time, which is dependent on the ratio between the permitted number of errors and the probe length. When processing even larger genomes, such as the human genome, one should take into account the fact that using a too large error threshold may lead to a situation where practically all oligonucleotides are non-unique. Unique and valid oligos can clearly be found from any part of the gene, however the termini are overrep- resented. The use of the criteria for valid oligos changes the overall properties of the oligonucleotides. By changing the criteria, the method could be easily modiﬁed for different purposes, e.g. to search for oligos functional in RNAi technology (37).