Disequilibrium between complementary bases ratio. A. Error rate at each cycle of sequencing. Each tag was aligned on the genome using align0 and the error rate defined by counting the number of differences between the bases called and the reference at the corresponding position. Black is the error rate for Solexa-called tags, blue for Rolexa tags called using only the ACGT alphabet and green for Rolexa-called tags with IUPAC codes. B. Proportion of bases A, C, G and T at each position in the tags for Solexa base calling (dashed lines) and Rolexa base calling (continuous line). The complementary A and T proportions are different (ratio is not 1) and are degrading along the sequences (lines drift apart). The proportions are less dependent on position with Rolexa base calling, although the ratios remain different from 1. Label on y-axis is wrong. Panels C-D focuses on tags "rescued" by Rolexa base calling, namely those tags that could not be mapped on the genome after Solexa base calling, but had a matching position via Rolexa base calling. C. The distribution of substitutions between the Solexa tags and the corresponding Rolexa tags shows a predominance of C to A and T to G substitutions which is consistent with a re-equilibration of the base complementarity.D. Introducing one to six mutations in the Solexa tags with the same frequencies as the Rolexa algorithm at random positions only rescues about 2% of the tags that were rescued by Rolexa with the same number of ambiguous bases (green bars).