To validate the software, we hybridized 14 patient samples in duplicate onto the microarray. The microarrays were analysed in parallel using Nimblescan (PBC algorithm) and EvolSTAR, and the sequences obtained were compared to Sanger capillary sequencing. We counted the number of true-non-mutation calls, true-mutation calls, error calls and ambiguous ('N') calls for both methods ( Table 2 ; Supplementary Data File 3). We also confirmed that the substitution bias in all 14 duplicate hybridization experiments (Table 3) were consistent with that found in Table 1 . Compared with the available capillary sequences for the 14 samples, EvolSTAR had an average error rate of 0.0029% and 12 ambiguous calls per sample (346 in total). This is far superior than Nimblescan PBC, where we obtained an average error rate of 0.083% and 158 ambiguous calls per sample (4434 in total). Furthermore, EvolSTAR called all true mutations correctly. The genome coverage attained by EvolSTAR (99.02 ± 0.82%) is also much higher than that of Nimblegen PBC (94.3 ± 6.06%).