EvolSTAR employs a two-step process for base-calling ( Figure 1 ). In the first step, each base query is scrutinized for signs of hybridization intensity abnormalities. If the gain-of-signal of the query base is strong and has no mutation, the base is called. In the second step, EvolSTAR then tries to recover base queries that have any hybridization intensity abnormalities with two analysis methods, namely neighbourhood hybridization intensity profile analysis and nucleotide substitution bias analysis.