The second-step processes the ambiguous bases. For bases confirmed to be mutations through NHIP analysis and satisfy the substitution bias rule, they are graded as high quality sequence calls and denoted in the FASTA sequence as UPPER CASE characters. For bases confirmed to be an isolated error through NHIP analysis and also satisfy the substitution bias rule, they are graded as low confidence sequence calls and denoted in the FASTA sequence in lower case characters. The rest of the bases are called 'N' in the FASTA sequence. The flowchart of EvolSTAR is shown in Figure 1 .