In particular, we first align 454 pyrosequencing reads to the reference sequence using the SEGEMEHL software [30] . Then we extend the reference sequence with a placeholder I for each nucleotide inserted by at least one uniquely aligned read. Similarly, we add a placeholder D to the read sequence for each reference nucleotide missing from the aligned read. Then we perform sequential multiple alignment of the previously aligned reads against this extended reference sequence. Finally, the consensus genome sequence is obtained by (1) replacing each nucleotide in the extended reference with the nucleotide or placeholder in the majority of the aligned reads and (2) removing all I and D placeholders, respectively corresponding to rare insertions and to deletions found in a majority of reads. Reads may contain a small portion of unidentified nucleotides denoted by N'swe treat N as a special allele value matching any of nucleotides A, C, T, G, as well as placeholders I, and D.