We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.

Users can check the guide tree by using the ––treeout option. In the case of ––addfragments, the estimated phylogenetic positions of new sequences are shown together with the estimated tree of the existing alignment. The alignment calculation is performed based on this phylogenetic estimation. It is also possible to compute such phylogenetic information only, without alignment, by the ––retree 0 option. An example of output is shown in Figure 3A.Fig. 3.