The paper presents improvements in the accuracy of multiple sequence alignment using MAFFT version 5.3. The new version introduces iterative refinement options (G-INS-i, H-INS-i, and F-INS-i) that incorporate pairwise alignment information into the objective function, enhancing alignment accuracy compared to previous methods like TCoffee and CLUSTAL W. These new options are particularly effective for alignments involving a large number of sequences and can handle hundreds of sequences on standard desktop computers. The study also evaluates the impact of including homologous sequences in the alignment, showing that adding close homologues can improve accuracy by 2-10 percentage points for alignments with low similarity. Additionally, the paper introduces a Ruby script (mafftE.rb) that automatically aligns input sequences with their close homologues from databases. The results demonstrate that the new options of MAFFT significantly improve alignment accuracy, especially for large and distantly related sequences.The paper presents improvements in the accuracy of multiple sequence alignment using MAFFT version 5.3. The new version introduces iterative refinement options (G-INS-i, H-INS-i, and F-INS-i) that incorporate pairwise alignment information into the objective function, enhancing alignment accuracy compared to previous methods like TCoffee and CLUSTAL W. These new options are particularly effective for alignments involving a large number of sequences and can handle hundreds of sequences on standard desktop computers. The study also evaluates the impact of including homologous sequences in the alignment, showing that adding close homologues can improve accuracy by 2-10 percentage points for alignments with low similarity. Additionally, the paper introduces a Ruby script (mafftE.rb) that automatically aligns input sequences with their close homologues from databases. The results demonstrate that the new options of MAFFT significantly improve alignment accuracy, especially for large and distantly related sequences.