The MAFFT online service provides multiple sequence alignment (MSA) tools for biological sequence analysis. As sequencing technologies advance, the need for accurate MSAs with large numbers of sequences is increasing. The service offers various options for aligning large datasets, including PartTree, DPartTree, FFT-NS-1, FFT-NS-2, mafft-sparsecore, G-INS-1, and Pileup. These methods vary in computational efficiency and accuracy, with some designed for speed and others for higher precision. The service also includes interactive tools for sequence selection, refinement, and visualization, allowing users to manually choose sequences and refine alignments. Users can add new sequences to existing alignments using different options, such as --add, --addfull, --addfragments, and --addlong. The service supports multithreading and low-memory modes for handling large datasets efficiently. Interactive sequence selection and visualization tools, such as Phylo.io and MSAViewer, are available for exploring and refining alignments. The service also includes features for phylogenetic tree inference and visualization. The effectiveness of MSAs depends on data size and sequence similarity, with some methods performing better for smaller or more similar datasets. The service is supported by the Japan Society for the Promotion of Science and the Japan Agency for Medical Research and Development. The online service provides a user-friendly interface for performing MSA, with detailed documentation and command-line options for advanced users.The MAFFT online service provides multiple sequence alignment (MSA) tools for biological sequence analysis. As sequencing technologies advance, the need for accurate MSAs with large numbers of sequences is increasing. The service offers various options for aligning large datasets, including PartTree, DPartTree, FFT-NS-1, FFT-NS-2, mafft-sparsecore, G-INS-1, and Pileup. These methods vary in computational efficiency and accuracy, with some designed for speed and others for higher precision. The service also includes interactive tools for sequence selection, refinement, and visualization, allowing users to manually choose sequences and refine alignments. Users can add new sequences to existing alignments using different options, such as --add, --addfull, --addfragments, and --addlong. The service supports multithreading and low-memory modes for handling large datasets efficiently. Interactive sequence selection and visualization tools, such as Phylo.io and MSAViewer, are available for exploring and refining alignments. The service also includes features for phylogenetic tree inference and visualization. The effectiveness of MSAs depends on data size and sequence similarity, with some methods performing better for smaller or more similar datasets. The service is supported by the Japan Society for the Promotion of Science and the Japan Agency for Medical Research and Development. The online service provides a user-friendly interface for performing MSA, with detailed documentation and command-line options for advanced users.