Two methods to add unaligned sequences into an existing multiple sequence alignment have been implemented as the '--add' and '--addfragments' options in the MAFFT package. The former option is a basic one and applicable only to full-length sequences, whereas the latter option is applicable even when the unaligned sequences are short and fragmentary. These methods internally infer the phylogenetic relationship among the sequences in the existing alignment and the phylogenetic positions of unaligned sequences. Benchmarks based on two independent simulations consistently suggest that the "--addfragments" option outperforms recent methods, PaPaRa and PAGAN, in accuracy for difficult problems and that these three methods appropriately handle easy problems.
Supplementary information: Supplementary data are available at Bioinformatics online.