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MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability
Open accessAbstract
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.
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“We conducted the analysis twice (e.g. the GUIDANCE2 filter and subsequent PAML analysis) using different aligners: (1) Muscle version 3.8.31 (Edgar, 2004) and (2) Mafft version X7.721 (Katoh and Standley, 2013).”
From Genomic and physiological mechanisms underlying skin plasticity during water to air transition in an amphibious fish (2020) · cites this paper as Katoh and Standley, 2013
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“Because the alignments produced by BLS are very different from the ones produced by state-of-the-art software, such as MUSCLE version 5.1 (ref. 28) or MAFFT version 7.490 (ref. 29), it is difficult to fairly compare them as alignments.”
From Improving microbial phylogeny with citizen science within a mass-market video game (2025) · cites this paper as [29]
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“The three mutations in S1 (N501Y, A570D, ΔH69/V70) did not appear to impact neutralisation in a pseudovirus assay, consistent with data on N501Y having little effect on nuetralisation by convalescent and post vaccination sera40.”
From Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies (2021) · cites this paper as [40]
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MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform
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“453 (19) software and manually edited using MEGA v6.”
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Analysis and benchmarking of small and large genomic variants across tandem repeats
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Mutations that adapt SARS-CoV-2 to mink or ferret do not increase fitness in the human airway
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The metabolic network of the last bacterial common ancestor
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Transposon-encoded nucleases use guide RNAs to promote their selfish spread
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Evolutionary Histories of Type III Polyketide Synthases in Fungi
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“…KOG2314 (translation initiation factor 3, subunit b eIF-3b) were retrieved from the JGI Mycocosm repository for all genomes that contain a type III PKS. Nucleotide sequences were aligned using MAFFT (Katoh and Standley, 2013) and phylogenetic trees were built using FastTree (Price et al., 2010).”
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Genetic diversity of Anopheles stephensi in Ethiopia provides insight into patterns of spread
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“In order to analyze sequence similarities among different ASVs, multiple sequence alignments were conducted by using the QIIME2 alignment MAFFT [35] against the NCBI database [36,37].”
MethodA phylogenomic supermatrix of Galliformes (Landfowl) reveals biased branch lengths
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Ancient DNA Reveals Evidence of Abundant Aurochs (Bos Primigenius) in Neolithic Northeast China
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Evolutionary dynamics of the chromatophore genome in three photosynthetic Paulinella species
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Evaluating the Accuracy and Efficiency of Multiple Sequence Alignment Methods
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Screening blood-fed mosquitoes for the diagnosis of filarioid helminths and avian malaria
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CRISPR-Cas systems in multicellular cyanobacteria
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Peng, J., Liu, J., Mann, S. A., Mitchell, A. M., Laurie, M. T., Sunshine, S., Pilarowski, G., Ayscue, P., Kistler, A., Vanaerschot, M., et al. (2022) · Clinical Infectious Diseases · doi:10.1093/cid/ciab283 · Cited by 41
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Coincident rapid expansion of two SARS-CoV-2 lineages with enhanced infectivity in Nigeria
Ozer, E. A., Simons, L. M., Adewumi, O. M., Fowotade, A. A., Omoruyi, E. C., Adeniji, J. A., Dean, T. J., Zayas, J., Bhimalli, P. P., Ash, M. K., et al. (2021) · doi:10.1101/2021.04.09.21255206 · Cited by 34
The origin and evolution of methanogenesis and Archaea are intertwined
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The transposon-encoded protein TnpB processes its own mRNA into ωRNA for guided nuclease activity
Nety, S. P., Altae-Tran, H., Kannan, S., Demircioglu, F. E., Faure, G., Hirano, S., Mears, K., Zhang, Y., Macrae, R. K., & Zhang, F. (2023) · The CRISPR Journal · doi:10.1089/crispr.2023.0015 · Cited by 35
“RNA sequences were aligned using mafft-xinsi.(21) RNAalifold(32) and R2R(33) were used to generate covariance models.”
MethodInfluentialFrom primordial clocks to circadian oscillators
Pitsawong, W., Pádua, R. A. P., Grant, T., Hoemberger, M., Otten, R., Bradshaw, N., Grigorieff, N., & Kern, D. (2023) · Nature · doi:10.1038/s41586-023-05836-9 · Cited by 37
Oxygen metabolism in descendants of the archaeal-eukaryotic ancestor
Appler, K. E., Lingford, J. P., Gong, X., Panagiotou, K., Leão, P., Langwig, M. V., Greening, C., Ettema, T. J. G., De Anda, V., & Baker, B. J. (2026) · Nature · doi:10.1038/s41586-026-10128-z · Cited by 36
Oxygen metabolism in descendants of the archaeal-eukaryotic ancestor
Appler, K. E., Lingford, J. P., Gong, X., Panagiotou, K., Leão, P., Langwig, M., Greening, C., Ettema, T. J. G., De Anda, V., & Baker, B. J. (2024) · doi:10.1101/2024.07.04.601786 · Cited by 36
Functional differences between rodent and human PD-1 linked to evolutionary divergence
Masubuchi, T., Chen, L., Marcel, N., Wen, G. A., Caron, C., Zhang, J., Zhao, Y., Morris, G. P., Chen, X., Hedrick, S. M., et al. (2025) · Science Immunology · doi:10.1126/sciimmunol.ads6295 · Cited by 36
Tomofuji, Y., Kishikawa, T., Maeda, Y., Ogawa, K., Otake-Kasamoto, Y., Kawabata, S., Nii, T., Okuno, T., Oguro-Igashira, E., Kinoshita, M., et al. (2022) · Cell Genomics · doi:10.1016/j.xgen.2022.100219 · Cited by 35
Michelini, S., Modesto, M., Filippini, G., Spiezio, C., Sandri, C., Biavati, B., Pisi, A., & Mattarelli, P. (2016) · Systematic and Applied Microbiology · doi:10.1016/j.syapm.2016.04.005 · Cited by 35
Nishimura, T., Fakim, H., Brandmann, T., Youn, J.-Y., Gingras, A.-C., Jinek, M., & Fabian, M. R. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky1011 · Cited by 31
Figueroa-Martinez, F., Nedelcu, A. M., Smith, D. R., & Reyes-Prieto, A. (2017) · Plant Physiology · doi:10.1104/pp.16.01628 · Cited by 34
“Multiple alignments of 16S rRNA and 18S rRNA sequences were generated with MAFFT version 7 (Katoh and Standley, 2013).”
MethodA MIR4646 associated methylation locus is hypomethylated in adolescent depression
Boström, A. E., Ciuculete, D.-M., Attwood, M., Krattinger, R., Nikontovic, L., Titova, O. E., Kullak-Ublick, G. A., Mwinyi, J., & Schiöth, H. B. (2017) · Journal of Affective Disorders · doi:10.1016/j.jad.2017.05.017 · Cited by 33
Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity
Guo, H., Stamper, E. L., Sato-Carlton, A., Shimazoe, M. A., Li, X., Zhang, L., Stevens, L., Tam, K. J., Dernburg, A. F., & Carlton, P. M. (2022) · eLife · doi:10.7554/elife.77956 · Cited by 32
Xie, R., Zan, X., Chu, L., Su, Y., Xu, P., & Liu, W. (2023) · BMC Bioinformatics · doi:10.1186/s12859-023-05237-9 · Cited by 32
Computed cancer interactome explains the effects of somatic mutations in cancers
Zhang, J., Pei, J., Durham, J., Bos, T., & Cong, Q. (2022) · Protein Science · doi:10.1002/pro.4479 · Cited by 31
Pittis, A. A., Goh, V., Cebrian-Serrano, A., Wettmarshausen, J., Perocchi, F., & Gabaldón, T. (2020) · Nature Communications · doi:10.1038/s41467-020-17705-4 · Cited by 29
Jones, B. R., Miller, R. L., Kinloch, N. N., Tsai, O., Rigsby, H., Sudderuddin, H., Shahid, A., Ganase, B., Brumme, C. J., Harris, M., et al. (2020) · Journal of Virology · doi:10.1128/jvi.01786-19 · Cited by 29
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Nori, S. R. C., Walsh, C. J., McAuliffe, F. M., Moore, R. L., Van Sinderen, D., Feehily, C., & Cotter, P. D. (2025) · npj Biofilms and Microbiomes · doi:10.1038/s41522-025-00682-1 · Cited by 29
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Samarra, A., Cabrera-Rubio, R., Martínez-Costa, C., & Collado, M. C. (2024) · Gut Microbes · doi:10.1080/19490976.2024.2357176 · Cited by 25
Boström, A. E., Chatzittofis, A., Ciuculete, D.-M., Flanagan, J. N., Krattinger, R., Bandstein, M., Mwinyi, J., Kullak-Ublick, G. A., Öberg, K. G., Arver, S., et al. (2020) · Epigenetics · doi:10.1080/15592294.2019.1656157 · Cited by 25
Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response
Ruperti, F., Becher, I., Stokkermans, A., Wang, L., Marschlich, N., Potel, C., Maus, E., Stein, F., Drotleff, B., Schippers, K. J., et al. (2024) · Current Biology · doi:10.1016/j.cub.2023.12.021 · Cited by 23
Bifidobacteriaceae diversity in the human microbiome from a large-scale genome-wide analysis
Pasolli, E., Mauriello, I. E., Avagliano, M., Cavaliere, S., De Filippis, F., & Ercolini, D. (2024) · Cell Reports · doi:10.1016/j.celrep.2024.115027 · Cited by 22
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Dudas, G., Hong, S. L., Potter, B., Calvignac-Spencer, S., Niatou-Singa, F. S., Tombolomako, T. B., Fuh-Neba, T., Vickos, U., Ulrich, M., Leendertz, F. H., et al. (2021) · doi:10.1101/2021.05.04.21256637 · Cited by 76
“These sequences were aligned in MAFFT (FFT-423 NS-2 setting; Katoh and Standley (2013)) with insertions relative to reference removed, and 5’ and 3’424 untranslated regions of the genome that were susceptible to sequencing and assembly error trimmed.”
MethodLugli, G. A., Calvete-Torre, I., Alessandri, G., Milani, C., Turroni, F., Laiolo, P., Ossiprandi, M. C., Margolles, A., Ruiz, L., & Ventura, M. (2021) · Systematic and Applied Microbiology · doi:10.1016/j.syapm.2021.126273 · Cited by 21
Socioeconomic position links circulatory microbiota differences with biological age
Craven, H., McGuinness, D., Buchanan, S., Galbraith, N., McGuinness, D. H., Jones, B., Combet, E., Mafra, D., Bergman, P., Ellaway, A., et al. (2021) · Scientific Reports · doi:10.1038/s41598-021-92042-0 · Cited by 21
Ploidy Variation and Spontaneous Haploid-Diploid Switching of Candida glabrata Clinical Isolates
Zheng, Q., Liu, J., Qin, J., Wang, B., Bing, J., Du, H., Li, M., Yu, F., & Huang, G. (2022) · mSphere · doi:10.1128/msphere.00260-22 · Cited by 20
Population genomics of the muskox' resilience in the near absence of genetic variation
Pečnerová, P., Lord, E., Garcia‐Erill, G., Hanghøj, K., Rasmussen, M. S., Meisner, J., Liu, X., van der Valk, T., Santander, C. G., Quinn, L., et al. (2024) · Molecular Ecology · doi:10.1111/mec.17205 · Cited by 19
Mamouei, Z., Singh, S., Lemire, B., Gu, Y., Alqarihi, A., Nabeela, S., Li, D., Ibrahim, A., & Uppuluri, P. (2021) · PLOS Biology · doi:10.1371/journal.pbio.3000957 · Cited by 19
Xue, Z., Brooks, J. T., Quart, Z., Stevens, E. T., Kable, M. E., Heidenreich, J., McLeod, J., & Marco, M. L. (2021) · mSystems · doi:10.1128/msystems.01114-20 · Cited by 17
Dong, Y.-W., Blanchard, T. S., Noll, A., Vasquez, P., Schmitz, J., Kelly, S. P., Wright, P. A., & Whitehead, A. (2020) · Journal of Experimental Biology · doi:10.1242/jeb.235515 · Cited by 18
Metadata
- Authors
- Katoh, K., & Standley, D. M.
- Affiliations
- Immunology Frontier Research Center, The University of Osaka (JP)
- Corresponding author
- Katoh, K.
- Venue
- Molecular Biology and Evolution
- Publisher
- Oxford University Press (OUP)
- Published
- 2013-01-16
- Volume
- 30
- Issue
- 4
- Pages
- 772-780
- ISSN
- 0737-4038 (ISSN-L)
- 1537-1719
- PMC
- PMC3603318
- OpenAlex
- W2160378127
- Cited by
- 46,209
- References
- 49
- Citations by year
- 2026: 5174 · 2025: 5549 · 2024: 5538 · 2023: 4824 · 2022: 5005 · 2021: 5291 · 2020: 4177 · 2019: 3143 · 2018: 2383 · 2017: 1916 · 2016: 1503 · 2015: 981 · 2014: 574 · 2013: 112
- FWCI
- 473.96
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by-nc
- Locations
- Molecular Biology and Evolution (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Funders
- National Institute of Advanced Industrial Science and Technology
- Ministry of Education, Culture, Sports, Science and Technology
- Agriculture and Agri-Food Canada
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fractal and DNA sequence analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- MAFFT, multiple sequence alignment, DNA sequence alignment, parallel computing, alignment errors, alignment software
- MeSH terms
- Algorithms, Amino Acid Sequence, Base Sequence, DNA, Fungal, DNA, Ribosomal, Fungi, Humans, Models, Genetic, Molecular Sequence Data, Phylogeny, Ribonucleases, RNA, Bacterial, Software, Transcription Factors, Sequence Alignment, Protein Structure, Tertiary, DNA, Ribosomal Spacer, Ribosome Subunits, Small, Bacterial, Quality Improvement
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