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Published online 19 March 2004

Nucleic Acids Research, 2004, Vol. 32, No. 5 1792-1797
Oxford University Press

MUSCLE: multiple sequence alignment with high accuracy and high throughput

Robert C. Edgar*

195 Roque Moraes Drive, Mill Valley, CA 94941, USA

*Email: bob{at}drive5.com

Received January 19, 2004; Revised January 30, 2004; Accepted February 24, 2004

We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.


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Y. Yin, F. Bangs, I. R. Paton, A. Prescott, J. James, M. G. Davey, P. Whitley, G. Genikhovich, U. Technau, D. W. Burt, et al.
The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation
Development, February 15, 2009; 136(4): 655 - 664.
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JDRHome page
J.M. ten Cate, F.M. Klis, T. Pereira-Cenci, W. Crielaard, and P.W.J. de Groot
Molecular and Cellular Mechanisms That Lead to Candida Biofilm Formation
Journal of Dental Research, February 1, 2009; 88(2): 105 - 115.
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Appl. Environ. Microbiol.Home page
S. E. Morales, T. F. Cosart, J. V. Johnson, and W. E. Holben
Extensive Phylogenetic Analysis of a Soil Bacterial Community Illustrates Extreme Taxon Evenness and the Effects of Amplicon Length, Degree of Coverage, and DNA Fractionation on Classification and Ecological Parameters
Appl. Envir. Microbiol., February 1, 2009; 75(3): 668 - 675.
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Mol. Cell. Biol.Home page
C. Dherin, E. Gueneau, M. Francin, M. Nunez, S. Miron, S. E. Liberti, L. J. Rasmussen, S. Zinn-Justin, B. Gilquin, J.-B. Charbonnier, et al.
Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair
Mol. Cell. Biol., February 1, 2009; 29(3): 907 - 918.
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Infect. Immun.Home page
P. A. Beare, N. Unsworth, M. Andoh, D. E. Voth, A. Omsland, S. D. Gilk, K. P. Williams, B. W. Sobral, J. J. Kupko III, S. F. Porcella, et al.
Comparative Genomics Reveal Extensive Transposon-Mediated Genomic Plasticity and Diversity among Potential Effector Proteins within the Genus Coxiella
Infect. Immun., February 1, 2009; 77(2): 642 - 656.
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Nucleic Acids ResHome page
V. Sorokin, K. Severinov, and M. S. Gelfand
Systematic prediction of control proteins and their DNA binding sites
Nucleic Acids Res., February 1, 2009; 37(2): 441 - 451.
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Nucleic Acids ResHome page
Y. Lu and S.-H. Sze
Improving accuracy of multiple sequence alignment algorithms based on alignment of neighboring residues
Nucleic Acids Res., February 1, 2009; 37(2): 463 - 472.
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Mol. Cell. ProteomicsHome page
B. L. Schulz and M. Aebi
Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency
Mol. Cell. Proteomics, February 1, 2009; 8(2): 357 - 364.
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Mol Biol EvolHome page
C. Brochier-Armanet, E. Talla, and S. Gribaldo
The Multiple Evolutionary Histories of Dioxygen Reductases: Implications for the Origin and Evolution of Aerobic Respiration
Mol. Biol. Evol., February 1, 2009; 26(2): 285 - 297.
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Mol Biol EvolHome page
C. Chen, T. Ara, and D. Gautheret
Using Alu Elements as Polyadenylation Sites: A Case of Retroposon Exaptation
Mol. Biol. Evol., February 1, 2009; 26(2): 327 - 334.
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Mol Biol EvolHome page
B. M. Hallstrom and A. Janke
Gnathostome Phylogenomics Utilizing Lungfish EST Sequences
Mol. Biol. Evol., February 1, 2009; 26(2): 463 - 471.
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J. Virol.Home page
D. C. Tully and M. A. Fares
Shifts in the Selection-Drift Balance Drive the Evolution and Epidemiology of Foot-and-Mouth Disease Virus
J. Virol., January 15, 2009; 83(2): 781 - 790.
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J. Virol.Home page
Y. Xu, L. Cong, C. Chen, L. Wei, Q. Zhao, X. Xu, Y. Ma, M. Bartlam, and Z. Rao
Crystal Structures of Two Coronavirus ADP-Ribose-1''-Monophosphatases and Their Complexes with ADP-Ribose: a Systematic Structural Analysis of the Viral ADRP Domain
J. Virol., January 15, 2009; 83(2): 1083 - 1092.
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J. Virol.Home page
D. J. Heslin, P. Murcia, F. Arnaud, K. Van Doorslaer, M. Palmarini, and J. Lenz
A Single Amino Acid Substitution in a Segment of the CA Protein within Gag That Has Similarity to Human Immunodeficiency Virus Type 1 Blocks Infectivity of a Human Endogenous Retrovirus K Provirus in the Human Genome
J. Virol., January 15, 2009; 83(2): 1105 - 1114.
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