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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
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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