MUSCLE (alignment software)

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MUSCLE (multiple sequence comparison by log-expectation) is public domain, multiple sequence alignment software for protein and nucleotide sequences. The method was published by Robert C. Edgar in two papers in 2004. The first paper, published in Nucleic Acids Research, introduced the sequence alignment algorithm.[1] The second paper, published in BMC Bioinformatics, presented more technical details.[2]


The MUSCLE algorithm proceeds in three stages: the 'draft progressive', 'improved progressive' and 'refinement' stages. In the 'draft progressive' stage, the algorithm produces a draft multiple alignment, with the emphasis on speed rather than accuracy. In the 'improved progressive' stage, the Kimura distance is used to reestimate the binary tree used to create the draft alignment, in turn producing a more accurate multiple alignment. The final 'refinement' stage refines the improved alignment produced in the second step. Multiple alignments are available at the end of each stage. The time complexity of the first two stages of the algorithm is O(N2L + NL2); the space complexity is O(N2 + NL + L2). The 'refinement' stage adds a further O(N3L) term to the time complexity.[1] MUSCLE is often used as a replacement for Clustal, since it typically (but not always) gives better sequence alignments, depending on the chosen options. In addition, MUSCLE is significantly faster than Clustal, especially for larger alignments.[1][2]


MUSCLE is integrated into DNASTAR's Lasergene software, Geneious, and MacVector and is available in Sequencher, MEGA and UGENE as a plugin. MUSCLE is also available as a web service provided by EMBL-EBI.[3] As of September 2016, the two papers describing MUSCLE have been cited more than 19,000 times in total.[4]

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