The MUSCLE algorithm is for gene level alignment of either protein or nucleotide sequences. To run a MUSCLE alignment, select two or more sequences and choose Align > (Re)Align Using MUSCLE.

If you wish to change method options, instead choose Align > Align with Options. Options vary depending whether the sequences are protein or nucleotide (protein version shown below).

Change settings as desired:

  • In the Using drop-down menu, choose MUSCLE.
  • Substitution matrix – (protein sequences only) Choose from a list that includes one or more members of the VTML, BLOSUM GONNET and PAM series of matrices.
  • Gap open penalty – This option affects the chance of gaps being introduced into alignments, and must be zero or a negative number. Decreasing the magnitude of the gap open penalty may lead to an excessive amount of gapping. The default is -400 for DNA; -420 for RNA; -2.9 for protein.
  • Gap extension penalty – This option affects the lengths of gaps, and must be zero or a negative number. Lowering the magnitude of the gap extension penalty may allow for longer gaps. The default is 0.
  • Center penalty – This must be zero or a negative number. The default is 0 for DNA; -300 for RNA; and -0.52 for protein.
  • Maximum iterations – This option, which must be a positive integer, affects the number of rounds of refinement used to produce an alignment. Depending on your data, the alignment might finish before the specified number of rounds have been performed. The default is 8.
  • Use Brenner’s Alignment – Checking the box causes MegAlign Pro to use Brenner’s method for the root alignment. Brenner’s method uses less memory than the default root alignment, but the alignment will not be as accurate as if the box were left unchecked. For this reason, it is not usually necessary to check the box unless you received certain types of error messages during a previous alignment attempt.
  • Disable anchors optimization – The default for MUSCLE is for anchor optimization to be enabled (i.e., for this box to be unchecked). One side effect of anchor optimization is that the selected number of Maximum iterations, if greater than two, may be over-ruled during sequence processing. If you instead wish to run the exact number of iterations selected in Maximum iterations (above), add a checkmark to this box.
  • Use the drop-down menus to choose the desired Cluster method for Initial iterations and Later iterations.
Cluster method Description
UPGMA Fast O(N2) approximation to the textbook UPGMA algorithm.
UPGMB (default) Variant of UPGMA that is used, by default, for both iteration 1 and 2.
Neighbor Joining Better at making phylogenetic trees, but slower, and usually worse for making guide trees for progressive alignment. Not recommended for most alignments.
  • Choose the desired Distance measures.
Distance measure Description
Initial iterations
Kmer 4-6 [default for DNA/RNA; not available for protein]
Kmer 6-6 [default for protein]
Kmer 20-3
Kmer 20-4
Kmer bit 20-3
Later iterations
Kimura % identity [default]
Log % identity
  • Select the desired Sequence weights for Initial iterations and Later iterations.
Sequence weight Description
None Do not weight sequences.
ClustalW (default)
Henikoff PSI-BLAST

The following table shows how the MegAlign Pro settings correspond with settings in MUSCLE online.

MegAlign Pro setting MUSCLE setting
Substitution matrix matrix
Gap open penalty gapopen
Gap extension penalty gapextend
Center parameter center
Maximum iterations maxiters
Use Brenner’s Alignment brenner
Disable anchors optimization anchors
Cluster method:
Initial iterations
Later iterations

Distance measure:
Initial iterations
Later iterations
Sequence weight:
Initial iterations
Later iterations


After making your changes:

  • Choose Align to use the entered options to perform a multiple sequence alignment.
  • Use the Reset to Default button if you would like to reset all values to the MegAlign Pro defaults.
  • Select Cancel to leave the dialog without saving any changes.

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