Which multiple alignment algorithm should I use?

Four different multiple alignment algorithms are available in Geneious Prime 2020 under Align/Assemble→Multiple Align. It is important to consider the size of your dataset when choosing which one to use.  Below is a brief overview of each algorithm. 

Geneious aligner

The Geneious aligner is a progressive pairwise aligner, similar to ClustalW (below).  It is the slowest algorithm in Geneious and recommended for small alignments (e.g. fewer than 50 sequences, less than 1 kb in length).


MUSCLE is  is a progressive aligner that features rapid sequence distance estimation using k-mer counting, progressive alignment using a profile function termed the log-expectation score, and refinement using tree-dependent restricted partitioning of the sequences.  The algorithm is described at http://nar.oxfordjournals.org/content/32/5/1792.full.pdf+html and a full manual is available at http://www.drive5.com/muscle/manual/index.html.  The defaults are optimized for best accuracy.  However, you can reduce the run-time on large alignments without too much reduction in accuracy by reducing the maximum number of iterations.  

  • Suitable for medium-large alignments up to 1000 sequences. 
  • Not suitable for sequences with low homology N-terminal and C-terminal extensions. 

Clustal Omega (replaces ClustalW in Geneious Prime 2020 onwards)

Clustal Omega is a fast, accurate aligner suitable for alignments of any size. It uses mBed guide trees and pair HMM-based algorithm which improves sensitivity and alignment quality.  A full description of the algorithms used by Clustal Omega is available in the Molecular Systems Biology paper Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Latest additions to Clustal Omega are described in Clustal Omega for making accurate alignments of many protein sciences.  See also the Clustal Omega website for further details.  

  • Suitable for very large datasets of over 2000 sequences
  • Multi-threaded for faster alignment.
  • Suitable for sequences with long, low homology N-terminal or C-terminal extensions
  • Not suitable for alignment of sequences with large internal indels

MAFFT (install by going to Tools -> Plugins)

MAFFT is a progressive-iterative aligner that uses guide tree re-estimation for obtaining more accurate distance measures.  Full details are given at http://mafft.cbrc.jp/alignment/software/

  • Fast and accurate, especially suited for large datasets (e.g. up to 30,000 sequences, or fewer long sequences*).
  • Multi-threaded for faster alignment. 
  • Suitable for sequences with long, low homology N-terminal or C-terminal extensions
  • Suitable for sequences with long internal gaps (use L-ins-i algorithm)

* For long sequences, the algorithm is faster if sequences are closely related.  For highly divergent sequences, a whole genome aligner like Mauve or LASTZ may be more efficient.  

ClustalW (available in Geneious Prime 2019 and earlier)

ClustalW is a progressive aligner similar to the Geneious aligner.  For details of the algorithm, see http://www.clustal.org/clustal2/.  ClustalW is a little faster than Geneious, but should still be restricted to small alignments.


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