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ClustalX is the graphical user interface version of Clustal. It is available for free for Windows, Mac OS and Unix/Linux.

Using ClustalX to build Phylogenetic trees

Note: This is only to give the user a very basic introduction to phylogenetic tree building techniques.

Calculating a phylogenetic tree using ClustalX involves the five steps listed below (all of the necessary commands are located in the 'Trees' menu):

  1. Remove the regions of the alignment with gaps: select 'delete positions with gaps'.
  2. If your sequences are less than ~95% identical, you should choose to correct your distance measures for multiple substitutions: 'correct for multiple substitutions'.
  3. Make sure you have the correct format for the output:
    • follow the 'output format link';
    • change 'bootstrap label options' from 'branches' to 'nodes'; and
    • close
  4. Calculate the tree by selecting 'bootstrap NJ tree'.
  5. View the results in a tree-viewing programme, such as Treeview or NJplot.


  • Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, and Thompson JD (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31:3497-3500.
  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, and Higgins DG (1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24:4876-4882.
  • Higgins DG, Thompson JD, and Gibson TJ (1996). Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 266:383-402.
  • Higgins DG and Sharp PM (1989). Fast and sensitive multiple sequence alignments on a microcomputer. CABIOS 5:151-153.
  • Higgins DG and Sharp PM (1988). CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237-244.


External links