When performing a multiple alignment, take the following tips into consideration.


Sequence order matters:

The order in which sequences appear in the Overview and Sequences view may affect the results of the multiple sequence alignment. If you are not satisfied with an alignment, try reordering sequences and running the alignment again.


32-Bit and 64-bit machines:

Mauve, MAFFT, and Clustal Omega automatically run 64-bit versions on 64-bit machines and 32-bit versions on other machines.


Divergent sequences

The MUSCLE multiple alignment algorithm assumes that the sequences to be aligned have a certain degree of relatedness. Groups of relatively divergent sequences, especially those with very large data sets, may require considerably more computer resources, particularly RAM. When working with these types of data, we recommend using Align > Align with Options and changing Maximum iterations to 1 or 2. This will both reduce the amount of memory needed, and ensure that the sequences are sufficiently related to allow for the alignment process to be successful.


Additional alignment methods:

Two additional multiple alignment methods are available in DNASTAR’s MegAlign application: Jotun Hein and Clustal V. Please open the classic MegAlign application and select Help > Contents for more information on these methods.


High-throughput sequencing reads:

We caution against attempting a multiple alignment in MegAlign Pro using high-throughput sequencing reads. Analysis of these reads should instead be handled through DNASTAR’s SeqMan NGen and ArrayStar applications.


Known issues with Mauve:

  • An issue in Mauve can cause an alignment to stall if sequences are ordered one way, but not another. If this issue occurs, you may see up to three error messages after beginning a Mauve alignment: “progressiveMauve-[OS].exe has stopped working,” “Do you want to send more information about the problem?,” and “Unexpected error while running the alignment.” The last message provides a Details button which reveals the text: “Mauve quit unexpectedly. Reported error: Unrecognized file format.” In addition, a progress bar will open, but the alignment will not finish. To circumvent this issue, Cancel the alignment, drag the sequences into a different order in the Sequences View or Overview, and then try the alignment again.
  • Sequences aligned with Mauve may give slightly different results on Windows and Macintosh. These differences originate with the Mauve algorithm and not with the implementation in MegAlign Pro.

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