The procedure described below is an informal way to visually compare other sequences in the alignment to one particular sequence in the Sequences view. This comparison is performed using settings in the Multiple alignment section of the Style panel and is a great way to visually highlight differences between the sequences.

Imagine that you have just spent several minutes aligning 1,000 CoV-2 genomes. All of the viral strains are similar, and none of them is “special” enough to merit using it as a formal reference sequence. You also don’t want to have to realign the entire set for each comparison. In this case, a visual comparison is probably all you need.


To visually compare sequences aligned with any algorithm except Mauve:

The following comparison applies to any alignment other than Mauve. Results are displayed in the Sequences view.

  1. Add sequences to the project and perform a multiple alignment using any algorithm except Mauve. If MAFFT is used, you can specify a reference (or not), but this will not affect the comparison.
  1. Choose how you want matches/mismatches to appear when viewed in the Sequences view. To do this, open the Multiple alignment section of the Style panel (View > Style > Multiple Alignment).



    • Use the Compare to drop-down menu to choose a comparison sequence. If you have previously specified a reference sequence, this will be pre-selected as the comparison sequence, but any sequence can be chosen from the menu. You can choose a different comparison sequence at any time without needing to realign the sequences.

    • Use the Alignment coloring drop-down menu to show what you want to color; for example, Color only differences from comparison sequence, Color only matches to comparison sequence, or Show only differences from comparison sequence.

    • If desired, check the box Differences from comparison sequence and choose one or more of the font style options (Bold, Italic, Underline) to accentuate the matches/mismatches even more.

In the Sequences view, similarities/differences will appear as you specified in the Multiple alignment section.

If you have specified a reference sequence and a comparison sequence, you may be wondering: Which takes precedence in the Sequences view?

The Variants view is always computed according to the chosen reference sequence and any comparison sequence is ignored. An active reference sequence also appears at the top of the Sequences view. However, once you choose a different comparison sequence (using the steps above), sequences in the Sequences view are compared to that sequence rather than to the reference sequence. Despite appearing at the top of the view, the reference sequence itself is also compared to the chosen comparison sequence.

In the example below (image), the alignment was done with MAFFT using “Rat” as the reference. “Red Bread Mold” was chosen as the comparison sequence, and Show only differences from comparison sequence was selected in the Style panel. Note that “Rat” continues to be used as the reference sequence (blue box). With this type of automatic coloring selected, it is easy to see that “Red Bread Mold” is the active comparison sequence (red), since it appears in normal black text throughout. In all other sequences, including the reference sequence, only differences from the comparison sequence are displayed (yellow).


To visually compare sequences aligned with Mauve:

The following comparison applies only to Mauve alignments. Results are displayed in the Overview.

  1. Add sequences to the project and perform a multiple alignment using Mauve.
  1. Choose how you want matches/mismatches to appear when viewed in the Overview. To do this, open the Overview section of the Style panel (View > Style > Overview).



    • Use the Compare to drop-down menu to choose a comparison sequence. If you have previously specified a reference sequence, this will be pre-selected as the comparison sequence, but any sequence can be chosen from the menu. You can choose a different comparison sequence at any time without needing to realign the sequences.

Choosing different Compare to sequences changes the lines that connect the Mauve blocks in the Overview. All blocks for the selected reference are placed on the forward strand. Blocks for non-reference sequences can appear on either strand.

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