Specify a reference sequence

This topic describes several reasons you might want to use a reference and procedures for specifying, changing, and removing the reference.

Note that a reference sequence in MegAlign Pro is different from a reference in SeqMan Ultra or SeqMan NGen. In SeqMan NGen or SeqMan Ultra, the reference is a long DNA sequence that is used as a template for guiding the assembly of small DNA fragment reads into contigs. By contrast, any sequence of any length or type (DNA, RNA, protein) can be used as a reference in MegAlign Pro.

Task	How To
Specify a reference sequence for guiding a MAFFT alignment	Use any of the following methods: Select the sequence you would like to use as the reference. Then choose Sequences > Use as Reference or right-click on the sequence name and choose the same command from the context menu. Then choose Align > Align Using MAFFT. Choose Align > Align with Options. In the Using menu, choose MAFFT. In the Reference Sequence menu, choose the sequence you want to use as the reference. Click Align. From the Welcome screen, choose the New Project tab on the left, then choose New alignment with options on the right. From the ensuing dialog, choose the sequences you wish to align and press Open. The Align Sequences dialog then pops up and is already set to MAFFT. In the Reference Sequence menu, choose the sequence you want to use as the reference. Click Align.
Specify a reference for any other reason	Select the sequence you would like to use as the reference. Then choose Sequences > Use as Reference or right-click on the sequence name and choose the same command from the context menu. Then choose the Align > Align Using command for the algorithm you want to use. You can specify the reference either before or after any alignment type. Below are two common reasons for specifying a reference sequence: For performing variant analysis for any type of alignment. For instance, you might download an annotated reference sequence that you found online and use this workflow to compare your experimental sequences to it. To map the features from an annotated reference sequence to other sequences in the alignment.
Switch to another reference	Use any of the methods above, but choose a different sequence. Note that if you perform a MAFFT alignment using “Sequence A” as the reference and later specify “Sequence B” as the reference, the variants calculated against “Sequence A” are not lost. You can redesignate “Sequence A” as the reference again without recomputing its variants.
Stop using a reference	One reason you might choose to stop using a reference is if you want to compute variants by comparing other sequences to the consensus, rather than to the reference. To remove the reference, select its name in a graphical view and choose Sequences > Stop Using as Reference. Alternatively, you can right-click on the reference sequence name and choose the same command from the context menu. The reference sequence will be removed from the References areas at the top of the Overview and Sequences view, but the sequence will still appear in blue bold-italic font in the list of sequence names. To remove this formatting, see the row just below.
Show/hide the reference used in the last alignment (after you have stopped using it as a reference)	If you align sequences with a reference selected and later stop using the reference (see the row just above), the reference sequence will be removed from the References area at the tops of the Overview and Sequences view, but the former reference sequence will still appear in blue bold-italic font in the list of sequence names. To remove this special formatting, choose Sequences > Unmark Reference of Last Alignment. To return to the special formatting, choose Sequences > Mark Reference of Last Alignment.
Change how the reference sequence is displayed in the views	After you specify a reference sequence using one of the methods above, the reference sequence, reference features and reference ruler can be displayed as tracks in the Sequences view and/or Overview. By default, the sequence that you specify as the reference is displayed in bold blue italics to differentiate it from the other sequences. To change the way the reference is displayed, you can select the corresponding Reference track in the Tracks panel, then modify the track options.
Visually compare sequences without changing the reference	See Visually compare other sequences to one sequence.