In this exercise you will create an alignment of 3 Mycobacterium genomes. Holding down shift, select NC_015758, NC_012943 and NC_009565. These are complete genomes downloaded from NCBI.
Then go to Align/Assemble → Align Whole Genomes . Ensure the Mauve Genome tab is selected, and the algorithm is set to progressiveMauve. All other settings should be kept at the default settings. For a full description of the parameters, see here. Click OK to run the alignment.
Note: in this example we are using complete genomes where each genome is a single contiguous sequence. It is also possible to align draft genomes, where each genome is represented by a list of contigs. If one or more genomes are represented by a sequence list, and progressiveMauve is chosen as the algorithm, Geneious will concatenate the contigs from each genome into a single sequence for the alignment. This option will not order contigs, and users should be aware that incorrectly ordered contigs will appear as genome rearrangements in the Mauve viewer. If you wish to align only two draft genomes, or align a draft genome against a complete genome, it is best to use the MCM algorithm, which is covered in Exercise 2.
The alignment may take a while to run. When it has finished a new Mauve alignment document will appear in your Document Table. Select the alignment and load it into the viewer.
Mauve alignments have a special viewer which enables you to see genome rearrangements and locally aligned blocks at a glance. Each sequence is represented by one horizontal panel of blocks. Each colored block represents a region of sequence that aligns to part of another genome, and is presumably homologous and free from internal rearrangements. These are called LCBs (locally collinear blocks). The colored blocks in each genome are connected by vertical lines. For a full description of the Mauve viewer, please see the Mauve user guide.
Your alignment should look as in the screenshot below:
In this example you can see there 3 LCBs denoted by the different colors. In the middle LCB, the block in sequence NC_012943 is below the line. This indicates that this region is inverted with respect to the other two sequences.
The Mauve viewer has its own controls for zooming in and out, and scrolling to the left and right above the viewer. Try zooming in by clicking the button 3 or 4 times. If your input sequences contained CDS, tRNA, rRNA or misc_RNA annotations, these should become visible as square blocks below the colored LCB block.
As you mouse over the colored blocks on any sequence in the alignment, you will see rectangular boxes showing the aligned region in the the other sequences. You can click on the alignment to center it on any given region.
To reset the alignment view at any time, click the button.
The LCB Weight slider has the effect of changing the resolution at which local collinear blocks are determined. The LCB weight sets the minimum number of matching nucleotides for a collinear region to be considered as having true homology rather than random similarity. When “automatically calculate the minimum LCB score” is checked in the Mauve setup options, an LCB weight of 3 times the seed size will be used for the alignment, and this will be set as the lowest value in the LCB weight slider. Try sliding the LCB weight up and see what effect is has on the LCB blocks.
To view the aligned sequences in a view that looks similar to the regular Geneious alignment viewer, click the Alignment View tab above the sequence viewer. This displays the alignment for one LCB at a time. To choose which LCB to display, use the drop down Alignment chooser in the Display tab next to the viewer (circled in the screenshot below).
Try changing the Alignment View to LCB 2. This is the middle block where the NC_012943 was inverted. Check the arrow directions on the Source annotations in this view and you will see that NC_012943 is in the opposite orientation to the other two sequences in the alignment. If you cannot see the blue Source annotations, make sure the annotation type "Source" is checked in the Annotations and Tracks panel.
This Alignment view works the same way as the regular Geneious alignment viewer, and has the same controls for zooming, sequence display and annotations. However, only annotations can be edited in this viewer, not the sequence itself. If you wish to edit the sequence you must create an editable copy, which will be a standard Geneious alignment document (you will be prompted to do this if you click Allow Editing).
Mauve alignment documents cannot be used for downstream processes such as tree building. You will need to extract the Mauve regions to a standard Geneious alignment document following the instructions in Exercise 3 of this tutorial in order to use it in downstream operations.
Exercise 2: Aligning draft genomes