Navigate to the Alignment Tab
To align sequences to a reference sequence, start by navigating to the “Alignment” tab in the top banner. Select the “Align to reference” button and the alignment modal box will open.
Selecting your Reference and Aligned Sequences
By default, the reference sequence is pre-selected to be the current open file. You can simply change this by removing it via the trash can icon. Once removed, you can select the “+ Add reference...” button then select your desired reference file. Note you can also choose the options to enter (paste) a sequence manually or upload a sequence from your local device if the desired file is not present in your “My files”.
After selecting the reference sequence you must select at least one sequence to align to the reference. Do this by using the “+ Add sequence” button in the aligned sequence(s) section. Note that it is possible to add multiple sequences at a time by holding ctrl or shift to select multiple files to align. Once you have finished selecting your files, select the “Next” button this will prompt you to rename your file. Note that the default name is “Reference sequence + Aligned sequence #1 + Aligned sequence #2 etc”. Note that the alignment will be saved as a new file (specifically for align to reference you will still see in map and sequence view that the reference plasmid is displayed). Once you have selected the name select “Align” to start the alignment.
Display and Edit Options in Alignment View
Once the alignment is completed, it will be displayed in the alignment view. In the top banner you will notice that this is set to the “View” tab and the options have slightly changed. The first option is the model indicator dropdown, for align to reference the only available algorithm is MAFFT. Next is the “Axis” toggle button, this will turn on and off the axis label below the sequences. The next button called “Chromatogram” is only available for sanger trace files (.ab1) as these contain chromatogram data, for other alignments this is disabled. The “Collapse all” and “Expand all” collapses the sequences into compact view where the features and alignment information is hidden. With the “Highlight when” button you can select which regions are highlighted in red in your sequence. By default all regions that are not matching are highlighted however you can select to switch this to only highlight matching regions or to not highlight at all. With the “Highlight color” dropdown you can select which color is used to highlight the matches or mismatches. Lastly the DNA colors button lets you toggle on the base pair colors (A = Green, T = Red, G = Yellow and C = Blue).
You can find more options for alignment under the “Alignment” tab in the top banner. Here you can find the option to “Add track” and “Delete track”. Select “Add track” to add new sequences to the alignment, this will open up the sequence selection box. Select “Delete track” to open up the deletion modal box, here you can select which sequences to remove.
Navigation in Alignment View
At the top of the alignment view, you will see the blue minimap of your alignment, additionally you will see a yellow focus window on the map, this can be used to scroll back and forth across your alignment by holding click in the yellow region and moving back and forth. If you would like to view a larger region, use the two handles on the outside of the yellow focus window to expand or decrease the view. Alternatively, you can scroll back and forth with your trackpad by scrolling horizontally with two fingers or if using a traditional mouse by holding down the shift key on your keyboard and using the scroll wheel on the mouse.
The reference track is always the top track in the large window, on the left hand side you can check which sequence is used as the reference. Below this on the left you can see the “Toggle tracks” option, under which all the aligned references are listed, to hide a given sequence just unselect it by clicking the checkbox next to its name under the “Toggle tracks” section. At the bottom of this section there is a “+ add track” button, use this to add more sequences to the alignment (note this is only available for align to reference not pairwise or multiple sequence alignment).
For each aligned track you will see some alignment information. The name is displayed with a colored arrow on the left side indicating the directionality of the aligned sequence. Under the name, the first line is the total length of the sequence, then the number of mismatches, insertion and deletions. To quickly navigate to the next mismatch, insertion or deletion you can use the arrow on the right side of the sequence name to jump to the next.
