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How to use Mauve to align genomes and identify orthologs?

[dropcap]B[/dropcap]ordetella species encode on their genomes multiple copies of insertion sequences (IS) elements. In species such as Bordetella pertussis, identical repeats of IS481 can be present in over 200 times and comprise over 6% of the total genome. These sequences provide sites for homologous recombination which are the origin of extensive genome remodeling. As a result, it is often difficult to identify orthologous genes.  This is where Mauve comes in. This tool allows you to compare multiple genomes at the same time and see to where exactly the genes have moved. Mauve was developed at the University of Wisconsin-Madison with a team including Aaron Darling, Bob Mau, and Nicole Perna and several others.

Now it’s time to get to work. Here you will find a step-by-step set of instructions to help guide you through the genome alignment process.

01 - Start Menu Mauve can be downloaded for free here.

Start Mauve by going to your computer’s program list and clicking the Mauve program icon.

02 - Mauve Home Page Once the home page for Mauve has opened, click ‘File’ and then ‘Align with progressiveMauve’.
03 - Align with Progressive Mauve You will now be prompted to add a sequence. Click the ‘Add Sequence’ button to browse for the sequence files.
04 - Add Sequence

05 - Selected Sequences in GBK Format

Be sure to select at least 2 sequences in .gbk format. Once you’ve chosen all the files you wish to align.

Important: you can only align .gbk files!

06 - Choose an Output Folder Now you must choose a space for the output file. An empty folder titled “Alignments” should be appropriate.
07 - Name the File For the Alignments Time to choose a file name for these alignments once they’re finished. A simple name like

“[Bacterial species name here] Alignments” Should work fine.

08 - Hit Save and Align Click the ‘Align’ button, and you’re ready to go!
09 - Wait for the Genomes to Align And now we wait. Be sure to wait til all the alignments have finished. Keep in mind, the more alignments running at a time, the longer this step will take.
10 - Alignment Finished Once the alignments are complete, hit the ‘close’ button.
11 - View Alignments in Mauve Window The alignments should now be visible in you Mauve program window. Take a glance at and compare the sequences aligned. Chances are they are very different! Hovering your mouse over the length of the alignments should show you where that gene is located in each of your strains.
12 - Tools Export Export Orthologs Now it’s time to export the orthologs. To do this, simply hit the ‘Tools’ tab, ‘Export’, and then ‘Export Orthologs’.
13 - Identity And Coverage Be sure to select the proper Identity and Coverage ranges. We suggest using a Min Identity of 60 and a Max of 100 as well as a Min Coverage of 70 and a Max of 100. Once the values are set, click the ‘Browse’ button.
15 - Export Within your newly created “Alignments” folder, save the file as “ [Bacterial species name here] Orthologs “.
14 - Name Orthologs in Alignments Folder Hit ‘Save’ and ‘Export’.
16 - Open Alignments The Orthologs file should now be accessible. Open the file.
17 - We Want Excel You may be asked to select a program in which to view these Orthologs. We suggest using Microsoft Excel.
18 - Read Only Once Excel has opened, you may be notified that the version you are about to view is a “Read-Only” version. This is fine. Open as a “Read-Only” file anyway.
19 - Sort Genes But Keep in Line You should now have your list of Orthologs in Excel. There is a chance the Orthologs have been jumbled. Each strain should occupy only one column. If multiple strains have mixed in a column, you will need to sort them. We suggest using the ‘Sort and Filter’ function in the upper right hand corner of the ‘Home’ tab.

CAUTION: Be very careful not to move an Ortholog vertically within a column. Only horizontal movement should occur.

More detailed information on the different parameters for alignments in Mauve can be found here.Mauve