Mauve for multiple genome alignment. Mauve is a free alignment tool with an interactive browser for visualising results [27, 41] (we used Mauve 2.3.1 on Mac OS X). • Launch Mauve and select File -> Align with progressiveMauve • Click ‘Add Sequence…’ to add your genome assembly (e.g. annotated E. coli O104:H4 contigs) and other reference genomes for comparison. • Specify a file for output, then click ‘Align…’ • When the alignment is finished, a visualization of the genome blocks and their homology will be displayed, as shown here. E. coli O104:H4 is on the top, red lines indicate contig boundaries within the assembly. Sequences outside coloured blocks do not have homologs in the other genomes.