Pairwise genome comparisons with ACT, the Artemis Comparison Tool. Artemis and ACT are free, interactive genome browsers [32, 40] (we used ACT 11.0.0 on Mac OS X). • Open the assembled E. coli O104:H4 contigs in Artemis and write out a single, concatenated sequence using File -> Write -> All Bases -> FASTA Format. • Generate a comparison file between the concatenated contigs and 2 alternative reference genomes using the website WebACT (http://www.webact.org/). • Launch ACT and load in the reference sequences, contigs and comparison files, to get a 3-way comparison like the one shown here. Here, the E. coli O104:H4 contigs are in the middle row, the enteroaggregative E. coli strain Ec55989 is on top and the enterohaemorrhagic E. coli strain EDL933 is below. Details of the comparison can be viewed by zooming in, to the level of genes or DNA bases.