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The Diagram window

The ‘Diagram’ tab takes you to a new blank window and the group number of interest (from the ‘Analysis’ window) is entered in the ‘Diagram Panel’ and on clicking the ‘Draw’ button the initial auto-edited eBURST diagram is produced. Where there is a predicted primary founder this ST is shown as the circle in the centre of the eBURST diagram and is coloured blue. The area of this circle, and of the circles that represent the other STs, is proportional to the abundance of the ST in the input dataset. The numbers are those of the STs.

All of the SLVs of the predicted primary founder are shown arranged in a circle, each linked (connected by a line) to the centrally positioned predicted founder. Unless the group is fairly simple, with a single founder and a set of linked SLVs, the initial auto-edited eBURST diagram will probably need some manual editing before it is saved (see here).

eBURST is conservative and only shows links between SLVs. It therefore differs from the original BURST in the way it displays the DLVs of the predicted founding ST. BURST showed all of these DLVs in an outer circle, whereas in eBURST, the DLVs of the founding ST are only shown if the linking SLV is present. Thus, if a SLV differs from the predicted founding ST by having allele 14 rather than allele 6 at locus 1, a DLV will only be shown linked to this SLV if it also has allele 14 at locus 1 and a second change at any of the other loci. eBURST therefore suggests pathways by which DLVs may have arisen from a founding ST through known SLVs. Similarly TLVs of the founding ST will be linked only if they connect through an existing DLV.

The default diagram (six out of seven group definition):
Using the default definition of a group, all isolates must have the same alleles at six or more of the seven loci with at least one other isolate in the group. Therefore, in the default eBURST diagram, all STs will be linked as a single cluster, which is considered to represent a clonal complex. This stringent default definition of the group means that some STs that may be quite closely related to other members of the group will not be included (e.g. even DLVs of the founding ST will be excluded from the group unless they are SLVs of some other ST in the group). Bootstrapping provides the level of support for the predicted primary founder of the clonal complex and for subgroup founders.

Relaxing the group definition to five out of seven:
When the definition of a group is relaxed to five out of seven shared alleles, the output from a large eBURST group will often show several unlinked clusters of STs, and individual unlinked STs (Figure 8). As only links between SLVs are shown, all directly linked STs within a cluster must differ at only one of the seven loci (i.e. they are links between SLVs). If there are two clusters in the eBURST diagram, the fact that the two clusters are not joined into a single cluster implies that no isolate in one cluster is a SLV of any isolate in the other cluster. Some of the isolates in different clusters are likely to be DLVs of each other but eBURST does show links between DLVs (N.B. all of the SLVs and DLVs of any ST can be displayed on the eBURST diagram; see section 9.7). A less conservative approach that allowed links to be drawn between DLVs would almost certainly connect these two clusters, but the validity of the link would be doubtful, as linkages between DLVs are expected to be less robust than those between SLVs. Two clusters within a single group may descend from a common founder, but without additional information the precise evolutionary relationship between them cannot be discerned with confidence. The clusters of linked STs are considered to be clonal complexes. Bootstrapping should be performed using the default group definition to provide the level of support for the predicted primary founder of each of the clonal complexes within a 5/7 eBURST diagram.

Several unlinked clusters can also occur using the original BURST algorithm, but to a lesser extent, as DLVs can be linked without the need for the intermediate SLV, and the unlinked clusters shown using eBURST provide a much more conservative view of the relationships between the STs in the group.

Further relaxation of the group definition:
Isolates that share alleles at five of seven loci with at least one other isolate in the group may be quite distantly related and a large group could even include isolates that differ at all loci. We recommend first using the default group definition (6/7) and then, if required, the relaxed definition (5/7). Relaxing the group definition further is not recommended, except to obtain the “population snapshot” (see here).

eBURSTv3 has been developed and is hosted at Imperial College London