Using GRIL to determine the rearrangement structure of five genomes

To give an example of how GRIL can be used, we have applied it to five enterobacterial genome sequences. The five genomes are those of E. coli K-12 MG1655, E. coli O157:H7 EDL933, E. coli O157:H7 Sakai VT-2, Salmonella enterica serovar Typhimurium LT2, and Salmonella enterica serovar Typhi CT18. The length of each genome ranges from 4.6 to 5.5MB. These genomes contain significant rearranged homologous regions and also numerous lineage specific regions.

To determine the locally collinear blocks, and hence the structure of rearrangement in these genomes, GRIL can be executed using the command line:

> gril -m 23 -f 100000 -r 10000 ecolim52.fas ecolim52.sml EDL933.fas EDL933.sml o157sakai.fas o157sakai.sml typhimurium.fas typhimurium.sml styphi.fas styphi.sml

The -m 23 parameter specifies that GRIL should use 23-bp exact and unique mers as the seed matches when searching for MUMs. The -f 100000 parameter specifies that 100000 is the maximum permissible difference in generalized offset between a MUM and neighboring MUMs. The -r 10000 parameter specifies that GRIL should remove LCBs that span less than 10000b.p. For a detailed description of the generalized offset, range, and identity filters, see the GRIL Algorithms web page. The remaining parameters specify the input sequence files and the sorted mer list files. If the sorted mer list (.sml) files do not exist, GRIL will create them.

The resulting list of LCBs generated by GRIL is:

Negative start and end coordinates signify a region that has been inverted relative to the first (the reference) sequence. Using the GenVision software package from DNAstar, the block structure can be graphically rendered as follows:

In this figure, each colored block represents a region of each bacteria's chromosome that has not undergone significant rearrangement. Blocks below a sequence's center line denote that the region has been inverted relative to the reference sequence, in this case E. coli K-12 MG1655. For example, the yellow block represents a region that is homologous in all sequences and is inverted in the Salmonella strains relative to E. coli K-12 MG1655. As shown in the above graphic, GRIL locates 10 LCBs using the parameters -r 10000 and -f 100000. By using different parameters, the blocks found can change. For example, if the minimum range (-r) parameter is reduced to 1000 GRIL reports the following 21 LCBs:

Through inspection of the sequence annotation, we can validate the existence of the additional LCBs reported by GRIL. Four new LCBs were found using the lower minimum range threshold and have been labeled according to their sequence annotation. Three of the new LCBs are 'mobile' operons that have relocated across the 5 genome sequences, and the fourth is ambiguously annotated. The four new LCBs break up the original 10 blocks into 17 collinear blocks, for a total of 21 LCBs. The original LCBs are labeled with the corresponding block number from the first comparison, and blocks that were broken are labeled as [original number]/[piece number]. The biological significance of the sequence contained in one of the rearrangements is unknown. Using GenVision, the position of the new rearrangements can be overlaid onto our visualization of the original set of 10 rearrangements:

Thus by modulating the minimum identity, maximum generalized offset distance, and especially the minimum range parameters, genome rearrangements at different scales can be selectively identified or ignored.