Figure 1.
The difference between positional homology alignment and glocal alignment.
Three example linear genomes are broken into genes labeled A,B,C,D, and R. R is a multi-copy (repetitive) gene, with different copies labeled using numeric subscripts. Each copy of R is assumed to be identical in sequence, so that orthology/paralogy is unknowable from nucleotide substitution (as is often the case with mobile DNA repeat elements). Genes shifted downward in a given genome are inverted (reverse complement) relative to the reference genome. The positional homology alignment would ideally create two local alignment blocks where each block has exactly one alignment row for each genome. Only positionally-conserved copies of the repetitive gene family R become aligned to each other. The glocal alignment would ideally create four local alignment blocks wherein all copies of the repetitive gene family become aligned to each other.
Figure 2.
Overview of the alignment algorithm using three example genomes A, B, and C.
Figure 3.
Illustration of terminology used for tree nodes.
Rotating the figure 90 degrees counterclockwise explains the descriptive use of left and right.
Figure 4.
The correct alignment is shown at left and four possible predicted alignments are shown as A, B, C, and D. Nucleotides have been assigned a numerical identifier. The correct alignment has a single indel which partitions the alignment into three sections: the left aligned block, the indel, and the right aligned block. Predicted alignments must have one correctly aligned nucleotide pair in each of the three sections to count a true positive indel prediction.
Figure 5.
The accuracy of aligners on sequences evolved without rearrangement and with increasing nucleotide substitution and indel rates.
Aligners were tested on 100 combinations of indel and substitution rate, with performance averaged over three replicates. All methods lose accuracy as mutation rates grow, and the most accurate alignment method depends on the particular mutation rates. progressiveMauve and MLAGAN exhibit the best indel sensitivity and positive predictive value (PPV), while TBA is more sensitive than other methods at extremely high mutation rates. MLAGAN did not align genomes without indels within the allotted 10 hours, resulting in the black row at the bottom. The asterisk in this figure indicates the combination of indel rate and substitution rate expected to be similar to our 23 target genomes.
Figure 6.
Accuracy of mauveAligner, progressiveMauve, and TBA when aligning genomes with inversions and segmental gain and loss.
In the experiments shown at top, the inversion rate increases along the -axis and the substitution rate along the
-axis. The most distant taxa have 0.05 indels per site. progressiveMauve clearly outperforms mauveAligner 1.3.0 over the entire space of inversion rates. It should be noted that in applications such as the UCSC browser alignments TBA was used in conjuction with a separate synteny-mapping method to identify rearrangements [66], so the performance results given here are not cause for alarm. Experiments at bottom quantify aligner performance in the presence of small- and large-scale gain and loss events. The
-axis gives the average number of large gain and loss events [length
Unif(10kbp, 50kbp)] between the most distant taxa, while the
-axis gives small gain and loss events [length
Geo(200bp)]. Substitution and indel rates are those indicated by the asterisk in Figure 5, and the most distant taxa have 42 inversions on average. The asterisk in this figure indicates a simulation scenario expected to be similar to our 23 target genomes. Once again progressiveMauve outperforms other methods, but all methods break down when faced with substantial large-scale gain and loss. Of note, when mauveAligner 1.3.0 attains high PPV it usually does so with very poor sensitivity.
Figure 7.
Frequency with which gaps are correctly predicted as a singular gap as a function of gap size.
Left Average size distribution of gaps in an alignment of the nine genomes evolved at mutation rates which correspond to previous estimates for the E. coli, Shigella, and Salmonella. The gap size distribution was averaged over 10 simulations. Right Fraction of TP indel predictions that are singular TP indel predictions by true gap size. Ten replicate simulations of evolution with gene gain, gene loss, indels, and nucleotide substitution were performed and alignments were computed using each aligner. Predicted indels were classified according to the definitions given in Figure 4, namely, a singular True Positive implies the true gap is predicted as a single gap. Remaining True Positive indels have the true gap broken up into two or more predicted gaps. For each aligner, the fraction of singular predicted gaps is shown as a function of gap size. Missing points indicate a lack of TP indel predictions in that size category. All aligners do well in predicting small gaps, but large gaps present problems. Most aligners, including Pecan which uses an extra pair-HMM state to model long gaps, tend to predict long gaps as a series of short gaps interspersed with alignments of unrelated sequence. We refer to such behavior as “gap dribble.” progressiveMauve was run with default parameters (proMauve), without the Homology HMM (proMauve_no_HMM), with the option to assume genomes are collinear (proMauve_col), and finally assuming collinearity and without the HMM (proMauve_col_no_HMM).
Figure 8.
Venn diagram of the pan-genome (left) and core genome (right) of E. coli/Shigella and S. enterica.
Figure 9.
A Mauve visualization of the hypervariable intergenic regions surrounding yhdE.
Each genome is laid out in a horizontal track, with annotated coding regions shown as white boxes. A colored similarity plot is shown for each genome, the height of which is proportional to the level of sequence identity in that region. When the similarity plot points downward it indicates an alignment to the reverse strand of the genome. Colors in the similarity plot indicate the combination of organisms containing a particular segment of the genome. Segments colored pink/mauve are conserved among all organisms, while purple segments are conserved in everything but Salmonella, and segments colored in olive green are conserved among non-uropathogenic E. coli. The visualization system is interactive and written in Java, and works on all computers supporting Java 1.4 or later.