Last modified on 23 December 2011.

Supplemental Figure Legend
Supplemental Figure S1. Pseudo code of phylogenetic event counting algorithm.
Supplemental Figure S2. Variation/covariation analysis of the secondary structure of the bacterial 16S rRNA sequence alignment. Total variation in each pairwise set of sequences (X-direction) is plotted vs. (1) the amount of variation in that set of sequences for the two positions that are base paired in the secondary structure (blue), (2) only one position of the two that are base paired in the secondary structure (red), and (3) variation in the unpaired region of the second structure (green) (Y-direction). The slope, Y-intercept, and R2 co-efficiency values of the linear regression line for each of the three analyses are at the right side of the line.
Supplemental Figure S3. Graphical representation of N-Best method. While the mutual-information (MIxy) covariation method compares all positions against all other positions, the N-best method ranks covariation scores for two positions for each individual position. The position numbers are in the X-axis and the MIxy values are in the Y-axis. (A) Left: The MIxy scores for position 3 with all 76 positions in tRNA; Right: The MIxy values for position 13 with all 76 positions are also displayed in the right side with the same manner. (B) Each nucleotide position in a tRNA is shown in the X-axis while the MIxy score are displayed in the Y-axis. The vertical bar is the MIxy value for position Z and each of the individual positions in the X-axis. When the positions with the best covariation scores for each position are base paired in the tRNA structure, that vertical bar is shown in red. The positions with lower MIxy values are shown as black vertical lines. This diagram illustrates that the majority of all positions that are base paired has a MIxy value significantly higher than the MIxy value for all of the other positions.
Supplemental Figure S4. The secondary (A) and three-dimensional structure (B) of S. cerevisiae Phe tRNA with neighbor effect identified in 1992.
Supplemental Figure S5. The underlying principle of coarse filter that reduce the number of pairwise comparison. (A) The conservation scores for all nucleotides that are base paired in the 16S rRNA comparative structure model. Each base pair is represented with a colored circle, where the color indicates the purity score (minimal value: 0.472; maximum value: 1). The vast majority of the dots representing base pairs are close to the diagonal. (B) The conservation scores for each nucleotide position from 138 to 205 which is under the shadow on the entire Escherichia coli 16S rRNA secondary structure (right). The red and blue lines indicate the outer and inner boundaries of the helices respectively while grey lines connect the positions that form a base pair.
Supplemental Figure S6. Base pairs in the Bacterial 16S rRNA structure model that are identified with the helix extension method using different nucleation pairs. Red: true positive base-pairs identified in Joint N-Best method, which are used as nucleation points in the helix extension Magenta: false positives in the nucleation pairs; Blue: true positive base-pairs identified with the helix-extension method; Yellow: false-positive pairs identified with the helix-extension method. Secondary base-pairs are represented by closed circles while tertiary base-pairs are represented by open circle and highlighted with arrows. (A) Using pairs identified in PEC/JN-Best as the nucleation pairs. (B) Using pairs identified in MI/JN-Best as the nucleation pairs.
Supplemental Figure S7. Example of the determination of a purity score. For a given pair of positions in the alignment, all base-pair types are ranked by their frequency, from the highest to the lowest as shown in the middle. Starting from the highest ranked base-pair type, each base-pair type is processed to determine if both positions change (ie. covariation). The sum of the percentages of the base pair types that are a covariation (red circles) are calculated as the purity score for this set of positions. The base pairing frequency matrix is rearranged during this process. The most frequent nucleotides are first placed as the top 3? nucleotide and leftmost 5? nucleotide. Subsequently the 5? and 3? nucleotides that form a covariation pair are placed in descending order, resulting in the placement of the base pairs that covary along a diagonal.
Supplemental Figure S8. The maximal distance between the positions defined to be a neighbor effect is determined from a comparison of the number of phylogenetic events. Different phylogenetic events and their number of positions with different physical distances were calculated. Those positions with at least 10 phylogenetic events contain a large number of positions that are very close in three-dimensional space and a very small number of positions with larger physical distances.