SynMap

Syntenic dotplot generated by SynMap between two substrains of Escherichia coli K12, DH10B and W3110.

Overview

SynMap allows you to generate a syntenic dotplot between two organisms and identify syntenic regions. This is done by:

1. Finding putative genes or regions of homology between two genomes
2. Identifying collinear sets of genes or regions of sequence similarity to infer synteny
3. Generating a dotplot of the results and coloring syntenic pairs.

If you choose, synonymous and non-synonymous site mutation data can be calculated for protein coding genes that are identified as syntenic. These genes will then be colored based on those values in the dotplot for rapid identification of different age-classes of syntenic regions.

Specifying genomes

Selecting organisms in SynMap is done under the "select organisms" tab.

It is easy to search and select organisms in SynMap. Just select the "select organisms" tab and search for two organisms by either name or organism description. Just type part of either in the text box and SynMap automatically searches for any organism that contains your text. The results are displayed below the search boxes. Some organisms may have multiple versions of their genome and different types of sequences (e.g. masked versus unmasked). These will be displayed in a drop-down menu from which you can select the correct genome. Also, this is where you can select for comparing CDS sequence or genomic sequence. If the genome does not have CDS features, the option won't be display and a warning will be printed in below these drop-down menus. When selected, a brief description of the genome will be displayed below the drop-down menus. This will include the full organism name and description, followed by an overview of the genome. If you click on "Genome Information:" it will link to OrganismView and give you a full description of the genome. Otherwise, it will display source of the genome, the number of chromosomes in the genome, the total length of the genome in nucleotides, and whether the genome contains plasmids or contigs.

DAGChainer options

SynMap's panel for specifying DAGChainer options.

DAGChainer is the algorithm used for finding series of collinear genes (or regions of sequence similarity) between two genomes in order to infer synteny. To specify DAGChainer options, select the "DAGChainer Options" tab.

Calculating and displaying synonymous/non-synonymous (Ks, Kn) data

This option is selected under the "SynMap Options" tab

To select, just set the option "Calculate substitution rates for syntenic protein coding gene pairs and color syntenic dots accordingly" to what rate you desire:

• Ks -- synonymous
• Kn -- non-synonymous
• Kn/Ks -- ratio of non-synonymous to synonymous changes. This is often used to detected neutral (Kn/Ks == 1), positive (Kn/Ks > 1), and purifying (Kn/Ks < 1) selection acting on a pair of genes.

Synonymous (Ks) and non-synonymous (Kn) site changes are calculated by:

1. Performing a global sequence alignment of the protein sequences using the using the Needleman-Wunsch algorithm implemented in nwalign and written and maintained by Brent Pedersen using the BLOSOM62 scoring matrix.
2. Back translating the protein sequence into aligned codons
3. Using codeml of the PAML software package written and maintained by Ziheng Yang. We modified codeml for implementation in SynMap in order to minimize the number of read/write cycles to the hard drives, as well as allow it to be more easily run in parallel on multi-core servers. For each pairwise comparison of aligned codon sequences, codeml is run 5 times using its default parameter sets, and the lowest Ks is kept.

Running SynMap

Just press "Generate SynMap" when everything is configured. While SynMap runs, messages will be displayed as to the stage of the analysis that is being processed.

Example Results

Diagram showing ancestral WGD events, alpha and beta, before lineage divergence in Arabidopsis

Figure 1a: Syntenic dotplot between Arabidopsis thaliana and Arabidopsis lyrata. Syntenic gene pairs identified by DAGChainer have been colored based on their synonymous rate change as calculated by CODEML. Results can be regenerated here.

Figure 1b: Histogram of synonymous rate change data for syntenic gene pairs between Arabidopsis thaliana and Arabidopsis lyrata. The two obvious peaks in the distribution are from syntenic gene pairs (syntelogs) derived from the speciation of these two taxa and from their shared most recent whole genome duplication event, known as alpha.

Figure 2: Syntenic dotplot and synonymous substitution histogram of Chromosome 1 of Arabidopsis thaliana and Arabidopsis lyrata. Syntenic gene pairs were identified by DAGChainer, and colored based on their synonymous substitution rate as calculated by CodeML. Syntenic regions derived from speciation (orthologs) or from the shared whole genome duplication events (alpha and beta) are labeled. These images were generated by SynMap and can be viewed at generated by SynMap at http://toxic.berkeley.edu/CoGe//diags/Arabidopsis_lyrata/Arabidopsis_thaliana_thale_cress/html/3068_8.CDS-CDS.blastn.dag_geneorder_D20_g10_A5.scaffold_1-1.w600.ks.html

Figure 3: GEvo analysis of syntenic regions from Arabidopsis thaliana and Arabidopsis lyrata. At1-Al1 and At2-At2 are derived from the speciation of the taxa. This is evidenced by the strong spatial syntenic signal as seen by the extensive collinear arrangement of orthologous gene pairs (pink and blue lines). These pairs of syntenic regions are also syntenic with respect to one another, and are derived from the most recent whole genome duplication event (WGD) shared by both taxa. This event is known as "alpha". Green lines connecting the out-paralogous gene pairs are shown between Al1 and At2. While also collinear, there is a lower density of syntenic gene pairs than between either At1-Al1 or At2-Al2. Al3 is also syntenic to these regions, but is derived from prior whole genome duplication event known as beta. Dark blue lines have been drawn connecting syntenic gene pairs between Al3 and Al2, and show the lowest density of all. Results can be regenerage at http://tinyurl.com/lvpzln .

Please refer to the appropriate images for this discussion, or you can regenerate this analysis here. This example shows a whole genome syntenic dotplot comparison of Arabidopsis thaliana (At) and Arabidopsis lyrata (Al). These taxa diverged from one another ~5MYA and share two sequential whole genome duplications events since the divergence of their lineage with Carica papaya's lineage . Each whole genome duplication event creates a contemporaneous copy of every chromosome and all the genetic information they contain. However, over evolutionary time, these duplicated homeologous chromosomes are fractionated, undergo rearrangement and inversions, gene transposition events, and other genomic changes. In addition, duplicated genes that are retained (as well as surrounding non-coding sequence) will diverge from one another. Coding sequence divergence can be measured by synonymous changes (Ks), and a population of contemporaneously created syntenic genes pairs from a whole genome duplication event will create characteristic peaks in a histogram of Ks values .

Shared whole genome duplication events can be detected through syntenic dotplot analysis (spacial analysis of gene order) and through synonymous change rate (Ks) histograms (temporal analysis of coding sequence divergence) for putative homologous gene pairs. SynMap can combine these approaches and can identify collinear sets of putatively homologous genes (spatial detection of synteny), calculate Ks values for these syntelogous gene pairs (temporal calculation of synteny), and use those Ks values to generate a color-metric histogram and paint the syntelogs the appropriate color on the dotplot. This combination of temporal and spatial syntenic analysis creates a final image that permits the rapid visual identification and evaluation of shared whole genome duplication events.

Figure 1a shows a syntenic dotplot between the genomes of At (y-axis) and Al (x-axis) laid on each axis. These plots are generated by comparing every coding sequence between these taxa using blastn in order to identify putatively homologous gene pairs. These results are used by DAGChainer to find collinear sets of genes shared between the taxa. The combined data-set is plotted according to their relative genomic position where each putative homologous gene pair is plotted with a gray dot, and syntenic gene pairs are plotted with a color based on their Ks value. The comparison of At and Al's genomes shows two significant patterns of synteny. First, these two genomes have syntenic regions identified by bright-green lines that are derived from the speciation divergence of these two taxa. Socond, there are smaller blocks of yellow-green lines that are derived from their shared whole genome duplication event (WGD) know as alpha. Comparison to the Ks histogram (Figure 1B) shows that the bright-green has smaller Ks values (fewer changes) than the yellow-green line, which is to be expected as their divergence post-dates their shared whole genome duplication event.

Generating a close-up view of the comparison of chromosome one from both taxa (Figure 2), reveals a similar pattern (light-blue orthologs, light-green out-paralogs derived from their shared most recent whole genome duplication event), with additional evidence of the more ancient shared whole genome duplication event known as beta. The beta whole genome duplication event is visualized by much smaller identified syntenic regions colored in yellow-orange. These syntenic gene-pairs correlate to a smaller peak in the Ks histogram with a larger mean Ks value than the subsequent whole genome duplication even (alpha) or the orthologs derived from the divergence of these taxa.

In order to validate and access the types and patterns of change at these genomic loci, high-resolution analysis of these syntenic regions can be performed using GEvo, and selecting the appropriate set of genomic regions using SynMap's interface. Such an analysis can be seen in figure 3 which compares five syntenic regions from these taxa. Two pairs of regions, At1-Al1 and At2-Al2, are orthologous and derived from the speciation of these lineages. This is evidenced by the high degree of spatial evidence for synteny between these regions (pink and blue lines) where nearly every gene in these regions has an orthologous partner in a collinear arrangement. These two pairs of regions are also syntenic with respect to one another (green lines) and are derived from their shared most recent whole genome duplication event (WGD) known as alpha. These four regions are syntenic to an additional region, Al3, which is derived from these lineages' shared second most recent whole genome duplication event known as beta. Syntenic genes are connected between Al2 and Al3 using dark blue lines, and note the lower density of syntenic gene pairs than for regions derived from the most recent WGD and the speciation of the lineages. While not shown in this figure, there is a syntenic region in At to Al3 from the speciation of these taxa, and two addition syntenic regions (one from each lineage) derived from the alpha whole genome duplication.

Please note that the Ks histogram is using log10 transformed Ks values. While many people set an upper cutoff for Ks values (usually at 2), these histograms show all values. The peak in the both these Ks histograms (Mean Ks ~ 65) at the far right and colored red is the result from mis-called syntenic gene pairs, genes whose alignments are very poor (e.g. due to a frame-shift mutation or pseudogenization), or from an error in the Ks calculation.