Ka/Ks Ratio - Limitations

Limitations

Although dN/dS is a good indicator of selective pressure at the sequence level, evolutionary change can often take in the regulatory region of gene which affect the level, timing or location of gene expression. Ka/Ks analysis will not detect such change. It will only calculate selective pressure within protein coding regions. In addition, selection that does not cause differences at an amino acid level—for instance, balancing selection -- cannot be detected by these techniques.

Another issue is that heterogeneity within a gene can make a result hard to interpret. For example, if Ka/Ks = 1, it could be due to relaxed selection, or to a chimera of positive and purifying selection at the locus. A solution to this limitation would be to apply Ka/Ks analysis across many species at individual codons.

The dN/dS method requires a rather strong signal in order to detect selection. In order to detect selection between lineages, then the selection, averaged over all sites in the sequence, must produce a dN/dS greater than one—quite a feat if regions of the gene are strongly conserved. In order to detect selection at specific sites, then the dN/dS ratio must be greater than one when averaged over all included lineages at that site—implying that the site must be under selective pressure in all sampled lineages. This limitation can be moderated by allowing the dN/dS rate to take multiple values across sites and across lineages; the inclusion of more lineages also increases the power of a sites-based approach.

Further, the method lacks the capability to distinguish between positive and negative nonsynonymous substitutions. Some amino acids are chemically similar to one another, whereas other substitutions may place an amino acid with wildly different properties to its precursor. In most situations, a smaller chemical change is more likely to allow the protein to continue to function, and a large chemical change is likely to disrupt the chemical structure and cause the protein to malfunction. However, incorporating this into a model is not straightforward as the relationship between a nucleotide substitution and the effects of the modified chemical properties is very difficult to determine.

An additional concern is that the effects of time must be incorporated into an analysis, if the lineages being compared are closely related; this is because it can take a number of generations for natural selection to "weed out" deleterious mutations from a population, especially if their effect on fitness is weak. This limits the usefulness of Ka/Ks for comparing closely related populations.