Neutrality analysis
Neutrality plot analysis was used to assess codon usage bias caused by
mutational pressure and natural selection. Codon neutrality is expressed
through the relationship between GC1,2 (average GC
content between GC1 and GC2) and
GC3, represented by a regression line (neutrality
plot)34,35. The slope ε of this regression
line, calculated using least squares regression analysis, is a
neutrality value that ranges from 0 when there is no effect of mutation
pressure (full selective constraints) to 1 when mutation bias is the
main force affecting the codon usage (complete neutrality of codon
usage). The equilibrium point Ep was also
defined. This point is at the intersection of the neutrality plot
(regression line) and the line of complete equilibrium between the
average GC content and the directional mutation pressure with neutrality
equal to one (ε = 1). The Ep value was
computed as\(\frac{\mathrm{A}_{\mathrm{0}}}{\left(\mathrm{1-\varepsilon}\right)}\),
where A0 is the value calculated from the
regression of GC1,2 and GC3 at
GC3 = 034. The direction of the
mutational pressure in the gene was defined according to the position of
this gene on the neutrality plot relative to the equilibrium point.