Effective Number of Codon (ENC)

The ENC value is a simple and absolute measure of codon usage bias in genes and genomes, which can quantify the degree of CUB and reflect the extent of preference of synonymous codons. ENC values range from 20 to 61. The value of 61 indicates that there is no CUB, and the value of 20 indicates that the codon bias is at a maximum while only one codon was used for each amino acid. It is generally considered that the gene has an obvious codon usage bias if the ENC value is equal to or less than 35.

The ENC value was calculated using the following equation:

where F_{i (i = 2, 3, 4, 6)} is the average of the F_i values for i-fold degenerate codon families. The F_i value is calculated using the following equation:

where n is the total number of the observed value of codons for one amino acid and n_j stands the numbers of the particular codon for that amino acid.

To determine the influencing factors of codon usage bias, an ENC-plot, between the GC3s and ENC values, was generated using Graph Pad Prism 6.0. The expected ENC for each GC3s was calculated as follows:

where s represents the frequency of G + C of synonymous codons in the third codon position (GC3s). In ENC-plot, if the codon usage is only constrained by the GC3s, the observed ENC values just lies on or around the expected curve, and it also indicates that mutation pressure is almost the only factor for evolution. Whereas if several factors constrain the codon usage, the observed ENC values lies far lower than the expected curve (Zhang et al. 2018b).