The General Average Neutrality Score (GRAVY)

Where, s represents the value of( G+C * 3%).
Ideally, the ENC should be in the range 20–61, so that when each of the amino acids is encoded by only one codon, ENC is 20 and when all the synonyms of codons have equal chances, ENC is 61. The more significant the codon usage bias, the lower the ENC value. The General Average hydrophobicity score (GRAVY) is the hypothetical translation of a gene product. GRAVY is the arithmetic mean of the hydropathic indices of each amino acid [40].
GRAVY has been used to quantitatively assess the major (PCA) trends in the usage of amino acid E. coli genes [30]. An aromaticity score (Aromo) determines the frequency of the aromatic amino acids (Phe, tyr and trp) in the hypothetical translation of a gene product. According to Lobry (2000), the strongest trend variation in the composition of amino acids in E. coli genes is associated with GRAVY, the second trend is correlated with gene expression, while the third is linked with the Aromo[30]. The change in the composition of amino acids may be useful to analyze codon usage.

The closer the plot to a slope of 0, the less impact from direct mutation pressure. When the slope is 1, the codon usage bias is fully formed by direct mutation pressure and neutrality. GC1 and GC2 contents were calculated using Perl