Accounting for background nucleotide composition when measuring codon usage bias: brilliant idea, difficult in practice

The effective number of codons used in a gene is a commonly used measure of codon usage. It varies between 20 and 61 (standard genetic code) and indicates to which degree the entire genetic code is used. It is a drawback of this method that it does not take background composition into account. This led Novembre to introduce a variant called Nc' (Novembre JA. 2002. Accounting for background nucleotide composition when measuring codon usage bias. Mol Biol Evol 19:1390-4). In this letter, its properties are under the loupe, with special emphasis on phenomena relating to codon homozygosity. A theoretical misunderstanding regarding this estimator is explained in detail, notably Nc varies between 0 and 61 instead of 20 and 61 (with the standard genetic code). Practical examples from the genome of Pseudomonas aeruginosa are given which demonstrate that the problem is not just theoretical.