Codon Usage

This calculator shows how often a gene uses a particular codon compared with its synonymous alternatives, so you can tell whether that codon is preferred or avoided, for example before codon-optimizing a gene for expression.

How the calculation works

The genetic code is degenerate: most amino acids are encoded by two, three, four or six synonymous codons (methionine and tryptophan have only one). The calculator returns three measures. Codon share = n / N_aa × 100%, where n is how many times the codon occurs and N_aa is the total count of codons for that amino acid. RSCU, relative synonymous codon usage (Sharp, Tuohy and Mosurski, 1986), = n / (N_aa / k), where k is the number of synonymous codons. Frequency per thousand = n / N_total × 1000, the format used in codon usage tables such as the Kazusa database.

RSCU compares the observed count with the count expected if all synonyms were used equally. RSCU = 1 means no bias, above 1 marks a preferred codon, below 1 an avoided one, and the maximum possible value is k. Enter the codon count, the total codons for its amino acid, the number of synonyms (1–6) and the gene length in codons.

Worked example

A four-codon amino acid such as alanine appears 100 times in a gene, and the codon of interest accounts for 30 of them. Share = 30 / 100 = 30%. Equal use would give 100 / 4 = 25, so RSCU = 30 / 25 = 1.2, a preferred codon. In a gene of 1000 codons its frequency is 30 per thousand. By contrast, a leucine codon (six synonyms) seen 5 times out of 100 has RSCU = 5 / (100/6) = 0.3, clearly avoided.

Things to keep in mind

• Small counts are noisy: 2 out of 4 gives RSCU 2.0 for a four-codon amino acid, which says almost nothing.
• For expression work, compare against the host organism’s codon table, not a single gene; what matters is how common the codon is in the cell making the protein.
• Stop codons form their own synonym group (three in the standard code) but are usually analyzed separately.
• To score a whole gene, use the Codon Adaptation Index (CAI; Sharp and Li, 1987), which is built from RSCU values.