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Background  Percentage Identity (PID) is frequently quoted in discussion of sequence alignments since it appears simple and easy to understand. However, although there are several different ways to calculate percentage identity and each may yield a different result for the same alignment, the method of calculation is rarely reported. Accordingly, quantification of the variation in PID caused by the different calculations would help in interpreting PID values in the literature. In this study, the variation in PID was quantified systematically on a reference set of 1028 alignments generated by comparison of the protein three-dimensional structures. Since the alignment algorithm may also affect the range of PID, this study also considered the effect of algorithm, and the combination of algorithm and PID method.    Results  The maximum variation in PID due to the calculation method was 11.5% while the effect of alignment algorithm on PID was up to 14.6% across three popular alignment methods. The combined effect of alignment algorithm and PID calculation gave a variation of up to 22% on the test data, with an average of 5.3% ± 2.8% for sequence pairs with < 30% identity. In order to see which PID method was most highly correlated with structural similarity, four different PID calculations were compared to similarity scores (Sc) from the comparison of the corresponding protein three-dimensional structures. The highest correlation coefficient for a PID calculation was 0.80. In contrast, the more sophisticated Z-score calculated by reference to randomized sequences gave a correlation coefficient of 0.84.    Conclusion  Although it is well known amongst expert sequence analysts that PID is a poor score for discriminating between protein sequences, the apparent simplicity of the percentage identity score encourages its widespread use in establishing cutoffs for structural similarity. This paper illustrates that not only is PID a poor measure of sequence similarity when compared to the Z-score, but that there is also a large uncertainty in reported PID values. Since better alternatives to PID exist to quantify sequence similarity, these should be quoted where possible in preference to PID. The findings presented here should prove helpful to those new to sequence analysis, and in warning those who seek to interpret the value of a PID reported in the literature.

Quantification of the variation in percentage identity for protein sequence alignments